@article{tandrup_lo leggio_meilleur_2023, title={Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature}, volume={79}, ISSN={["2053-230X"]}, url={https://doi.org/10.1107/S2053230X22011335}, DOI={10.1107/S2053230X22011335}, abstractNote={Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state refined against X-ray and neutron data at 2.1 and 2.8 Å resolution, respectively, is presented. The results provide an experimental determination of the protonation states of the copper(II)-coordinating residues and second-shell residues in LsAA9_A, paving the way for future neutron crystallographic studies of LPMO–carbohydrate complexes.}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Tandrup, Tobias and Lo Leggio, Leila and Meilleur, Flora}, year={2023}, month={Jan}, pages={1–7} }
 @article{meilleur_2023, title={Neutron Scattering in the Biological Sciences: Techniques And Applications}, volume={1}, ISSN={["1940-087X"]}, DOI={10.3791/64806}, abstractNote={ARTICLES DISCUSSED
Vahdatahar, E., Junius, N., Budayova-Spano, M. Optimization of crystal growth for neutron macromolecular crystallography. Journal of Visualized Experiments. (169), e61685 (2021). Schröder, G. C., Meilleur, F. Neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures. Journal of Visualized Experiments. (166), e61903 (2020). Kelley, E. G., Nguyen, M. H. L., Marquardt, D., Maranville, B. B., Murphy, R. P. Measuring the time-evolution of nanoscale materials with stopped-flow and small-angle neutron scattering. Journal of Visualized Experiments. (174), e62873 (2021). Bilheux, H. Z. et al. Neutron radiography and computed tomography of biological systems at the Oak Ridge National Laboratory's high flux isotope reactor. Journal of Visualized Experiments. (171), e61688 (2021). Stingaciu, L.-R. Study of protein dynamics via neutron spin echo spectroscopy. Journal of Visualized Experiments. (182), e61862 (2022). Kumarage, T., Nguyen, J., Ashkar, R. Neutron spin echo spectroscopy as a unique probe for lipid membrane dynamics and membrane-protein interactions. Journal of Visualized Experiments. (171), e62396 (2021).}, number={191}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={Meilleur, Flora}, year={2023}, month={Jan} }
 @article{dagher_vaishnav_stanley_meilleur_edwards_bruno-barcena_2023, title={Structural analysis and functional evaluation of the disordered ß-hexosyltransferase region from <i>Hamamotoa (Sporobolomyces) singularis</i>}, volume={11}, ISSN={["2296-4185"]}, url={http://dx.doi.org/10.3389/fbioe.2023.1291245}, DOI={10.3389/fbioe.2023.1291245}, abstractNote={Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1–110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region’s impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%–97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.}, journal={FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY}, author={Dagher, Suzanne F. and Vaishnav, Asmita and Stanley, Christopher B. and Meilleur, Flora and Edwards, Brian F. P. and Bruno-Barcena, Jose M.}, year={2023}, month={Dec} }
 @article{schroder_william b. o'dell_webb_agarwal_meilleur_2022, title={Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase}, volume={11}, ISSN={["2041-6539"]}, url={https://doi.org/10.1039/D2SC05031E}, DOI={10.1039/D2SC05031E}, abstractNote={Superoxo and hydroperoxo intermediates were cryotrapped at the copper active site of lytic polysaccharide monooxygenase using neutron protein crystallography.}, journal={CHEMICAL SCIENCE}, author={Schroder, Gabriela C. and William B. O'Dell and Webb, Simon P. and Agarwal, Pratul K. and Meilleur, Flora}, year={2022}, month={Nov} }
 @article{tandrup_muderspach_banerjee_santoni_ipsen_hernandez-rollan_norholm_johansen_meilleur_lo leggio_2022, title={Changes in active-site geometry on X-ray photo-reduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding}, volume={9}, ISSN={["2052-2525"]}, url={https://doi.org/10.1107/S2052252522007175}, DOI={10.1107/S2052252522007175}, abstractNote={The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu2+ to Cu+. Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis (LsAA9_A) and one from Thermoascus aurantiacus (TaAA9_A), as the active-site Cu is photoreduced in the X-ray beam. For LsAA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu2+ form use a total X-ray dose below 3 × 104 Gy, while the Cu+ form can be attained using 1 × 106 Gy. In all cases, we observe the transition from a hexacoordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ2 parameter and a decrease of the θ3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θT. Thus, the θ2 and θ3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to LsAA9_A, regardless of the production source, the θT parameter increases, making the Cu site less planar, while the active-site Tyr—Cu distance decreases reproducibly for the Cu2+ form. Thus, the θT increase found on copper reduction may bring LsAA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced LsAA9_A for cellulosic substrates.}, number={5}, journal={IUCRJ}, publisher={International Union of Crystallography (IUCr)}, author={Tandrup, Tobias and Muderspach, Sebastian J. and Banerjee, Sanchari and Santoni, Gianluca and Ipsen, Johan O. and Hernandez-Rollan, Cristina and Norholm, Morten H. H. and Johansen, Katja S. and Meilleur, Flora and Lo Leggio, Leila}, year={2022}, month={Sep}, pages={666–681} }
 @article{moreno-chicano_carey_axford_beale_doak_duyvesteyn_ebrahim_henning_monteiro_myles_et al._2022, title={Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature}, volume={9}, ISSN={["2052-2525"]}, DOI={10.1107/S2052252522006418}, abstractNote={Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.}, journal={IUCRJ}, author={Moreno-Chicano, Tadeo and Carey, Leiah M. and Axford, Danny and Beale, John H. and Doak, R. Bruce and Duyvesteyn, Helen M. E. and Ebrahim, Ali and Henning, Robert W. and Monteiro, Diana C. F. and Myles, Dean A. and et al.}, year={2022}, month={Sep}, pages={610–624} }
 @article{david_setzler_sorescu_lieberman_meilleur_petty_2022, title={Mapping H+ in the Nanoscale (A(2)C(4))(2)-Ag-8 Fluorophore}, volume={12}, ISSN={["1948-7185"]}, url={https://doi.org/10.1021/acs.jpclett.2c03161}, DOI={10.1021/acs.jpclett.2c03161}, abstractNote={When strands of DNA encapsulate silver clusters, supramolecular optical chromophores develop. However, how a particular structure endows a specific spectrum remains poorly understood. Here, we used neutron diffraction to map protonation in (A2C4)2-Ag8, a green-emitting fluorophore with a "Big Dipper" arrangement of silvers. The DNA host has two substructures with distinct protonation patterns. Three cytosines from each strand collectively chelate handle-like array of three silvers, and calorimetry studies suggest Ag+ cross-links. The twisted cytosines are further joined by hydrogen bonds from fully protonated amines. The adenines and their neighboring cytosine from each strand anchor a dipper-like group of five silvers via their deprotonated endo- and exocyclic nitrogens. Typically, exocyclic amines are strongly basic, so their acidification and deprotonation in (A2C4)2-Ag8 suggest that silvers perturb the electron distribution in the aromatic nucleobases. The different protonation states in (A2C4)2-Ag8 suggest that atomic level structures can pinpoint how to control and tune the electronic spectra of these nanoscale chromophores.}, journal={JOURNAL OF PHYSICAL CHEMISTRY LETTERS}, author={David, Fred and Setzler, Caleb and Sorescu, Alexandra and Lieberman, Raquel L. and Meilleur, Flora and Petty, Jeffrey T.}, year={2022}, month={Dec} }
 @article{dawe_manuel garcia-ruiz_hadju_mcintyre_meilleur_stephenson_2022, title={Teaching and Education highlighted}, volume={55}, ISSN={["1600-5767"]}, url={https://doi.org/10.1107/S1600576722003661}, DOI={10.1107/S1600576722003661}, abstractNote={The development of the Teaching and Education (T&E) category of articles in Journal of Applied Crystallography, leading to the recent appointment of Louise Dawe as an additional T&E Editor, is described. Louise and ongoing T&E Editor Juan Manuel García-Ruiz outline their vision for the category.}, number={2}, journal={JOURNAL OF APPLIED CRYSTALLOGRAPHY}, publisher={International Union of Crystallography (IUCr)}, author={Dawe, Louise N. and Manuel Garcia-Ruiz, Juan and Hadju, Janos and McIntyre, Garry J. and Meilleur, Flora and Stephenson, Lisa}, year={2022}, month={Apr}, pages={215–217} }
 @article{sacco_hu_gongora_meilleur_kemp_zhang_wang_chen_2022, title={The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition}, volume={3}, ISSN={["1748-7838"]}, DOI={10.1038/s41422-022-00640-y}, journal={CELL RESEARCH}, author={Sacco, Michael Dominic and Hu, Yanmei and Gongora, Maura Verenice and Meilleur, Flora and Kemp, Michael Trent and Zhang, Xiujun and Wang, Jun and Chen, Yu}, year={2022}, month={Mar} }
 @article{correy_kneller_phillips_pant_russi_cohen_meigs_holton_gahbauer_thompson_et al._2022, title={The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature}, volume={8}, ISSN={["2375-2548"]}, DOI={10.1126/sciadv.abo5083}, abstractNote={The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose–bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.}, number={21}, journal={SCIENCE ADVANCES}, author={Correy, Galen J. and Kneller, Daniel W. and Phillips, Gwyndalyn and Pant, Swati and Russi, Silvia and Cohen, Aina E. and Meigs, George and Holton, James M. and Gahbauer, Stefan and Thompson, Michael C. and et al.}, year={2022}, month={May} }
 @article{schröder_o'dell_webb_agarwal_meilleur_2021, title={Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase}, url={https://doi.org/10.26434/chemrxiv-2021-n4c1x}, DOI={10.26434/chemrxiv-2021-n4c1x}, abstractNote={Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory (DFT) calculations indicate that both active site states are stable. The hydroperoxo formed is potentially an intermediate in the mechanism of hydrogen peroxide formation in the absence of substrate. We establish that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by mining minima free energy calculations we establish His157 conformational flexibility in solution that is abolished by steric hindrance in the crystal. A neutron crystal structure of NcLPMO9D at low pH supports occlusion of the active site which prevents protonation of His157 at acidic conditions.}, author={Schröder, Gabriela C. and O'Dell, William B. and Webb, Simon P. and Agarwal, Pratul K. and Meilleur, Flora}, year={2021}, month={Dec} }
 @article{schroeder_meilleur_2021, title={Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography}, volume={77}, ISSN={["2059-7983"]}, url={https://doi.org/10.1107/S2059798321009025}, DOI={10.1107/S2059798321009025}, abstractNote={Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.}, number={10}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, publisher={International Union of Crystallography (IUCr)}, author={Schroeder, Gabriela C. and Meilleur, Flora}, year={2021}, month={Oct}, pages={1251–1269} }
 @article{schroder_william b. o'dell_swartz_meilleur_2021, title={Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions}, volume={77}, ISSN={["2053-230X"]}, url={https://doi.org/10.1107/S2053230X21002399}, DOI={10.1107/S2053230X21002399}, abstractNote={Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper–oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.}, number={4}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={Schroder, Gabriela C. and William B. O'Dell and Swartz, Paul D. and Meilleur, Flora}, year={2021}, month={Apr}, pages={128–133} }
 @article{meilleur_2020, title={A beginner’s guide to neutron macromolecular crystallography}, volume={42}, url={https://doi.org/10.1042/BIO20200078}, DOI={10.1042/BIO20200078}, abstractNote={Hydrogen atoms drive biological structure and function, but the lightest element is often unseen in three-dimensional macromolecule structures, hampering our understanding of biochemical processes. This guide will i) present how neutron crystallography uniquely reveals the experimental positions of hydrogen atoms and resolves mechanical controversies, ii) briefly introduce beamlines at neutron facilities, iii) discuss sample requirements and preparation and iv) familiarize the reader with neutron data and refinement statistics.}, number={6}, journal={The Biochemist}, publisher={Portland Press Ltd.}, author={Meilleur, Flora}, year={2020}, month={Dec}, pages={16–20} }
 @article{pierce_cuneo_jennings_li_meilleur_zhao_myles_2020, title={Dynamic nuclear polarization enhanced neutron crystallography: Amplifying hydrogen in biological crystals}, volume={634}, ISSN={["0076-6879"]}, DOI={10.1016/bs.mie.2019.11.018}, abstractNote={Dynamic nuclear polarization (DNP) can provide a powerful means to amplify neutron diffraction from biological crystals by 10–100-fold, while simultaneously enhancing the visibility of hydrogen by an order of magnitude. Polarizing the neutron beam and aligning the proton spins in a polarized sample modulates the coherent and incoherent neutron scattering cross-sections of hydrogen, in ideal cases amplifying the coherent scattering by almost an order of magnitude and suppressing the incoherent background to zero. This chapter describes current efforts to develop and apply DNP techniques for spin polarized neutron protein crystallography, highlighting concepts, experimental design, labeling strategies and recent results, as well as considering new strategies for data collection and analysis that these techniques could enable.}, journal={NEUTRON CRYSTALLOGRAPHY IN STRUCTURAL BIOLOGY}, author={Pierce, Joshua and Cuneo, Matthew J. and Jennings, Anna and Li, Le and Meilleur, Flora and Zhao, Jinkui and Myles, Dean A. A.}, year={2020}, pages={153–175} }
 @article{meilleur_kovalevsky_myles_2020, title={IMAGINE: The neutron protein crystallography beamline at the high flux isotope reactor}, volume={634}, ISSN={["0076-6879"]}, DOI={10.1016/bs.mie.2019.11.016}, abstractNote={IMAGINE is a high intensity, quasi-Laue neutron crystallography beamline developed at the 85 MW High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory (ORNL). This state-of-the-art facility for neutron-diffraction enables neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of < 1 mm3 and unit cell edges of < 150 Å. The beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. The beamline is equipped with a single-axis goniometer, neutron-sensitive cylindrical image plate detector and room temperature and cryogenic sample environments. This article describes the beamline components, the diffractometer and the data collection and data analysis protocols that are used, and outlines the protein deuteration, crystallization and conventional crystallography capabilities that are available to users at ORNL's neutron facilities. We also present examples of the scientific questions being addressed at this beamline and highlight important findings in enzyme chemistry that have been made possible by IMAGINE.}, journal={NEUTRON CRYSTALLOGRAPHY IN STRUCTURAL BIOLOGY}, author={Meilleur, Flora and Kovalevsky, Andrey and Myles, Dean A. A.}, year={2020}, pages={69–85} }
 @article{schroeder_meilleur_2020, title={Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures}, volume={12}, ISSN={["1940-087X"]}, DOI={10.3791/61903}, abstractNote={Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam rapidly induces radiation damage of photosensitive cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (> 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.}, number={166}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={Schroeder, Gabriela C. and Meilleur, Flora}, year={2020}, month={Dec} }
 @article{pierce_crow_cuneo_edwards_herwig_jennings_jones_li_meilleur_myles_et al._2019, title={A prototype system for dynamically polarized neutron protein crystallography}, volume={940}, ISSN={["1872-9576"]}, DOI={10.1016/j.nima.2019.06.023}, abstractNote={The sensitivity of Neutron Macromolecular Crystallography to the presence of hydrogen makes it a powerful tool to complement X-ray crystallographic studies using protein crystals. The power of this technique is currently limited by the relative low neutron flux provided by even the most powerful neutron sources. The strong polarization dependence of the neutron scattering cross section of hydrogen will allow us to use Dynamic Nuclear Polarization to dramatically improve the signal to noise ratio of neutron diffraction data, delivering order of magnitude gains in performance, and enabling measurements of radically smaller crystals of larger protein systems than are possible today. We present a prototype frozen spin system, built at Oak Ridge National Laboratory to polarize single protein crystals on the IMAGINE beamline at the High Flux Isotope Reactor (HFIR). Details of the design and construction will be described, as will the performance of the system offline and during preliminary tests at HFIR.}, journal={NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH SECTION A-ACCELERATORS SPECTROMETERS DETECTORS AND ASSOCIATED EQUIPMENT}, author={Pierce, J. and Crow, L. and Cuneo, M. and Edwards, M. and Herwig, K. W. and Jennings, A. and Jones, A. and Li, L. and Meilleur, F. and Myles, D. A. A. and et al.}, year={2019}, month={Oct}, pages={430–434} }
 @article{lu_selvaraj_ghimire-rijal_orf_meilleur_blankenship_cuneo_myles_2019, title={Neutron and X-ray analysis of the Fenna-Matthews-Olson photosynthetic antenna complex from Prosthecochloris aestuarii}, volume={75}, ISSN={["2053-230X"]}, url={https://doi.org/10.1107/S2053230X19000724}, DOI={10.1107/S2053230X19000724}, abstractNote={The Fenna–Matthews–Olson protein from Prosthecochloris aestuarii (PaFMO) has been crystallized in a new form that is amenable to high-resolution X-ray and neutron analysis. The crystals belonged to space group H3, with unit-cell parameters a = b = 83.64, c = 294.78 Å, and diffracted X-rays to ∼1.7 Å resolution at room temperature. Large PaFMO crystals grown to volumes of 0.3–0.5 mm3 diffracted neutrons to 2.2 Å resolution on the MaNDi neutron diffractometer at the Spallation Neutron Source. The resolution of the neutron data will allow direct determination of the positions of H atoms in the structure, which are believed to be fundamentally important in tuning the individual excitation energies of bacteriochlorophylls in this archetypal photosynthetic antenna complex. This is one of the largest unit-cell systems yet studied using neutron diffraction, and will allow the first high-resolution neutron analysis of a photosynthetic antenna complex.}, number={3}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={Lu, Xun and Selvaraj, Brinda and Ghimire-Rijal, Sudipa and Orf, Gregory S. and Meilleur, Flora and Blankenship, Robert E. and Cuneo, Matthew J. and Myles, Dean A. A.}, year={2019}, month={Mar}, pages={171–175} }
 @article{knihtila_volmar_meilleur_mattos_2019, title={Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras}, volume={75}, ISSN={["2053-230X"]}, url={https://doi.org/10.1107/S2053230X18018125}, DOI={10.1107/S2053230X18018125}, abstractNote={Neutron protein crystallography (NPC) reveals the three-dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X-ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in enzyme catalysis, NPC has the advantage over X-ray crystallography that the crystals do not suffer radiation damage. The lack of radiation damage can be exploited to conduct in crystallo parametric studies. Here, the use of a single crystal of the small GTPase Ras to collect three neutron data sets at pD 8.4, 9.0 and 9.4 is reported, enabling an in crystallo titration study using NPC. In addition to revealing the behavior of titratable groups in the active site, the data sets will allow the analysis of allosteric water-mediated communication networks across the molecule, particularly regarding Cys118 and three tyrosine residues central to these networks, Tyr32, Tyr96 and Tyr137, with pK
                  a values expected to be in the range sampled in our experiments.}, number={2}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={Knihtila, Ryan and Volmar, Alicia Y. and Meilleur, Flora and Mattos, Carla}, year={2019}, month={Feb}, pages={111–115} }
 @misc{coates_cao_chakoumakos_frontzek_hoffmann_kovalevsky_liu_meilleur_santos_myles_et al._2018, title={A suite-level review of the neutron single-crystal diffraction instruments at Oak Ridge National Laboratory}, volume={89}, ISSN={["1089-7623"]}, url={https://doi.org/10.1063/1.5030896}, DOI={10.1063/1.5030896}, abstractNote={The nascent suite of single-crystal neutron diffractometers at the Oak Ridge National Laboratory has no equal at any other neutron scattering facility worldwide and offers the potential to re-assert single-crystal diffraction using neutrons as a significant tool to study nuclear and magnetic structures of small unit cell crystals, nuclear structures of macromolecules, and diffuse scattering. Signature applications and features of single-crystal neutron diffraction are high resolution nuclear structure analysis, magnetic structure and spin density determinations, contrast variation (particularly D2O/H2O) for nuclear structural studies, lack of radiation damage when using crystals of biological molecules such as proteins, and the fidelity to measure nuclear and magnetic diffuse scattering with elastic discrimination.}, number={9}, journal={REVIEW OF SCIENTIFIC INSTRUMENTS}, author={Coates, L. and Cao, H. B. and Chakoumakos, B. C. and Frontzek, M. D. and Hoffmann, C. and Kovalevsky, A. Y. and Liu, Y. and Meilleur, F. and Santos, A. M. and Myles, D. A. A. and et al.}, year={2018}, month={Sep} }
 @article{duff_borreguero_cuneo_ramanathan_he_kamath_chennubhotla_meilleur_howell_herwig_et al._2018, title={Modulating Enzyme Activity by Altering Protein Dynamics with Solvent}, volume={57}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/ACS.BIOCHEM.8B00424}, DOI={10.1021/ACS.BIOCHEM.8B00424}, abstractNote={Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aqueous mixtures of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent khydride rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational substates, explaining the decreased khydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.}, number={29}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Duff, Michael R., Jr and Borreguero, Jose M. and Cuneo, Matthew J. and Ramanathan, Arvind and He, Junhong and Kamath, Ganesh and Chennubhotla, S. Chakra and Meilleur, Flora and Howell, Elizabeth E. and Herwig, Kenneth W. and et al.}, year={2018}, month={Jun}, pages={4263–4275} }
 @article{ashkar_bilheux_bordallo_briber_callaway_cheng_chu_curtis_dadmun_fenimore_et al._2018, title={Neutron scattering in the biological sciences: progress and prospects}, volume={74}, ISSN={["2059-7983"]}, DOI={10.1107/S2059798318017503}, abstractNote={The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Ashkar, Rana and Bilheux, Hassina Z. and Bordallo, Heliosa and Briber, Robert and Callaway, David J. E. and Cheng, Xiaolin and Chu, Xiang-Qiang and Curtis, Joseph E. and Dadmun, Mark and Fenimore, Paul and et al.}, year={2018}, month={Dec}, pages={1129–1168} }
 @article{haberl_dissanayake_wu_myles_santos_loguillo_rucker_armitage_cochran_andrews_et al._2018, title={Next-generation diamond cell and applications to single-crystal neutron diffraction}, volume={89}, ISSN={["1089-7623"]}, DOI={10.1063/1.5031454}, abstractNote={A diamond cell optimized for single-crystal neutron diffraction is described. It is adapted for work at several of the single-crystal diffractometers of the Spallation Neutron Source and the High Flux Isotope Reactor at the Oak Ridge National Laboratory (ORNL). A simple spring design improves portability across the facilities and affords load maintenance from offline pressurization and during temperature cycling. Compared to earlier prototypes, pressure stability of polycrystalline diamond (Versimax®) has been increased through double-conical designs and ease of use has been improved through changes to seat and piston setups. These anvils allow ∼30%-40% taller samples than possible with comparable single-crystal anvils. Hydrostaticity and the important absence of shear pressure gradients have been established with the use of glycerin as a pressure medium. Large single-crystal synthetic diamonds have also been used for the first time with such a clamp-diamond anvil cell for pressures close to 20 GPa. The cell is made from a copper beryllium alloy and sized to fit into ORNL’s magnets for future ultra-low temperature and high-field studies. We show examples from the Spallation Neutron Source’s SNAP and CORELLI beamlines and the High Flux Isotope Reactor’s HB-3A and IMAGINE beamlines.}, number={9}, journal={REVIEW OF SCIENTIFIC INSTRUMENTS}, author={Haberl, Bianca and Dissanayake, Sachith and Wu, Yan and Myles, Dean A. A. and Santos, Antonio M. and Loguillo, Mark and Rucker, Gerald M. and Armitage, Douglas P. and Cochran, Malcolm and Andrews, Katie M. and et al.}, year={2018}, month={Sep} }
 @article{bodenheimer_odell_oliver_qian_stanley_meilleur_2018, title={Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms}, volume={1862}, ISSN={["1872-8006"]}, DOI={10.1016/j.bbagen.2018.01.016}, abstractNote={Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized.Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa.The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation.These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D.The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.}, number={4}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS}, author={Bodenheimer, Annette M. and ODell, William B. and Oliver, Ryan C. and Qian, Shuo and Stanley, Christopher B. and Meilleur, Flora}, year={2018}, month={Apr}, pages={1031–1039} }
 @misc{meilleur_coates_cuneo_kovalevsky_myles_2018, title={The Neutron Macromolecular Crystallography Instruments at Oak Ridge National Laboratory: Advances, Challenges, and Opportunities}, volume={8}, ISSN={["2073-4352"]}, DOI={10.3390/cryst8100388}, abstractNote={The IMAGINE and MaNDi instruments, located at Oak Ridge National Laboratory High Flux Isotope Reactor and Spallation Neutron Source, respectively, are powerful tools for determining the positions of hydrogen atoms in biological macromolecules and their ligands, orienting water molecules, and for differentiating chemical states in macromolecular structures. The possibility to model hydrogen and deuterium atoms in neutron structures arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction studies at the 1.5–2.5 Å resolutions, which are typical for protein crystals. Neutrons have the additional benefit for structural biology of not inducing radiation damage to protein crystals, which can be critical in the study of metalloproteins. Here we review the specifications of the IMAGINE and MaNDi beamlines and illustrate their complementarity. IMAGINE is suitable for crystals with unit cell edges up to 150 Å using a quasi-Laue technique, whereas MaNDi provides neutron crystallography resources for large unit cell samples with unit cell edges up to 300 Å using the time of flight (TOF) Laue technique. The microbial culture and crystal growth facilities which support the IMAGINE and MaNDi user programs are also described.}, number={10}, journal={CRYSTALS}, author={Meilleur, Flora and Coates, Leighton and Cuneo, Matthew J. and Kovalevsky, Andrey and Myles, Dean A. A.}, year={2018}, month={Oct} }
 @article{golden_yu_meilleur_blakeley_duff_karton_vrielink_2017, title={An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase}, volume={7}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/SREP40517}, DOI={10.1038/srep40517}, abstractNote={Abstract}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Golden, Emily and Yu, Li-Juan and Meilleur, Flora and Blakeley, Matthew P. and Duff, Anthony P. and Karton, Amir and Vrielink, Alice}, year={2017}, month={Jan}, pages={40517,} }
 @article{william b. o'dell_swartz_weiss_meilleur_2017, title={Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction}, volume={73}, ISSN={["2053-230X"]}, DOI={10.1107/s2053230x16020318}, abstractNote={Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.}, number={2}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={William B. O'Dell and Swartz, Paul D. and Weiss, Kevin L. and Meilleur, Flora}, year={2017}, month={Feb}, pages={70–78} }
 @article{o'dell_agarwal_meilleur_2017, title={Innentitelbild: Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase (Angew. Chem. 3/2017)}, url={https://doi.org/10.1002/ange.201611987}, DOI={10.1002/ange.201611987}, abstractNote={Die oxidative Spaltung glykosidischer Bindungen ermöglicht es lytischen Polysaccharid-Monooxygenasen (LPMOs), die Polysaccharidketten aufzubrechen. Diese Aktivität verstärkt die enzymatische Hydrolyse von hartnäckiger Kohlenhydrat-Biomasse wie Zellulose oder Chitin. F. Meilleur und Mitarbeiter liefern in ihrer Zuschrift auf S. 785 eine strukturelle Beschreibung der Sauerstoffaktivierung am einkernigen Kupferzentrum einer LPMO auf der Grundlage von Röntgen- und Neutronenkristallographie sowie DFT-Rechnungen. Bild: J. Hemman.}, journal={Angewandte Chemie}, author={O'Dell, William B. and Agarwal, Pratul K. and Meilleur, Flora}, year={2017}, month={Jan} }
 @article{li_shukla_meilleur_standaert_pierce_myles_cuneo_2017, title={Neutron crystallographic studies of T4 lysozyme at cryogenic temperature}, volume={26}, ISSN={["1469-896X"]}, DOI={10.1002/pro.3231}, abstractNote={Abstract}, number={10}, journal={PROTEIN SCIENCE}, author={Li, Le and Shukla, Shantanu and Meilleur, Flora and Standaert, Robert F. and Pierce, Josh and Myles, Dean A. A. and Cuneo, Matthew J.}, year={2017}, month={Oct}, pages={2098–2104} }
 @article{hiromoto_meilleur_shimizu_shibazaki_adachi_tamada_kuroki_2017, title={Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type}, volume={26}, ISSN={["1469-896X"]}, DOI={10.1002/pro.3230}, abstractNote={Abstract}, number={10}, journal={PROTEIN SCIENCE}, author={Hiromoto, Takeshi and Meilleur, Flora and Shimizu, Rumi and Shibazaki, Chie and Adachi, Motoyasu and Tamada, Taro and Kuroki, Ryota}, year={2017}, month={Oct}, pages={1953–1963} }
 @article{william b. o'dell_agarwal_meilleur_2017, title={Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase}, volume={56}, ISSN={["1521-3773"]}, url={https://doi.org/10.1002/anie.201610502}, DOI={10.1002/anie.201610502}, abstractNote={Abstract}, number={3}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, publisher={Wiley}, author={William B. O'Dell and Agarwal, Pratul K. and Meilleur, Flora}, year={2017}, month={Jan}, pages={767–770} }
 @article{o'dell_agarwal_meilleur_2017, title={Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase}, volume={129}, url={https://doi.org/10.1002/ange.201610502}, DOI={10.1002/ange.201610502}, abstractNote={Abstract}, number={3}, journal={Angewandte Chemie}, publisher={Wiley}, author={O'Dell, William B. and Agarwal, Pratul K. and Meilleur, Flora}, year={2017}, month={Jan}, pages={785–788} }
 @article{bodenheimer_william b. o'dell_stanley_meilleur_2017, title={Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering}, volume={448}, ISSN={["1873-426X"]}, DOI={10.1016/j.carres.2017.03.001}, abstractNote={Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.}, journal={CARBOHYDRATE RESEARCH}, publisher={Elsevier BV}, author={Bodenheimer, Annette M. and William B. O'Dell and Stanley, Christopher B. and Meilleur, Flora}, year={2017}, month={Aug}, pages={200–204} }
 @article{bodenheimer_meilleur_2016, title={Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states}, volume={590}, ISSN={["1873-3468"]}, DOI={10.1002/1873-3468.12464}, abstractNote={Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild‐type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product‐binding site mutants.}, number={23}, journal={FEBS LETTERS}, publisher={Wiley-Blackwell}, author={Bodenheimer, Annette M. and Meilleur, Flora}, year={2016}, month={Dec}, pages={4429–4438} }
 @article{mascarelli_tartara_pereyra_maggi_2016, title={Detection of Mycoplasma haemocanis, Mycoplasma haematoparvum, Mycoplasma suis and other vector-borne pathogens in dogs from Córdoba and Santa Fé, Argentina}, volume={9}, ISSN={1756-3305}, url={http://dx.doi.org/10.1186/s13071-016-1920-8}, DOI={10.1186/s13071-016-1920-8}, abstractNote={In Argentina, only very few reports are available for canine tick-borne diseases where most are related to parasitic diseases. The objective of this survey was to investigate the prevalence of tick-borne pathogens in 70 dogs from Santa Fé and Córdoba, Argentina. Microscopic blood smear examination as well as polymerase chain reaction (PCR) amplification using species-specific markers of Anaplasma, Babesia, Bartonella, Borrelia, Ehrlichia, Francisella, Mycoplasma (hemotropic group) and Rickettsia, followed by DNA sequencing were used to establish the prevalence of each infecting pathogen. Blood smear analysis showed 81% (57/70) prevalence of structures morphologically compatible with hemotropic mycoplasmas. No structures resembling either piroplasms or Anaplasma/Ehrlichia were detected. Hemotropic mycoplasma species (Mycoplasma haematoparvum, Mycoplasma haemocanis and Mycoplasma suis) were the most prevalent pathogens detected with an overall prevalence of 77.1%. Anaplasma platys was detected and identified in 11 of the 70 dogs (15.7%), meanwhile two Bartonella spp. (B. clarridgeiae and an uncharacterized Bartonella sp.) and Babesia vogeli were detected at 3 and 7% prevalence, respectively. The work presented here describes a high molecular prevalence for hemotropic mycoplasma species in each of the five locations selected. Three Mycoplasma spp., including Mycoplasma suis, reported for the first time in dogs have been identified by DNA amplification and sequencing. This study highlights the risk that these bacterial pathogens represent for companion animals and, due to their potential zoonotic nature, also for people.}, number={1}, journal={Parasites & Vectors}, publisher={Springer Nature}, author={Mascarelli, Patricia E. and Tartara, Gustavo P. and Pereyra, Norma B. and Maggi, Ricardo G.}, year={2016}, month={Dec} }
 @article{o'dell_agarwal_meilleur_2016, title={Inside Cover: Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase (Angew. Chem. Int. Ed. 3/2017)}, volume={56}, ISSN={1433-7851}, url={http://dx.doi.org/10.1002/ANIE.201611987}, DOI={10.1002/anie.201611987}, abstractNote={Oxidative cleavage of glycosidic bonds allows lytic polysaccharide monooxygenases (LPMOs) to break polysaccharide chains. This activity enhances the enzymatic hydrolysis of recalcitrant carbohydrate biomass, such as cellulose or chitin. In their Communication on page 767 ff., F. Meilleur and co-workers provide a structural description of molecular-oxygen activation by a fungal LPMO at the mononuclear copper active site as revealed by X-ray and neutron crystallography and DFT calculations. Credit: J. Hemman.}, number={3}, journal={Angewandte Chemie International Edition}, publisher={Wiley}, author={O'Dell, William B. and Agarwal, Pratul K. and Meilleur, Flora}, year={2016}, month={Dec}, pages={658–658} }
 @article{william b. o'dell_bodenheimer_meilleur_2016, title={Neutron protein crystallography: A complementary tool for locating hydrogens in proteins}, volume={602}, ISSN={["1096-0384"]}, url={https://doi.org/10.1016/j.abb.2015.11.033}, DOI={10.1016/j.abb.2015.11.033}, abstractNote={Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, publisher={Elsevier BV}, author={William B. O'Dell and Bodenheimer, Annette M. and Meilleur, Flora}, year={2016}, month={Jul}, pages={48–60} }
 @article{zhuravleva_lindsey_chakoumakos_custelcean_meilleur_hughes_kriven_melcher_2015, title={Crystal structure and thermal expansion of a CsCe2Cl7 scintillator}, volume={227}, ISSN={0022-4596}, url={http://dx.doi.org/10.1016/J.JSSC.2015.03.032}, DOI={10.1016/J.JSSC.2015.03.032}, abstractNote={We used single-crystal X-ray diffraction data to determine crystal structure of CsCe2Cl7. It crystallizes in a P1121/b space group with a=19.352(1) Å, b=19.352(1) Å, c=14.838(1) Å, γ=119.87(2)°, and V=4818.6(5) Å3. Differential scanning calorimetry measurements combined with the structural evolution of CsCe2Cl7 via X-ray diffractometry over a temperature range from room temperature to the melting point indicates no obvious intermediate solid–solid phase transitions. The anisotropy in the average linear coefficient of thermal expansion of the a axis (21.3×10–6/°C) with respect to the b and c axes (27.0×10–6/°C) was determined through lattice parameter refinement of the temperature dependent diffraction patterns. These findings suggest that the reported cracking behavior during melt growth of CsCe2Cl7 bulk crystals using conventional Bridgman and Czochralski techniques may be largely attributed to the anisotropy in thermal expansion.}, journal={Journal of Solid State Chemistry}, publisher={Elsevier BV}, author={Zhuravleva, M. and Lindsey, A. and Chakoumakos, B.C. and Custelcean, R. and Meilleur, F. and Hughes, R.W. and Kriven, W.M. and Melcher, C.L.}, year={2015}, month={Jul}, pages={142–149} }
 @article{knihtila_holzapfel_weiss_meilleur_mattos_2015, title={Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP gamma-Phosphate}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.679860}, abstractNote={Background: The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated γ-phosphate. Conclusion: The active site of RAS significantly increases the pKa of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases. RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.}, number={52}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Knihtila, Ryan and Holzapfel, Genevieve and Weiss, Kevin and Meilleur, Flora and Mattos, Carla}, year={2015}, month={Dec}, pages={31025–31036} }
 @article{golden_attwood_duff_meilleur_vrielink_2015, title={Production and characterization of recombinant perdeuterated cholesterol oxidase}, volume={485}, ISSN={["1096-0309"]}, DOI={10.1016/j.ab.2015.06.008}, abstractNote={Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.}, journal={ANALYTICAL BIOCHEMISTRY}, publisher={Elsevier BV}, author={Golden, Emily and Attwood, Paul V. and Duff, Anthony P. and Meilleur, Flora and Vrielink, Alice}, year={2015}, month={Sep}, pages={102–108} }
 @article{bodenheimer_cuneo_swartz_he_o'neill_myles_evans_meilleur_section_2014, title={Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms}, volume={70}, ISSN={["2053-230X"]}, url={http://europepmc.org/abstract/med/24915091}, DOI={10.1107/s2053230x14008851}, abstractNote={Cel7A (previously known as cellobiohydrolase I) fromHypocrea jecorinawas crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space groupC2, with unit-cell parametersa= 152.5,b= 44.9,c= 57.6 Å, β = 101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space groupP6322, with unit-cell parametersa=b≃ 155,c≃ 138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.}, number={Pt 6}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Bodenheimer, A.M. and Cuneo, M.J. and Swartz, P.D. and He, J. and O'Neill, H.M. and Myles, D.A. and Evans, B.R. and Meilleur, Flora and Section, F.}, year={2014}, month={Jun}, pages={773–776} }
 @article{munshi_snell_woerd_mj_ra_da_z_f_d._2014, title={Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant}, volume={70}, ISSN={["2059-7983"]}, url={http://europepmc.org/abstract/med/24531475}, DOI={10.1107/s1399004713029684}, abstractNote={Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl groupviaan activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.}, number={Pt 2}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Munshi, P. and Snell, E.H. and Woerd and Mj, Judge and Ra, Myles and Da, Ren and Z, Meilleur and F, Acta Crystallographica Section and D.}, year={2014}, month={Feb}, pages={414–420} }
 @article{meilleur_2014, title={ORNL hosts the participants of the 4thNeutrons in Structural Biology Workshop and the IMAGINE single crystal neutron diffractometer first external users}, volume={25}, DOI={10.1080/10448632.2014.870435}, abstractNote={Volume 25 • Number 1 • 2014 Neutron News 12 O Ridge National Laboratory (ORNL) hosted the fourth workshop on the applications of neutron scattering in structural biology during June 24–28, 2013. Since the inaugural event in 2010, the workshop has increased in popularity and broadened to include graduate students, post-doctoral fellows and university faculty with diverse backgrounds and interests in structural biology. The workshop is structured to introduce the participants to neutron scattering techniques, instrumentation and data collection, analysis and interpretation and to expose them to cuttingedge research in neutron structural biology. The 2013 workshop symposium opened with an introduction to neutrons in biology by Dean Myles (ORNL), and featured research presentations by Dennis Brown (North Carolina State University) on the solution structure of the prototype virus of genus alphavirus in the family Togaviridae Sindbis investigated by Small Angle Neutron Scattering (SANS), Zimei Bu (City College of New York) on the structure and dynamics of the interfacial adapter proteins NHERF1 and Ezrin upon heterodimer formation investigated by SANS and Neutron Spin Echo (NSE), Alice Vrielink (University of Western Australia) on cholesterol oxidase enzyme structure and function in fl avin mediated oxidative catalysis investigated by ultra-high resolution X-ray and neutron crystallography, Jan Kmetko (Kenyon College) on the interaction of an amyloid peptide with biomimetic membrane studied by neutron refl ectometry and Frederick Heberle (ORNL) on membrane raft mixtures investigated by SANS. The workshop program included lectures and tutorials on neutron scattering techniques and on practical applications of SANS, neutron crystallography, refl ectometry, spectroscopy and imaging. Particpants also toured ORNL’s fl agship neutron and computational facilities: the High Flux Isotope Reactor (HFIR), the Spallation Neutron Source (SNS), the historic Graphite Reactor and the Titan supercomputer. This year’s workshop had an additional special fl avor as the course also provided several participants with the opportunity to become the fi rst external users of the new single crystal diffractometer at the HFIR (Figures 1 and 2). The IMAGINE instrument is designed to provide neutron data at or near atomic resolutions (1.5 Å) from crystals with volume <1.0 mm3 and with unit cell edges <100 Å. Beam line features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm2 focal spot at the sample position with full width vertical and horizontal divergence of 0.5° and 0.6°, respectively, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength confi gurations (λmin = 2.0 Å, 2.8 Å, 3.3 Å – λmax = 3.0 Å, 4.0 Å, 4.5 Å, ~20 Å) (Meilleur et al., 2013). The course sponsors included ORNL (Neutron Sciences Directorate), the Tennessee Department of Energy (DOE) Experimental Program to Stimulate Competitive Research (EPSCoR) and the Joint Institute for Neutron Scattering (JINS).}, number={1}, journal={Neutron News}, publisher={Informa UK Limited}, author={Meilleur, Flora}, year={2014}, month={Jan}, pages={12–12} }
 @article{gruene_hahn_luebben_meilleur_sheldrick_2014, title={Refinement of macromolecular structures against neutron data with SHELXL2013}, volume={47}, ISSN={["1600-5767"]}, DOI={10.1107/s1600576713027659}, abstractNote={Some of the improvements inSHELX2013makeSHELXLconvenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to useSHELXLfor refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst.(1991), A47, 392–400] restraints to H atoms and discusses several of the features ofSHELXLthat make the program particularly useful for the investigation of H atoms with neutron diffraction.SHELXL2013is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.}, number={1}, journal={JOURNAL OF APPLIED CRYSTALLOGRAPHY}, publisher={International Union of Crystallography (IUCr)}, author={Gruene, Tim and Hahn, Hinrich W. and Luebben, Anna V. and Meilleur, Flora and Sheldrick, George M.}, year={2014}, month={Jan}, pages={462–466} }
 @article{meilleur_2014, title={The Neutrons in Structural Biology Workshop celebrates its 5th edition}, volume={25}, DOI={10.1080/10448632.2014.955418}, abstractNote={"The Neutrons in Structural Biology Workshop celebrates its 5th edition." Neutron News, 25(4), p. 11}, number={4}, journal={Neutron News}, publisher={Informa UK Limited}, author={Meilleur, Flora}, year={2014}, month={Oct}, pages={11–11} }
 @article{ankner_heller_herwig_meilleur_myles_2013, title={Neutron Scattering Techniques and Applications in Structural Biology}, DOI={10.1002/0471140864.ps1716s72}, abstractNote={Abstract}, journal={Current Protocols in Protein Science}, author={Ankner, John F. and Heller, William T. and Herwig, Kenneth W. and Meilleur, Flora and Myles, Dean A.A.}, year={2013}, month={Apr} }
 @article{martin_he_meilleur_guenther_sit_lommel_heller_2013, title={New insight into the structure of RNA in red clover necrotic mosaic virus and the role of divalent cations revealed by small-angle neutron scattering}, volume={158}, ISSN={["0304-8608"]}, url={http://europepmc.org/abstract/med/23483344}, DOI={10.1007/s00705-013-1650-6}, number={8}, journal={ARCHIVES OF VIROLOGY}, publisher={Springer Science and Business Media LLC}, author={Martin, Stanton L. and He, Lilin and Meilleur, Flora and Guenther, Richard H. and Sit, Tim L. and Lommel, Steven A. and Heller, William T.}, year={2013}, month={Aug}, pages={1661–1669} }
 @article{meilleur_munshi_robertson_stoica_crow_kovalevsky_koritsanszky_chakoumakos_blessing_myles_et al._2013, title={The IMAGINE instrument: first neutron protein structure and new capabilities for neutron macromolecular crystallography.}, volume={69}, url={http://europepmc.org/abstract/med/24100333}, DOI={10.1107/s0907444913019604}, abstractNote={The first high-resolution neutron protein structure of perdeuterated rubredoxin fromPyrococcus furiosus(PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported. Neutron diffraction data extending to 1.65 Å resolution were collected from a relatively small 0.7 mm3PfRd crystal using 2.5 d (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the D atoms of the protein and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolution (1.5 Å) from crystals with volume <1.0 mm3and with unit-cell edges <100 Å. Beamline features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position with full-width vertical and horizontal divergences of 0.5 and 0.6°, respectively. Variable short- and long-wavelength cutoff optics provide automated exchange between multiple-wavelength configurations (λmin= 2.0, 2.8, 3.3 Å to λmax= 3.0, 4.0, 4.5, ∼20 Å). These optics produce a more than 20-fold increase in the flux density at the sample and should help to enable more routine collection of high-resolution data from submillimetre-cubed crystals. Notably, the crystal used to collect thesePfRd data was 5–10 times smaller than those previously reported.}, number={Pt 10}, journal={Biological crystallography}, author={Meilleur, F. and Munshi, P. and Robertson, L. and Stoica, A.D. and Crow, L. and Kovalevsky, A. and Koritsanszky, T. and Chakoumakos, B.C. and Blessing, R. and Myles, D.A. and et al.}, year={2013}, month={Oct}, pages={2157–2160,} }
 @article{meilleur_2013, title={Third School on the Applications of Neutron Scattering Techniques in Structural Biology, Oak Ridge, TN}, volume={24}, DOI={10.1080/10448632.2013.750140}, abstractNote={"Third School on the Applications of Neutron Scattering Techniques in Structural Biology, Oak Ridge, TN." Neutron News, 24(1), p. 4}, number={1}, journal={Neutron News}, publisher={Informa UK Limited}, author={Meilleur, Flora}, year={2013}, month={Feb}, pages={4–4} }
 @article{he_piper_meilleur_hernandez_heller_brown_2012, title={Conformational Changes in Sindbis Virus Induced by Decreased pH Are Revealed by Small-Angle Neutron Scattering}, volume={86}, ISSN={["0022-538X"]}, url={http://europepmc.org/abstract/med/22156534}, DOI={10.1128/jvi.06569-11}, abstractNote={ABSTRACT}, number={4}, journal={JOURNAL OF VIROLOGY}, author={He, Lilin and Piper, Amanda and Meilleur, Flora and Hernandez, Raquel and Heller, William T. and Brown, Dennis T.}, year={2012}, month={Feb}, pages={1982–1987} }
 @article{myles_dauvergne_blakeley_meilleur_2012, title={Neutron protein crystallography at ultra-low (< 15 K) temperatures}, volume={45}, ISSN={["1600-5767"]}, DOI={10.1107/s0021889812019784}, abstractNote={Techniques and equipment have been developed that enable large protein crystals (1–6 mm3) flash-cooled in liquid nitrogen at 77 K to be transferred and mounted on a liquid helium Displex cryorefrigerator and cooled to temperatures down to 15 K for accurate neutron diffraction analysis. In preliminary experiments, it was possible to collect high-quality high-resolution neutron diffraction data to 1.55 Å resolution from several large crystals of triclinic hen egg white lysozyme cooled to 15 K. This enabled the subsequent cryogenic analysis of two further proteins, rubredoxin and concanavalin A, at 1.7 and 2.5 Å, respectively, demonstrating the generality of the approach. The ability to flash-cool such large crystals for cryogenic neutron analysis should significantly broaden the range of scientific questions examined by neutron protein crystallography, allowing the analysis of structures and transitions as a function of temperature and enabling freeze-trapped capture of kinetic intermediates in protein systems.}, number={4}, journal={JOURNAL OF APPLIED CRYSTALLOGRAPHY}, publisher={International Union of Crystallography (IUCr)}, author={Myles, Dean A. A. and Dauvergne, Francois and Blakeley, Matthew P. and Meilleur, Flora}, year={2012}, month={Aug}, pages={686–692} }
 @article{jayasundar_ju_he_liu_meilleur_zhao_callaway_bu_2012, title={Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering}, volume={287}, ISSN={["1083-351X"]}, url={http://europepmc.org/abstract/med/22927432}, DOI={10.1074/jbc.m112.380972}, abstractNote={Background: The structure of activated ezrin is not known. Results: We have determined the conformation of activated ezrin upon binding to PIP2 and to F-actin. Conclusion: Activated ezrin forms more extensive contacts with F-actin than generally depicted. Significance: This study provides new insight into the mechanisms by which ezrin assembles signaling complexes at the membrane-cytoskeleton interface. Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP2. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP2 binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP2 binding. The study provides the first structural view of the activated ezrin bound to PIP2 and to F-actin.}, number={44}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Jayasundar, Jayant James and Ju, Jeong Ho and He, Lilin and Liu, Dazhi and Meilleur, Flora and Zhao, Jinkui and Callaway, David J. E. and Bu, Zimei}, year={2012}, month={Oct}, pages={37119–37133} }
 @article{munshi_chung_blakeley_weiss_myles_meilleur_section_2012, title={Rapid visualization of hydrogen positions in protein neutron crystallographic structures}, volume={68}, ISSN={["0907-4449"]}, url={http://europepmc.org/abstract/med/22194331}, DOI={10.1107/s0907444911048402}, abstractNote={Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.}, number={Pt 1}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY}, author={Munshi, P. and Chung, S.L. and Blakeley, M.P. and Weiss, K.L. and Myles, D.A. and Meilleur, Flora and Section, D.}, year={2012}, month={Jan}, pages={35–41} }
 @article{pérez_maggi_diniz_breitschwerdt_2011, title={Molecular and Serological Diagnosis of Bartonella Infection in 61 Dogs from the United States}, volume={25}, ISSN={0891-6640}, url={http://dx.doi.org/10.1111/j.1939-1676.2011.0736.x}, DOI={10.1111/j.1939-1676.2011.0736.x}, abstractNote={Background:Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA.}, number={4}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Pérez, C. and Maggi, R.G. and Diniz, P.P.V.P. and Breitschwerdt, E.B.}, year={2011}, month={May}, pages={805–810} }
 @article{borreguero_he_meilleur_weiss_brown_myles_herwig_agarwal_2011, title={Redox-Promoting Protein Motions in Rubredoxin}, volume={115}, ISSN={["1520-6106"]}, url={http://europepmc.org/abstract/med/21608980}, DOI={10.1021/jp201346x}, abstractNote={Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This study reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf). Computational modeling allows detailed investigations of protein motions as a function of temperature, and neutron scattering experiments are used to compare to computational results. Just above the dynamical transition temperature which marks the onset of significant anharmonic motions of the protein, the computational simulations show both a significant reorientation of the average electrostatic force experienced by the coordinated Fe(3+) ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated and dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of P. furiosus, simulations show that three anharmonic modes including motions of two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe(3+) ion. The motions of these residues undergo displacements which may facilitate solvent access to the ion.}, number={28}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Borreguero, Jose M. and He, Junhong and Meilleur, F. and Weiss, Kevin L. and Brown, Craig M. and Myles, Dean A. and Herwig, Kenneth W. and Agarwal, Pratul K.}, year={2011}, month={Jul}, pages={8925–8936} }
 @article{li_shew_he_meilleur_myles_liu_zhang_smith_herwig_pynn_et al._2011, title={Scattering functions of Platonic solids}, volume={44}, ISSN={["0021-8898"]}, DOI={10.1107/s0021889811011691}, abstractNote={The single-particle small-angle scattering properties of five Platonic solids, including the tetrahedron, hexahedron, octahedron, dodecahedron and icosahedron, are systematically investigated. For each given geometry, the Debye spatial autocorrelation function, pair distance distribution function and intraparticle structure factor (form factor) are calculated and compared with the corresponding scattering function of a spherical reference system. From the theoretical models, the empirical relationship between the dodecahedral and icosahedral structural characteristics and those of the equivalent spheres is found. Moreover, the single-particle scattering properties of icosahedral and spherical shells with identical volume are investigated, and the prospect of using different data analysis approaches to explore their structural differences is presented and discussed.}, journal={JOURNAL OF APPLIED CRYSTALLOGRAPHY}, author={Li, Xin and Shew, Chwen-Yang and He, Lilin and Meilleur, Flora and Myles, Dean A. A. and Liu, Emily and Zhang, Yang and Smith, Gregory S. and Herwig, Kenneth W. and Pynn, Roger and et al.}, year={2011}, month={Jun}, pages={545–557} }
 @article{martin_guenther_sit_swartz_meilleur_lommel_rose_section_2010, title={Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus}, volume={66}, ISSN={["2053-230X"]}, url={http://europepmc.org/abstract/med/21045294}, DOI={10.1107/s1744309110032483}, abstractNote={Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Å resolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Å in the analysis presented here.}, number={Pt 11}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Martin, S.L. and Guenther, R.H. and Sit, T.L. and Swartz, P.D. and Meilleur, Flora and Lommel, S.A. and Rose, Robert and Section, F.}, year={2010}, month={Nov}, pages={1458–1462} }
 @article{he_piper_meilleur_myles_hernandez_brown_heller_2010, title={The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering}, volume={84}, ISSN={["1098-5514"]}, url={http://europepmc.org/abstract/med/20219936}, DOI={10.1128/jvi.00044-10}, abstractNote={ABSTRACT}, number={10}, journal={JOURNAL OF VIROLOGY}, author={He, Lilin and Piper, Amanda and Meilleur, Flora and Myles, Dean A. A. and Hernandez, Raquel and Brown, Dennis T. and Heller, William T.}, year={2010}, month={May}, pages={5270–5276} }
 @article{gardberg_castillo_ar_kl_f_mp_da_d._2010, title={Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography}, volume={66}, ISSN={["2059-7983"]}, url={http://europepmc.org/abstract/med/20445231}, DOI={10.1107/s0907444910005494}, abstractNote={The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 Å resolution neutron diffraction studies of fully perdeuterated and selectively CH3-protonated perdeuterated crystals ofPyrococcus furiosusrubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 Å resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the σ level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 Å resolution RT neutron data for perdeuterated rubredoxin are ∼8 times more likely overall to provide high-confidence positions for D atoms than 1.1 Å resolution X-ray data at 100 K or RT. At or above the 1.0σ level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 Å resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0σ level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.}, number={Pt 5}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Gardberg, A.S. and Castillo, Del and Ar, Weiss and Kl, Meilleur and F, Blakeley and Mp, Myles and Da, Acta Crystallographica Section and D.}, year={2010}, month={May}, pages={558–567} }
 @article{meilleur_weiss_myles_2009, title={Deuterium Labeling for Neutron Structure-Function-Dynamics Analysis}, DOI={10.1007/978-1-59745-483-4_18}, abstractNote={Neutron scattering and diffraction provide detailed information on the structure and dynamics of biological materials across time and length scales that range from picoseconds to nanoseconds and from 1 to 10,000 A, respectively. The particular sensitivity of neutrons to the isotopes of hydrogen makes selective deuterium labeling of biological systems an essential tool for maximizing the return from neutron scattering experiments. In neutron protein crystallography, the use of fully deuterated protein crystals improves the signal-to-noise ratio of the data by an order of magnitude and enhances the visibi-lity of the molecular structure (Proc Natl Acad Sci U S A 97:3872-3877, 2000; Acta Crystallogr D Biol Crystallogr 61:1413-1417, 2005; Acta Crystallogr D Biol Crystallogr 61:539-544, 2005). In solution and surface scattering experiments, the incorporation of deuterium-labeled subunits or components into complex assemblies or structures makes it possible to deconvolute the scattering of the labeled and unlabeled subunits and to determine their relative dispositions within the complex (J Mol Biol 93:255-265, 1975). With multiple labeling patterns, it is also possible to reconstruct the locations of multiple subunits in ternary and higher-order complexes (Science 238:1403-1406, 1987; J Mol Biol 271:588-601, 1997; J Biol Chem 275:14432-14439, 2000; Biochemistry 42:7790-7800, 2003). In inelastic neutron scattering experiments, which probe hydrogen dynamics in biological materials, the application of site, residue, or region-specific hydrogen-deuterium-labeling patterns can be used to distinguish and highlight the specific dynamics within a system (Proc Natl Acad Sci U S A 95:4970-4975, 1998).Partial, selective, or fully deuterated proteins can be readily produced by endogenous expression of recombinant proteins in bacterial systems that are adapted to growth in D(2)O solution and using selectively deuterated carbon sources. Adaptation can be achieved either by gradual step-wise increase in D(2)O concentration or, more directly, by plating cells on media of choice and selecting colonies that perform best for subsequent culture and inoculation. Scale-up growth and expression is typically performed in standard shaker flasks using either commercial or "home-grown" rich media (derived, for example, from cell lysates produced from algae grown in D(2)O) or under more controlled conditions in defined minimal media. Cell growth is typically slower in deuterated media (>5 times slower) and yields are correspondingly lower. Once the target protein has been expressed, purification proceeds by the protocols developed for the hydrogenated protein. The deuteration levels of the final product are determined by mass spectrometry.}, journal={Micro and Nano Technologies in Bioanalysis}, publisher={Springer Nature}, author={Meilleur, Flora and Weiss, Kevin L. and Myles, Dean A.A.}, year={2009}, pages={281–292} }
 @article{teixeira_zaccai_ankner_bellissent-funel_bewley_blakeley_callow_coates_dahint_dalgliesh_et al._2008, title={Erratum to “New sources and instrumentation for neutrons in biology” [Chem. Phys. 345 (2008) 133–151]}, volume={351}, ISSN={0301-0104}, url={http://dx.doi.org/10.1016/j.chemphys.2008.04.007}, DOI={10.1016/j.chemphys.2008.04.007}, number={1-3}, journal={Chemical Physics}, publisher={Elsevier BV}, author={Teixeira, S.C.M. and Zaccai, G. and Ankner, J. and Bellissent-Funel, M.C. and Bewley, R. and Blakeley, M.P. and Callow, P. and Coates, L. and Dahint, R. and Dalgliesh, R. and et al.}, year={2008}, month={Jul}, pages={170} }
 @article{teixeira_zaccai_ankner_bellissent-funel_bewley_blakeley_callow_coates_dahint_dalgliesh_et al._2008, title={New sources and instrumentation for neutrons in biology}, volume={345}, ISSN={["1873-4421"]}, url={http://europepmc.org/abstract/med/19132140}, DOI={10.1016/j.chemphys.2008.02.030}, abstractNote={Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.}, number={2-3}, journal={CHEMICAL PHYSICS}, author={Teixeira, S. C. M. and Zaccai, G. and Ankner, J. and Bellissent-Funel, M. C. and Bewley, R. and Blakeley, M. P. and Callow, P. and Coates, L. and Dahint, R. and Dalgliesh, R. and et al.}, year={2008}, month={Apr}, pages={133–151} }
 @article{teixeira_zaccai_ankner_bellissent-funel_bewley_blakeley_callow_coates_dahint_dalgliesh_et al._2008, title={New sources and instrumentation for neutrons in biology (vol 345, pg 133, 2008)}, volume={351}, number={1-3}, journal={Chemical Physics}, author={Teixeira, S. C. M. and Zaccai, G. and Ankner, J. and Bellissent-Funel, M. C. and Bewley, R. and Blakeley, M. P. and Callow, P. and Coates, L. and Dahint, R. and Dalgliesh, R. and et al.}, year={2008}, pages={170–170} }
 @article{weiss_meilleur_blakeley_myles_section_2008, title={Preliminary neutron crystallographic analysis of selectively CH3-protonated deuterated rubredoxin from Pyrococcus furiosus}, volume={64}, ISSN={["2053-230X"]}, url={http://europepmc.org/abstract/med/18540070}, DOI={10.1107/S1744309108013997}, abstractNote={Neutron crystallography is used to locate H atoms in biological materials and can distinguish between negatively scattering hydrogen-substituted and positively scattering deuterium-substituted positions in isomorphous neutron structures. Recently, Hauptman & Langs (2003; Acta Cryst. A59, 250-254) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering H atoms in the structure. Selective-labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of H to D atoms can be precisely controlled. Methyl selective-labeling protocols were applied to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here, the production, crystallization and preliminary neutron analysis of a selectively CH3-protonated deuterated PfRd sample, which provided a high-quality neutron data set that extended to 1.75 A resolution using the new LADI-III instrument at the Institut Laue-Langevin, are reported. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of H atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.}, number={Pt 6}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Weiss, K.L. and Meilleur, Flora and Blakeley, M.P. and Myles, D.A. and Section, F.}, year={2008}, month={Jun}, pages={537–540} }
 @article{blakeley_ruiz_cachau_hazemann_meilleur_mitschler_ginell_afonine_ventura_cousido-siah_et al._2008, title={Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase.}, volume={105}, url={http://europepmc.org/abstract/med/18250329}, DOI={10.1073/pnas.0711659105}, abstractNote={We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.}, number={6}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Blakeley, MP and Ruiz, F and Cachau, R and Hazemann, I and Meilleur, F and Mitschler, A and Ginell, S and Afonine, P and Ventura, ON and Cousido-Siah, A and et al.}, year={2008}, month={Feb}, pages={1844–1848,} }
 @article{di costanzo_moulin_haertlein_meilleur_christianson_2007, title={Expression, purification, assay, and crystal structure of perdeuterated human arginase I}, volume={465}, ISSN={["1096-0384"]}, DOI={10.1016/j.abb.2007.04.036}, abstractNote={Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to yield l-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) at 1.90A resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeuteration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study.}, number={1}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, publisher={Elsevier BV}, author={Di Costanzo, Luigi and Moulin, Martine and Haertlein, Michael and Meilleur, Flora and Christianson, David W.}, year={2007}, month={Sep}, pages={82–89} }
 @article{budayova-spano_bonneté_ferté_el hajji_meilleur_blakeley_castro_acta crystallographica. section_2006, title={A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin.}, volume={62}, url={http://europepmc.org/abstract/med/16511330}, DOI={10.1107/s1744309106006439}, abstractNote={Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 A resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 A) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.}, number={Pt 3}, journal={Structural biology and crystallization communications}, author={Budayova-Spano, M. and Bonneté, F. and Ferté, N. and El Hajji, M. and Meilleur, F. and Blakeley, M.P. and Castro, B. and Section, F. Acta crystallographica.}, year={2006}, month={Mar}, pages={306–309,} }
 @article{meilleur_snell_woerd_judge_myles_2006, title={A quasi-Laue neutron crystallographic study of d-xylose isomerase}, volume={35}, DOI={10.1007/s00249-006-0066-6}, abstractNote={The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.}, number={7}, journal={European Biophysics Journal}, publisher={Springer Nature}, author={Meilleur, Flora and Snell, Edward H. and Woerd, Mark J. and Judge, Russell A. and Myles, Dean A. A.}, year={2006}, month={May}, pages={601–609} }
 @article{blakeley_mitschler_hazemann_meilleur_myles_podjarny_2006, title={Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase-inhibitor complex.}, volume={35}, url={http://europepmc.org/abstract/med/16622654}, DOI={10.1007/s00249-006-0064-8}, abstractNote={Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.}, number={7}, journal={European biophysics journal : EBJ}, author={Blakeley, MP and Mitschler, A and Hazemann, I and Meilleur, F and Myles, DA and Podjarny, A}, year={2006}, month={Sep}, pages={577–583,} }
 @article{meilleur_myles_blakeley_2006, title={Neutron Laue macromolecular crystallography}, volume={35}, DOI={10.1007/s00249-006-0074-6}, abstractNote={Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.}, number={7}, journal={European Biophysics Journal}, publisher={Springer Nature}, author={Meilleur, Flora and Myles, Dean A. A. and Blakeley, Matthew P.}, year={2006}, month={Aug}, pages={611–620} }
 @article{hazemann_dauvergne_blakeley_meilleur_haertlein_van dorsselaer_mitschler_myles_podjarny_acta crystallographica. section_2005, title={High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.}, volume={61}, url={http://europepmc.org/abstract/med/16204895}, DOI={10.1107/s0907444905024285}, abstractNote={Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.}, number={Pt 10}, journal={Biological crystallography}, author={Hazemann, I. and Dauvergne, M.T. and Blakeley, M.P. and Meilleur, F. and Haertlein, M. and Van Dorsselaer, A. and Mitschler, A. and Myles, D.A. and Podjarny, A. and Section, D. Acta crystallographica.}, year={2005}, month={Oct}, pages={1413–1417,} }
 @article{bennett_meilleur_myles_howell_dealwis_acta crystallographica. section_crystallography_2005, title={Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate.}, volume={61}, url={http://europepmc.org/abstract/med/15858267}, DOI={10.1107/s0907444905004804}, abstractNote={The contribution of H atoms in noncovalent interactions and enzymatic reactions underlies virtually all aspects of biology at the molecular level, yet their 'visualization' is quite difficult. To better understand the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR), a neutron diffraction study is under way to directly determine the accurate positions of H atoms within its active site. Despite exhaustive investigation of the catalytic mechanism of DHFR, controversy persists over the exact pathway associated with proton donation in reduction of the substrate, dihydrofolate. As the initial step in a proof-of-principle experiment which will identify ligand and residue protonation states as well as precise solvent structures, a neutron diffraction data set has been collected on a 0.3 mm(3) D(2)O-soaked crystal of ecDHFR bound to the anticancer drug methotrexate (MTX) using the LADI instrument at ILL. The completeness in individual resolution shells dropped to below 50% between 3.11 and 3.48 A and the I/sigma(I) in individual shells dropped to below 2 at around 2.46 A. However, reflections with I/sigma(I) greater than 2 were observed beyond these limits (as far out as 2.2 A). To our knowledge, these crystals possess one of the largest primitive unit cells (P6(1), a = b = 92, c = 73 A) and one of the smallest crystal volumes so far tested successfully with neutrons.}, number={Pt 5}, author={Bennett, B.C. and Meilleur, F. and Myles, D.A. and Howell, E.E. and Dealwis, C.G. and Section, D. Acta crystallographica. and crystallography, Biological}, year={2005}, month={May}, pages={574–579,} }
 @article{meilleur_dauvergne_schlichting_myles_acta crystallographica. section_2005, title={Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam.}, volume={61}, url={http://europepmc.org/abstract/med/15858263}, DOI={10.1107/s0907444905003872}, abstractNote={Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 A. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O(2) via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 A, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.}, number={Pt 5}, journal={Biological crystallography}, author={Meilleur, F. and Dauvergne, M.T. and Schlichting, I. and Myles, D.A. and Section, D. Acta crystallographica.}, year={2005}, month={May}, pages={539–544,} }
 @article{meilleur_contzen_myles_jung_2004, title={Structural Stability and Dynamics of Hydrogenated and Perdeuterated Cytochrome P450cam (CYP101)†}, volume={43}, DOI={10.1021/bi049418q}, abstractNote={Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns). Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.}, number={27}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Meilleur, Flora and Contzen, Jörg and Myles, Dean A. A. and Jung, Christiane}, year={2004}, month={Jul}, pages={8744–8753} }
 @article{adamo_heitzmann_meilleur_rega_scalmani_grand_cadet_barone_2001, title={Interplay of intrinsic and environmental effects on the magnetic properties of free radicals issuing from H-atom addition to cytosine.}, volume={123}, url={http://europepmc.org/abstract/med/11459491}, DOI={10.1021/ja004284z}, abstractNote={Possible radical reaction products issuing from H-atom addition to cytosine have been characterized and analyzed by means of a comprehensive quantum mechanical approach including density functional computations (B3LYP), together with simulation of the solvent by the polarizable continuum model (PCM), and averaging of spectroscopic properties over the most important vibrational motions. The hyperfine couplings of the semirigid 5,6-dihydrocytos-6yl radical computed at the optimized geometry are in good agreement with their experimental counterparts. On the other hand, vibrational averaging is mandatory for obtaining an effectively planar structure for the 5,6-dihydrocytos-5yl radical with the consequent equivalence of beta-hydrogens. Finally, only proper consideration of environmental effects restores the agreement between computed and experimental couplings for the base anion protonated at N3.}, number={29}, journal={Journal of the American Chemical Society}, author={Adamo, C and Heitzmann, M and Meilleur, F and Rega, N and Scalmani, G and Grand, A and Cadet, J and Barone, V}, year={2001}, month={Jul}, pages={7113–7117,} }