| 1. |
- Aggarwal, Swati, et al.
(författare)
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A protocol for production of perdeuterated OmpF porin for neutron crystallography
- 2021
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 188
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
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| 2. |
- Bergmann, Justin, et al.
(författare)
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Can the results of quantum refinement be improved with a continuum-solvation model?
- 2021
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Ingår i: Acta Crystallographica. Section B: Structural Science. - 0108-7681. ; 77:6, s. 906-918
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Tidskriftsartikel (refereegranskat)abstract
- Quantum refinement has repeatedly been shown to be a powerful approach to interpret and improve macromolecular crystal structures, allowing for the discrimination between different interpretations of the structure, regarding the protonation states or the nature of bound ligands, for example. In this method, the empirical restraints, used to supplement the crystallographic raw data in standard crystallographic refinement, are replaced by more accurate quantum mechanical (QM) calculations for a small, but interesting, part of the structure. Previous studies have shown that the results of quantum refinement can be improved if the charge of the QM system is reduced by adding neutralizing groups. However, this significantly increases the computation time for the refinement. In this study, we show that a similar improvement can be obtained if the original highly charged QM system is instead immersed in a continuum solvent in the QM calculations. The best results are typically obtained with a high dielectric constant (ε). The continuum solvent improves real-space it Z values, electron-density difference maps and strain energies, and it normally does not affect the discriminatory power of the calculations between different chemical interpretations of the structure. However, for structures with a low charge in the QM system or with a low crystallographic resolution (>2Å), no improvement of the structures is seen.
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| 3. |
- Bergmann, Justin, et al.
(författare)
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Combining crystallography with quantum mechanics
- 2022
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Ingår i: Current Opinion in Structural Biology. - : Elsevier BV. - 0959-440X. ; 72, s. 18-26
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Forskningsöversikt (refereegranskat)abstract
- In standard crystallographic refinement of biomacromolecules, the crystallographic raw data are supplemented by empirical restraints that ensure that the structure makes chemical sense. These restraints are typically accurate for amino acids and nucleic acids, but less so for cofactors, substrates, inhibitors, ligands and metal sites. In quantum refinement, this potential is replaced by more accurate quantum mechanical (QM) calculations. Several implementations have been presented, differing in the level of QM and whether it is used for the entire structure or only for a site of particular interest. It has been shown that the method can improve and correct errors in crystal structures and that it can be used to determine protonation and tautomeric states of various ligands and to decide what is really seen in the structure by refining different interpretations and using standard crystallographic and QM quality measures to decide which fits the structure best.
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| 4. |
- Bergmann, Justin, et al.
(författare)
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Critical evaluation of a crystal structure of nitrogenase with bound N2 ligands
- 2021
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 26:2-3, s. 341-353
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Tidskriftsartikel (refereegranskat)abstract
- Recently, a 1.83 Å crystallographic structure of nitrogenase was suggested to show N2-derived ligands at three sites in the catalytic FeMo cluster, replacing the three μ2 bridging sulfide ligands (two in one subunit and the third in the other subunit) (Kang et al. in Science 368: 1381–1385, 2020). Naturally, such a structure is sensational, having strong bearings on the reaction mechanism of the enzyme. Therefore, it is highly important to ensure that the interpretation of the structure is correct. Here, we use standard crystallographic refinement and quantum refinement to evaluate the structure. We show that the original crystallographic raw data are strongly anisotropic, with a much lower resolution in certain directions than others. This, together with the questionable use of anisotropic B factors, give atoms an elongated shape, which may look like diatomic atoms. In terms of standard electron-density maps and real-space Z scores, a resting-state structure with no dissociated sulfide ligands fits the raw data better than the interpretation suggested by the crystallographers. The anomalous electron density at 7100 eV is weaker for the putative N2 ligands, but not lower than for several of the μ3 bridging sulfide ions and not lower than what can be expected from a statistical analysis of the densities. Therefore, we find no convincing evidence for any N2 binding to the FeMo cluster. Instead, a standard resting state without any dissociated ligands seems to be the most likely interpretation of the structure. Likewise, we find no support that the homocitrate ligand should show monodentate binding. Graphic abstract: [Figure not available: see fulltext.].
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| 5. |
- Bergmann, Justin, et al.
(författare)
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Quantum-refinement studies of the bidentate ligand of V‑nitrogenase and the protonation state of CO-inhibited Mo‑nitrogenase
- 2021
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Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 0162-0134. ; 219
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Tidskriftsartikel (refereegranskat)abstract
- Nitrogenase is the only enzyme that can cleave the triple bond in N2, making nitrogen available to plants (although the enzyme itself is strictly microbial). It has been studied extensively with both experimental and computational methods, but many details of the reaction mechanism are still unclear. X-ray crystallography is the main source of structural information for biomacromolecules, but it has problems to discern hydrogen atoms or to distinguish between elements with the same number of electrons. These problems can sometimes be alleviated by introducing quantum chemical calculations in the refinement, providing information about the ideal structure (in the same way as the empirical restraints used in standard crystallographic refinement) and comparing different interpretations of the structure with normal crystallographic and quantum mechanical quality measures. We have performed such quantum-refinement calculations to address two important issues for nitrogenase. First, we show that the bidentate ligand of the active-site FeV cluster in V‑nitrogenase is carbonate, rather than bicarbonate or nitrate. Second, we study the CO-inhibited structure of Mo‑nitrogenase. CO binds to a reduced and protonated state of the enzyme by replacing one of the sulfide ions (S2B) in the active-site FeMo cluster. We examined if it is possible to deduce from the crystal structure the location of the protons. Our results indicates that the crystal structure is best modelled as fully deprotonated.
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| 6. |
- Caldararu, Octav, et al.
(författare)
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Are crystallographic : B-factors suitable for calculating protein conformational entropy?
- 2019
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 21:33, s. 18149-18160
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Caldararu, Octav, et al.
(författare)
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Exploring ligand dynamics in protein crystal structures with ensemble refinement
- 2021
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1099-1115
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and R free values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.
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| 8. |
- Caldararu, Octav, et al.
(författare)
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Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase
- 2019
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 10:2, s. 576-586
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Tidskriftsartikel (refereegranskat)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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| 9. |
- Caldararu, Octav, et al.
(författare)
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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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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 368-380
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Caldararu, Octav, et al.
(författare)
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Water structure in solution and crystal molecular dynamics simulations compared to protein crystal structures
- 2020
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 10, s. 8435-8443
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Tidskriftsartikel (refereegranskat)abstract
- The function of proteins is influenced not only by the atomic structure but also by the detailed structure of the solvent surrounding it. Computational studies of protein structure also critically depend on the water structure around the protein. Herein we compare the water structure obtained from molecular dynamics (MD) simulations of galectin-3 in complex with two ligands to crystallographic water molecules observed in the corresponding crystal structures. We computed MD trajectories both in a water box, which mimics a protein in solution, and in a crystallographic unit cell, which mimics a protein in a crystal. The calculations were compared to crystal structures obtained at both cryogenic and room temperature. Two types of analyses of the MD simulations were performed. First, the positions of the crystallographic water molecules were compared to peaks in the MD density after alignment of the protein in each snapshot. The results of this analysis indicate that all simulations reproduce the crystallographic water structure rather poorly. However, if we define the crystallographic water sites based on their distances to nearby protein atoms and follow these sites throughout the simulations, the MD simulations reproduce the crystallographic water sites much better. This shows that the failure of MD simulations to reproduce the water structure around proteins in crystal structures observed both in this and previous studies is caused by the problem of identifying water sites for a flexible and dynamic protein (traditionally done by overlaying the structures). Our local clustering approach solves the problem and shows that the MD simulations reasonably reproduce the water structure observed in crystals. Furthermore, analysis of the crystal MD simulations indicates a few water molecules that are close to unmodeled electron density peaks in the crystal structures, suggesting that crystal MD could be used as a complementary tool for identifying and modelling water in protein crystallography.
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