Strategies for crystallization and structure determination of very large macromolecular assemblies

High-resolution structures of macromolecular assemblies are pivotal for our understanding of their biological functions in fundamental cellular processes. In the field of X-ray crystallography, recent methodological and instrumental advances have led to the structure determinations of macromolecular assemblies of increased size and complexity, such as those of ribosomal complexes, RNA polymerases, and large multifunctional enzymes. These advances include the use of robotic screening techniques that maximize the chances of obtaining well-diffracting crystals of large complexes through the fine sampling of crystallization space. Sophisticated crystal optimization and cryoprotection techniques and the use of highly brilliant X-ray beams from third-generation synchrotron light sources now allow data collection from weakly diffracting crystals with large asymmetric units. Combined approaches are used to derive phase information, including phases calculated from electron microscopy (EM) models, heavy atom clusters, and density modification protocols. New crystallographic software tools prove valuable for structure determination and model refinement of large macromolecular complexes.     

Protein crystallization in the structural genomics era

There are five broad areas where noteworthy advances have occurred in the field of macromolecular crystallization in the past 10 years, though some areas have seen the major part of those advances in only the last two years. This is largely a consequence of the international structural genomics initiative and its early results. The five areas are: (1) Physical studies and characterization of the protein crystallization process; (2) Development of new practical approaches and procedures; (3) The implementation of protein engineering by genetic means to enhance both purification and crystallization; (4) The creation of new screening conditions based on information and databases emerging from structural genomics; and (5) Development and implementation of automation, robotics, and mass screening of crystallization conditions using very small amounts of protein. A brief summary is provided here of the progress in the past few years and the influence of the structural genomics project.     

Analysis of conformational variation in macromolecular structural models

Experimental conditions or the presence of interacting components can lead to variations in the structural models of macromolecules. However, the role of these factors in conformational selection is often omitted by in silico methods to extract dynamic information from protein structural models. Structures of small peptides, considered building blocks for larger macromolecular structural models, can substantially differ in the context of a larger protein. This limitation is more evident in the case of modeling large multi-subunit macromolecular complexes using structures of the individual protein components. Here we report an analysis of variations in structural models of proteins with high sequence similarity. These models were analyzed for sequence features of the protein, the role of scaffolding segments including interacting proteins or affinity tags and the chemical components in the experimental conditions. Conformational features in these structural models could be rationalized by conformational selection events, perhaps induced by experimental conditions. This analysis was performed on a non-redundant dataset of protein structures from different SCOP classes. The sequence-conformation correlations that we note here suggest additional features that could be incorporated by in silico methods to extract dynamic information from protein structural models.     

Mineralization and non-ideality: on nature’s foundry

Understanding how ions, ion-clusters and particles behave in non-ideal environments is a fundamental question concerning planetary to atomic scales. For biomineralization phenomena wherein diverse inorganic and organic ingredients are present in biological media, attributing biomaterial composition and structure to the chemistry of singular additives may not provide a holistic view of the underlying mechanisms. Therefore, in this review, we specifically address the consequences of physico-chemical non-ideality on mineral formation. Influences of different forms of non-ideality such as macromolecular crowding, confinement and liquid-like organic phases on mineral nucleation and crystallization in biological environments are presented. Novel prospects for the additive-controlled nucleation and crystallization are accessible from this biophysical view. In this manner, we show that non-ideal conditions significantly affect the form, structure and composition of biogenic and biomimetic minerals.     

水/乙醇混合溶剂中的阿奇霉素结晶动力学

利用粒数密度和粒度之间的关系判别晶体生长模型;采用间歇动态法,以粒数衡算方程、溶质质量守恒和McCabe定律为基础,利用Beer-Lambert定律,借助光学关联的方法,建立了包含透光率变量的伴有成核和晶体生长的动力学模型;通过在线测量溶液密度与透光率数据,采用非线性最小二乘法拟合得到了晶体成核和生长动力学经验方程,并以实时浓度为目标验证了动力学参数的准确性以及模型表达式的正确性。     

Carboxylic acids in crystallization of macromolecules: learning from successful crystallization experiments

The production of macromolecular crystals suitable for structural analysis is one of the most important and limiting steps in the structure determination process. Often, preliminary crystallization trials are performed using hundreds of empirically selected conditions. Carboxylic acids and/or their salts are one of the most popular components of these empirically derived crystallization conditions. Our findings indicate that almost 40 % of entries deposited to the Protein Data Bank (PDB) reporting crystallization conditions contain at least one carboxylic acid. In order to analyze the role of carboxylic acids in macromolecular crystallization, a large-scale analysis of the successful crystallization experiments reported to the PDB was performed. The PDB is currently the largest source of crystallization data, however it is not easily searchable. These complications are due to a combination of a free text format, which is used to capture information on the crystallization experiments, and the inconsistent naming of chemicals used in crystallization experiments. Despite these difficulties, our approach allows for the extraction of over 47,000 crystallization conditions from the PDB. Initially, the selected conditions were investigated to determine which carboxylic acids or their salts are most often present in crystallization solutions. From this group, selected sets of crystallization conditions were analyzed in detail, assessing parameters such as concentration, pH, and precipitant used. Our findings will lead to the design of new crystallization screens focused around carboxylic acids.     

Crystallization of the most active RNA-cleaving deoxyribozyme.

The structural compositions of the most active deoxyribozyme and its derivatives have been examined by electrophoresis, and their crystallization conditions were surveyed for X-ray analysis. It has been found that Mg2+ ion is essential to form the active binary complex between the catalytic DNA and the substrate, and that heat-treatment is effective to prevent formation of the inactive quaternary complex between the two enzymes and the two substrates. Crystals obtained by the hanging drop vapor diffusion method are composed of the active binary complex.     

De novo high-resolution protein structure determination from sparse spin-labeling EPR data

As many key proteins evade crystallization and remain too large for nuclear magnetic resonance spectroscopy, electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling offers an alternative approach for obtaining structural information. Such information must be translated into geometric restraints to be used in computer simulations. Here, distances between spin labels are converted into distance ranges between beta carbons by using a "motion-on-a-cone" model, and a linear-correlation model links spin-label accessibility to the number of neighboring residues. This approach was tested on T4-lysozyme and alphaA-crystallin with the de novo structure prediction algorithm Rosetta. The results demonstrate the feasibility of obtaining highly accurate, atomic-detail models from EPR data by yielding 1.0 A and 2.6 A full-atom models, respectively. Distance restraints between amino acids far apart in sequence but close in space are most valuable for structure determination. The approach can be extended to other experimental techniques such as fluorescence spectroscopy, substituted cysteine accessibility method, or mutational studies.     

Automatic Classification and Pattern Discovery in High-throughput Protein Crystallization Trials

Conceptually, protein crystallization can be divided into two phases search and optimization. Robotic protein crystallization screening can speed up the search phase, and has a potential to increase process quality. Automated image classification helps to increase throughput and consistently generate objective results. Although the classification accuracy can always be improved, our image analysis system can classify images from 1536-well plates with high classification accuracy (85%) and ROC score (0.87), as evaluated on 127 human-classified protein screens containing 5600 crystal images and 189472 non-crystal images. Data mining can integrate results from high-throughput screens with information about crystallizing conditions, intrinsic protein properties, and results from crystallization optimization. We apply association mining, a data mining approach that identifies frequently occurring patterns among variables and their values. This approach segregates proteins into groups based on how they react in a broad range of conditions, and clusters cocktails to reflect their potential to achieve crystallization. These results may lead to crystallization screen optimization, and reveal associations between protein properties and crystallization conditions. We also postulate that past experience may lead us to the identification of initial conditions favorable to crystallization for novel proteins.     

Crystallization of intrinsic membrane proteins

After many years of discouraging failures, it is now possible to crystallize the intrinsic membrane proteins and to obtain structural information from diffraction studies on the crystals. The strategy for the crystallization consists of depletion of boundary phospholipids from the protein and complex formation with specific ligands.     

Structure of cation channels, revealed by single particle electron microscopy

A large barrier in the way to obtaining high-resolution structures of eukaryotic ion channels remains the expression and purification of sufficient amounts of channel protein to carry out crystallization trials. In the absence of crystals, the main available source of structural information has been electron microscopy (EM), which is well suited to the visualization of isolated macromolecular complexes and their conformational changes. The recently published EM structures outline native conformations of eukaryotic cation channels that until now have eluded crystallization. According to these results, homo-tetrameric K(+) channels have a distinct two-layer architecture with connectors conjoining the two layers, while the pseudo-tetrameric Ca(2+) or Na(+) channels are more globular and have flexible surface loops, which makes the identification of subunits complicated. Subunits can be identified using atomic structure docking into the EM maps, labeling, or deletion studies.     

单颗粒电子显微学的研究进展

单颗粒电子显微学是一种新型的结构生物学技术和方法,一方面,其解析生物大分子复合体结构的分辨率日益提高,可以达到近原子分辨率,提供大蛋白分子或复合体的精细结构;另一方面,还可以解析生物大分子在不同功能状态下的结构及变化,对于揭示生物大分子复合体结构的作用机理具有重要作用。本文就单颗粒电子显微学的研究进展作一综述。     

Crystallization mechanisms of hemoglobin C in the R state

Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.     

Purification,characterization and crystallization of the human 80S ribosome

Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.     

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Growth and disorder of macromolecular crystals: insights from atomic force microscopy and X-ray diffraction studies

The growth processes and defect structures of protein and virus crystals have been studied in situ by atomic force microscopy (AFM), X-ray diffraction topography, and high-resolution reciprocal space scanning. Molecular mechanisms of macromolecular crystallization were visualized and fundamental kinetic and thermodynamic parameters, which govern the crystallization process of a number of macromolecular crystals, have been determined. High-resolution AFM imaging of crystal surfaces provides information on the packing of macromolecules within the unit cell and on the structure of large macromolecular assemblies. X-ray diffraction techniques provide a bulk probe with poorer spatial resolution but excellent sensitivity to mosaicity and strain. Defect structures and disorder created in macromolecular crystals during growth, seeding, and post-growth treatments including flash cooling were characterized and their impacts on the diffraction properties of macromolecular crystals have been analyzed. The diverse and dramatic effects of impurities on growth and defect formation have also been studied. Practical implications of these fundamental insights into the improvement of macromolecular crystallization protocols are discussed.     

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.     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

Interferometric Studies of Growth Kinetics and Surface Morphology in Macromolecular Crystal Growth: Canavalin, Thaumatin, and Turnip Yellow Mosaic Virus

In situ laser Michelson interferometry was utilized to investigate mechanisms of growth and surface morphology in protein and virus crystallization, These included plant proteins canavalin and thaumatin and turnip yellow mosaic virus. The experimental apparatus allowed us to obtain interferometric patterns and investigate growth kinetics from growing macromolecular crystals as small as 20 μm. For the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces. Supersaturation and time dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were measured. The kinetic coefficient β (rate of incorporation of protein molecules into the growing crystal) was estimated for canavalin to be 9 × 10^-4 cm/sec. This is among the first estimates of such fundamental kinetic parameters for macromolecular crystallization. The change in the activities of dislocation sources under different growth conditions was also analyzed. Michelson interferometry was clearly demonstrated to be a useful tool for quantitative studies of macromolecular crystal growth.     

Cryocrystallography of metalloprotein reaction intermediates

Freeze-trapping reaction intermediates in macromolecular crystals is now a proven technique for obtaining their high-resolution structures by X-ray crystallography. The structural study of metalloprotein mechanisms has spearheaded this work, mainly because of the increased availability of single-crystal UV/visible spectrophotometry that enables reaction monitoring in the crystalline state. In particular, through formation of the frozen glass state, the stabilization of intermediates involving dissolved gases has yielded some of the most spectacular results. Metalloprotein systems still dominate this field, and the most recent successes, along with the accompanying advances in methodology, are presented.