| 1. |
- Caldararu, Octav, et al.
(author)
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Are crystallographic : B-factors suitable for calculating protein conformational entropy?
- 2019
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In: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 21:33, s. 18149-18160
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Journal article (peer-reviewed)abstract
- Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.
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| 2. |
- Caldararu, Octav, et al.
(author)
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Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase
- 2019
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In: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 10:2, s. 576-586
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Journal article (peer-reviewed)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are copper-containing metalloenzymes that can cleave the glycosidic link in polysaccharides. This could become crucial for production of energy-efficient biofuels from recalcitrant polysaccharides. Although LPMOs are considered oxygenases, recent investigations have shown that H2O2 can also act as a co-substrate for LPMOs. Intriguingly, LPMOs generate H2O2 in the absence of a polysaccharide substrate. Here, we elucidate a new mechanism for H2O2 generation starting from an AA10-LPMO crystal structure with an oxygen species bound, using QM/MM calculations. The reduction level and protonation state of this oxygen-bound intermediate has been unclear. However, this information is crucial to the mechanism. We therefore investigate the oxygen-bound intermediate with quantum refinement (crystallographic refinement enhanced with QM calculations), against both X-ray and neutron data. Quantum refinement calculations suggest a Cu(ii)-O-2 system in the active site of the AA10-LPMO and a neutral protonated -NH2 state for the terminal nitrogen atom, the latter in contrast to the original interpretation. Our QM/MM calculations show that H2O2 generation is possible only from a Cu(i) center and that the most favourable reaction pathway is to involve a nearby glutamate residue, adding two electrons and two protons to the Cu(ii)-O-2 system, followed by dissociation of H2O2.
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| 3. |
- Caldararu, Octav, et al.
(author)
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Refinement of protein structures using a combination of quantum-mechanical calculations with neutron and X-ray crystallographic data
- 2019
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In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 368-380
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Journal article (peer-reviewed)abstract
- Neutron crystallography is a powerful method to determine the positions of H atoms in macromolecular structures. However, it is sometimes hard to judge what would constitute a chemically reasonable model, and the geometry of H atoms depends more on the surroundings (for example the formation of hydrogen bonds) than heavy atoms, so that the empirical geometry information for the H atoms used to supplement the experimental data is often less accurate. These problems may be reduced by using quantum-mechanical calculations. A method has therefore been developed to combine quantum-mechanical calculations with joint crystallographic refinement against X-ray and neutron data. A first validation of this method is provided by re-refining the structure of the galectin-3 carbohydrate-recognition domain in complex with lactose. The geometry is improved, in particular for water molecules, for which the method leads to better-resolved hydrogen-bonding interactions. The method has also been applied to the active copper site of lytic polysaccharide monooxygenase and shows that the protonation state of the amino-terminal histidine residue can be determined.
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| 4. |
- Fisher, Zoe, et al.
(author)
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Biological Structures
- 2017
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In: Experimental Methods in the Physical Sciences. - 1079-4042. ; 49, s. 1-75
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Book chapter (peer-reviewed)abstract
- Neutron scattering methods are excellent for probing the detailed structure of biological systems, which rely on the intricate interplay of a large number of molecules from proteins and nucleic acids to lipids, hormones, and metabolites. With recent instrument developments and emergence of both new neutron sources and techniques, many biological systems that are not yet amenable to characterization by neutron scattering will become accessible in the near future, which will allow new experiments to be developed with a range of biologically relevant samples, offering new insights in life science. In this chapter, we will describe neutron methods for biological structure characterization on different length scales from atomic resolution to macromolecular length scales-up to micrometers. The dynamics of biological molecules are described by Seydel in Chapter 2 of this thematic volume.
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| 5. |
- Kelpšas, Vinardas, et al.
(author)
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Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant
- 2019
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In: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 75:4, s. 260-269
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Journal article (peer-reviewed)abstract
- Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.
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| 6. |
- Manzoni, Francesco, et al.
(author)
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Elucidation of Hydrogen Bonding Patterns in Ligand-Free, Lactose- and Glycerol-Bound Galectin-3C by Neutron Crystallography to Guide Drug Design
- 2018
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 61:10, s. 4412-4420
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Journal article (peer-reviewed)abstract
- The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.
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| 7. |
- Manzoni, Francesco, et al.
(author)
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Perdeuteration, crystallization, data collection and comparison of five neutron diffraction data sets of complexes of human galectin-3C
- 2016
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In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 72:11, s. 1194-1202
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Journal article (peer-reviewed)abstract
- Galectin-3 is an important protein in molecular signalling events involving carbohydrate recognition, and an understanding of the hydrogen-bonding patterns in the carbohydrate-binding site of its C-terminal domain (galectin-3C) is important for the development of new potent inhibitors. The authors are studying these patterns using neutron crystallography. Here, the production of perdeuterated human galectin-3C and successive improvement in crystal size by the development of a crystal-growth protocol involving feeding of the crystallization drops are described. The larger crystals resulted in improved data quality and reduced data-collection times. Furthermore, protocols for complete removal of the lactose that is necessary for the production of large crystals of apo galectin-3C suitable for neutron diffraction are described. Five data sets have been collected at three different neutron sources from galectin-3C crystals of various volumes. It was possible to merge two of these to generate an almost complete neutron data set for the galectin-3C-lactose complex. These data sets provide insights into the crystal volumes and data-collection times necessary for the same system at sources with different technologies and data-collection strategies, and these insights are applicable to other systems.Perdeuteration, purification and the growth of large crystals of the carbohydrate-recognition domain of galectin-3C are described. Five neutron diffraction data sets have been collected at four neutron sources; these are compared and two are merged.
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| 8. |
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| 9. |
- Oksanen, Esko, et al.
(author)
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Neutron crystallography for the study of hydrogen bonds in macromolecules
- 2017
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In: Molecules. - : MDPI AG. - 1420-3049. ; 22:4
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Research review (peer-reviewed)abstract
- The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.
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| 10. |
- Pfeiffer, Dorothea, et al.
(author)
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The mu TPC method: improving the position resolution of neutron detectors based on MPGDs
- 2015
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In: Journal of Instrumentation. - : IOP Publishing: Hybrid Open Access. - 1748-0221. ; 10
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Journal article (peer-reviewed)abstract
- Due to the He-3 crisis, alternatives to the standard neutron detection techniques are becoming urgent. In addition, the instruments of the European Spallation Source (ESS) require advances in the state of the art of neutron detection. The instruments need detectors with excellent neutron detection efficiency, high rate capabilities and unprecedented spatial resolution. The Macromolecular Crystallography instrument (NMX) requires a position resolution in the order of 200 mu m over a wide angular range of incoming neutrons. Solid converters in combination with Micro Pattern Gaseous Detectors (MPGDs) are proposed to meet the new requirements. Charged particles rising from the neutron capture have usually ranges larger than several millimetres in gas. This is apparently in contrast with the requirements for the position resolution. In this paper, we present an analysis technique, new in the field of neutron detection, based on the Time Projection Chamber (TPC) concept. Using a standard Single-GEM with the cathode coated with (B4C)-B-10, we extract the neutron interaction point with a resolution of better than sigma = 200 mu m.
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