Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

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Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase

Depression, Somatization and Anxiety in Female Patients with Temporomandibular Disorders (TMD)

The aim of this research was to determine the possible differences in degrees of depression, somatization and anxiety between the acute and chronic female patients with temporomandibular disorders (TMD), and whether these differences exist in healthy female patients. Ninety female patients were involved in this research; 60 of them were TMD patients of the Dental Polyclinic, while other 30 females came for a rutine recall visit and had no problem related to TMD. Patients were aged 22 to 67 years, the average age being 38.5±12 years. All patients were asked to fill in the RDC/TMD protocol and three psychological tests (Emotions Profile Index, Somatization Scale and life Events Scale). Following the analysis of the RDC/TMD protocol and psychological tests, it was determined that the chronic female patients had higher depression and somatization scores in comparison with the acute patients (p<0.01); the acute patients self-perceive higher levels of anxiety in relation to the control group; furthermore, the patients reporting higher levels of depression were more inclined to somatization and had experienced a greater number of stress events in the past six months. It is beyond doubt that patients suffering from the TMD’s exhibit higher levels of depression, somatization and anxiety compared to the healthy ones, which proves that physiological factors may play a predisposing role in combination with reduced level of body tolerance to pain, and a decreased tolerance to stress

A preliminary neutron crystallographic study of proteinase K at pD 6.5

Preliminary neutron crystallographic data from the serine protease proteinase K have been recorded using the LADI-III diffractometer at the Institut Laue–Langevin. The results illustrate the feasibility of a full neutron structural analysis aimed at further understanding the catalytic mechanism of proteinase K

Use of neutron scattering techniques for Antifreeze Protein mechanistic studies

Antifreeze proteins (AFP) have evolved in organisms living in sub-zero temperatures to avoid freezing of internal fl uids. They bind to ice nuclei lowering the freezing point and inhibiting recrystallization [1]. Even though this protein has been thoroughly studied, including several structures determined by X-ray crystallography, the exact mechanism of binding of ice to the largely hydrophobic Ice Binding Surface (IBS), (i.e. the region of the protein involved in the ice recognition) has remained unclear. In particular, the study of the hydration layers around the protein using X-ray crystallography did not provide a model of the IBS-ice interface.Fil: Howard, Eduardo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Blakeley, Matthew P.. Institut Laue Langevin; FranciaFil: Salvay, Andrés Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Podjarny, Alberto Daniel. Centre National de la Recherche Scientifique; Franci

Production, crystallization and neutron diffraction of fully deuterated human myelin peripheral membrane protein P2

The molecular details of the formation of the myelin sheath, a multilayered membrane in the nervous system, are to a large extent unknown. P2 is a peripheral membrane protein from peripheral nervous system myelin, which is believed to play a role in this process. X-ray crystallographic studies and complementary experiments have provided information on the structure–function relationships in P2. In this study, a fully deuterated sample of human P2 was produced. Crystals that were large enough for neutron diffraction were grown by a ten-month procedure of feeding, and neutron diffraction data were collected to a resolution of 2.4 Å from a crystal of 0.09 mm(3) in volume. The neutron crystal structure will allow the positions of H atoms in P2 and its fatty-acid ligand to be visualized, as well as shedding light on the fine details of the hydrogen-bonding networks within the P2 ligand-binding cavity

The neutron structure of urate oxidase resolves a long-standing mechanistic conundrum and reveals unexpected changes in protonation.

International audienceUrate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion