Crystal structure of polymerization-competent actin

All actin crystal structures reported to date represent actin complexed or chemically modified with molecules that prevent its polymerization. Actin cleaved with ECP32 protease at a single site between Gly42 and Val43 is virtually non-polymerizable in the Ca-ATP bound form but remains polymerization-competent in the Mg-bound form. Here, a crystal structure of the true uncomplexed ECP32-cleaved actin (ECP-actin) solved to 1.9 A resolution is reported. In contrast to the much more open conformation of the ECP-actin's nucleotide binding cleft in solution, the crystal structure of uncomplexed ECP-actin contains actin in a typical closed conformation similar to the complexed actin structures. This unambiguously demonstrates that the overall structure of monomeric actin is not significantly affected by a multitude of actin-binding proteins and toxins. The invariance of actin crystal structures suggests that the salt and precipitants necessary for crystallization stabilize actin in only one of its possible conformations. The asymmetric unit cell contains a new type of antiparallel actin dimer that may correspond to the "lower dimer" implicated in F-actin nucleation and branching. In addition, symmetry-related actin-actin contacts form a head to tail dimer that is strikingly similar to the longitudinal dimer predicted by the Holmes F-actin model, including a rotation of the monomers relative to each other not observed previously in actin crystal structures.     

Enhancing protein crystallization through precipitant synergy

Suitable conditions for protein crystallization are commonly identified by screening combinations of independent factors that affect crystal formation. Because precipitating agents are prime determinants of crystallization, we investigated whether a systematic exploration of combinations of mechanistically distinct precipitants would enhance crystallization. A crystallization screen containing 64 precipitant mixtures was devised. Tests with ten HIV envelope-related proteins demonstrated that use of precipitant mixtures significantly enhanced both the probability of crystallization as well as the quality of optimized crystals. Tests with hen egg white lysozyme generated a novel C2 crystal from a salt/organic solvent mixture; structure solution at 2 A resolution revealed a lattice held together by both hydrophobic and electrostatic dyad interactions. The results indicate that mechanistically distinct precipitants can synergize, with precipitant combinations adding unique dimensions to protein crystallization.     

Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations

Cryo-cooling is routinely performed before x-ray diffraction image collection to reduce the damage to crystals due to ionizing radiation. It has been suggested that although backbone structures are usually very similar between room temperature and cryo-temperature, cryo-cooling may hamper biologically relevant dynamics. In this study, the crystal of Escherichia coli dihydrofolate reductase is studied with replica-exchange molecular dynamics simulation, and the results are compared with the crystal structure determined at cryo-temperature and room temperature with the time-averaged ensemble method. Although temperature dependence of unit cell compaction and root mean-square fluctuation of Cα is found in accord with experiment, it is found that the protein structure at low temperature can be more heterogeneous than the ensemble of structures reported by using the time-averaged ensemble method, encouraging further development of the time-averaged ensemble method and indicating that data should be examined carefully to avoid overinterpretation of one average structure.     

沉淀剂类型对蛋白质晶体分子堆积的影响

以不对称单位只有一个分子的牛胰核糖核酸酶和T4溶菌酶晶体为材料,着重研究了无机盐、有机溶剂和PEG三类不同的沉淀剂对晶体分子堆积的影响,经研究发现两种蛋白质中用无机盐做沉淀剂的晶型几乎都含有面积较大的二次轴对称接触面和较少的相邻分子数,同时其含有的参与接触的非极性残基集中分布于二次轴对称接触面,而盐键则在二次轴对称接触面上分布稀少。用有机溶剂作沉淀剂的晶型却含有面积较小的非二次轴对称接触面和较多的相含分子数,而参与接触的非极性残基和直键在各个非二次轴对称接触面上随机分布,用PEG作沉淀剂的晶型其分子堆积特征总体上类似于用有机溶剂作沉淀剂的晶型,但个别晶型具有与用无机盐做沉淀剂的晶型相似的分子堆积特征,以上结果提示,用三类沉淀剂得到的不同的分子堆积特征可能与三类沉淀剂不同的诱导结晶机理密切相关。     

Simulations of a protein crystal: explicit treatment of crystallization conditions links theory and experiment in the streptavidin-biotin complex

A 250 ns molecular dynamics simulation of the biotin-liganded streptavidin crystal lattice, including cryoprotectant molecules and crystallization salts, is compared to a 250 ns simulation of the lattice solvated with pure water. The simulation using detailed crystallization conditions preserves the initial X-ray structure better than the simulation using pure water, even though the protein molecules display comparable mobility in either simulation. Atomic fluctuations computed from the simulation with crystallization conditions closely reproduce fluctuations derived from experimental temperature factors (correlation coefficient of 0.88, omitting two N-terminal residues with very high experimental B-factors). In contrast, fluctuations calculated from the simulation with pure water were less accurate, particularly for two of the streptavidin loops exposed to solvent in the crystal lattice. Finally, we obtain good agreement between the water and cryoprotectant densities obtained from the simulated crystallization conditions and the electron density due to solvent molecules in the X-ray structure. Our results suggest that detailed lattice simulations with realistic crystallization conditions can be used to assess potential function parameters, validate simulation protocols, and obtain valuable insights that solution-phase simulations do not easily provide. We anticipate that this will prove to be a powerful strategy for molecular dynamics simulations of biomolecules.     

Crystal structure of peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro)

The crystal and molecular structure of the rubidium/picrate complex of the peptide cyclo-(D-val-L-pro-L-val-D-pro)₃, called prolinomycin, has been determined by X-ray crystallography and found to be similar to the well known ion-carrier valinomycin. Prolinomycin crystallizes in the triclinic system with two prolinomycin molecules and two each rubidium cations and picrate anions in the unit cell. There are also ordered toluene and chloroform molecules, which are the solvents of crystallization, in the unit cell. The conformation of the two crystallographically independent prolinomycin molecules in the unit cell are very similar. Potential energy calculations show that the cation is bound more strongly in prolinomycin compared to valinomycin. This was also observed in solution (7).     

3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures.

Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.     

Molecular dynamics simulations of a double unit cell in a protein crystal: volume relaxation at constant pressure and correlation of motions between the two unit cells

Eight molecular dynamics simulations of a double crystal unit cell of ubiquitin were performed to investigate the effects of simulating at constant pressure and of simulating two unit cells compared to a single unit cell. To examine the influence of different simulation conditions, the constant-pressure and constant-volume simulations were each performed with and without counterions and using two different treatments of the long-range electrostatic interactions (lattice-sum and reaction-field methods). The constant-pressure simulations were analyzed in terms of unit cell deformation and accompanying protein deformations. Energetic and structural properties of the proteins in the simulations of the double unit cell were compared to the results of previously reported one-unit-cell simulations. Correlation between the two unit cells was also investigated based on relative translational and rotational movements of the proteins and on dipole fluctuations. The box in the constant-pressure simulations is found to deform slowly to reach convergence only after 5-10 ns. This deformation does not result from a distortion in the structure of the proteins but rather from changes in protein packing within the unit cell. The results of the double-unit-cell simulations are closely similar to the results of the single-unit-cell simulations, and little motional correlation is found between the two unit cells.     

Crystal packing in six crystal forms of pancreatic ribonuclease.

We compare the molecular packing of bovine pancreatic ribonuclease A (RNase A) in six crystal forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which bury 1570 A2 to 2790 A2 of the RNase surface. Regions of the protein surface involved in the six packings cover almost the whole RNase molecule. The abundance of polar interactions, about one per 200 A2, is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large interface covering 1800 A2 and eight to ten polar interactions. Its presence in the three salt-grown crystal forms suggests that it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms contain only small interfaces, implying a different mechanism of nucleation.     

膜蛋白晶体的生长及其进展

生物膜中与脂双层结合的蛋白质称为膜蛋白.由于它们具有很大的疏水表面以及既亲水又疏水的两性特点致使其纯化与结晶都十分困难.在膜蛋白晶体生长系统中引入小分子去污剂与小的两性分子获得突破性进展.迄今为止,结晶出来的膜蛋白为数不多.其中只有光合细菌绿色红假单胞菌及球型红假单胞菌的反应中心得到3分辨率的晶体结构与解析.一系列膜蛋白形成二维晶体,可用电子显微镜与像重构技术获得三维结构信息.     

Generation of initial molecular dynamics configurations in arbitrary geometries and in parallel

A computational pre-processing tool for generating initial configurations of molecules for molecular dynamics simulations in geometries described by a mesh of unstructured arbitrary polyhedra is described. The mesh is divided into separate zones and each can be filled with a single crystal lattice of atoms. Each zone is filled by creating an expanding cube of crystal unit cells, initiated from an anchor point for the lattice. Each unit cell places the appropriate atoms for the user-specified crystal structure and orientation. The cube expands until the entire zone is filled with the lattice; zones with concave and disconnected volumes may be filled. When the mesh is spatially decomposed into portions for distributed parallel processing, each portion may be filled independently, meaning that the entire molecular system never needs to fit onto a single processor, allowing very large systems to be created. The computational time required to fill a zone with molecules scales linearly with the number of cells in the zone for a fixed number of molecules, and better than linearly with the number of molecules for a fixed number of mesh cells. Our tool, molConfig, has been implemented in the open source C++ code OpenFOAM.     

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.     

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.     

Single crystals of alpha-chitin.

Single crystals of alpha-chitin were grown by the addition of precipitants to dilute solutions of low molecular weight chitin fractions dissolved in aqueous LiSCN. At temperatures around 200 degrees C, bundles of thin needle-shaped crystals were obtained. Each of these needles was an alpha-chitin single crystal, characterized by a spot electron diffraction pattern which could be indexed along the hk0 reciprocal net corresponding to the Minke and Blackwell unit cell [a = 0.474 nm, b = 1.88 nm, c (fibre axis) = 1.032 nm, space group P2(1)2(1)2(1)]. In a crystal, the a* parameter was along the crystal axis and the b* perpendicular to it.     

Promotion of Crystal Phase Transitions by Mass-of-cell Control in Molecular Dynamics Simulations: Phase Transitions in Benzene Crystals

Abstract

The promotion of crystal phase transitions in molecular dynamics (MD) simulations was realized by controlling the momentum of the MD cell. It was implemented by increasing the mass or velocity of the MD cell instantaneously during simulations within the framework of the constant-pressure method by Parrinello and Rahman. This method induced phase transitions in benzene crystals which have not been obtained in conventional MD simulations. This method is useful for the global search of stable (and metastable) crystal structures.     

Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens

Recent efforts to collect and mine crystallization data from structural genomics (SG) consortia have led to the identification of minimal screens and novel screening strategies that can be used to streamline the crystallization process. Two groups, the Joint Center for Structural Genomics and the University of Toronto, carried out large-scale crystallization trials on different sets of bacterial targets (539, JCSG and 755, Toronto), using different sample processing and crystallization methods, and then analyzed their results to identify the smallest subset of conditions that would have crystallized the maximum number of protein targets. The JCSG Core Screen contains 67 conditions (from 480) while the Toronto Minimal Screen contains 6 (from 48). While the exact conditions included in the two screens do not overlap, the major precipitants of the conditions are similar and thus both screens can be used to determine if a protein has a natural propensity to crystallize. In addition, studies from other groups including the University of Queensland, the Mycobacterium tuberculosis SG group, the Southeast Collaboratory for SG, and the York Structural Biology Laboratory indicate that alternative crystallization strategies may be more successful at identifying initial crystallization conditions than typical sparse matrix screens. These minimal screens and alternative screening strategies are already being used to optimize the crystallization processes within large SG efforts. The differences between these results, however, demonstrate that additional studies which examine the influence of protein biophysical properties and sample preparation methods on crystal formation must also be carried out before more robust screens can be identified.     

Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

Currently, the best existing molecular dynamics (MD) force fields cannot accurately reproduce the global free‐energy minimum which realizes the experimental protein structure. As a result, long MD trajectories tend to drift away from the starting coordinates (e.g., crystallographic structures). To address this problem, we have devised a new simulation strategy aimed at protein crystals. An MD simulation of protein crystal is essentially an ensemble simulation involving multiple protein molecules in a crystal unit cell (or a block of unit cells). To ensure that average protein coordinates remain correct during the simulation, we introduced crystallography‐based restraints into the MD protocol. Because these restraints are aimed at the ensemble‐average structure, they have only minimal impact on conformational dynamics of the individual protein molecules. So long as the average structure remains reasonable, the proteins move in a native‐like fashion as dictated by the original force field. To validate this approach, we have used the data from solid‐state NMR spectroscopy, which is the orthogonal experimental technique uniquely sensitive to protein local dynamics. The new method has been tested on the well‐established model protein, ubiquitin. The ensemble‐restrained MD simulations produced lower crystallographic R factors than conventional simulations; they also led to more accurate predictions for crystallographic temperature factors, solid‐state chemical shifts, and backbone order parameters. The predictions for ¹⁵N relaxation rates are at least as accurate as those obtained from conventional simulations. Taken together, these results suggest that the presented trajectories may be among the most realistic protein MD simulations ever reported. In this context, the ensemble restraints based on high‐resolution crystallographic data can be viewed as protein‐specific empirical corrections to the standard force fields.     

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.     

Molecular resolution imaging of macromolecular crystals by atomic force microscopy.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).     

The dynamics of camphor in the cytochrome P450 CYP101D2

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.