Protein crystallization: from purified protein to diffraction-quality crystal

Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth.     

Models of protein crystal growth

The growth of large and well ordered protein crystals remains the major obstacle in protein structure determination by means of X-ray crystallography. One of the reasons is that the physico-chemical aspect of protein crystallization process is not understood. This article reviews efforts towards formulation of models that could become theoretical frameworks for the interpretation of voluminous experimental data collected on protein crystal growth. Special attention is devoted to microscopic models that recognize the role of the shape of protein molecules in crystal formation.     

Crystallization of RNA and RNA-protein complexes

RNA plays a direct role in a variety of cellular activities, and in many cases its biological function is conferred by the RNA three-dimensional structure. X-ray crystallography is the method of choice for determining high resolution structures of large RNA molecules, and can also be used to compare related RNAs and identify conformational changes that may accompany biochemical activity. However, crystallization remains the rate-limiting step in RNA structure determination due to the difficulty in obtaining well-ordered crystals for X-ray diffraction analysis. Several approaches to sample preparation, crystallization, and crystal handling are presented that have been used successfully in the structure determination of RNA and RNA-protein complexes in our laboratory, and should be generally applicable to RNAs in other systems.     

Evaluation of a model for seeded isothermal batch protein crystallization

Bulk protein crystallization, unlike small molecule crystallization, has found very limited use in biopharmaceutical manufacture. Most work in this area targets obtaining single large crystals for molecular structure determination by crystallography. Design and optimization of bulk crystallization for protein recovery and purification is much less common, and requires a mathematical model for analysis of laboratory data suitable for scale-up purposes. Traditionally, the crystal size distribution and method of moments is used to characterize the crystallization process. A simpler method is presented in this paper that utilizes the desupersaturation curve. The method uses an approach that does not require expensive instrumentation or characterization of the seed crystal size distribution. The method is extended to allow determination of both the mass deposition rate constant and the growth rate order from a single desuperaturation curve. Experimental data for the bulk crystallization of ovalbumin are used to validate the method. The rate constants and rate order obtained using the new method compare well with literature values. Scale-up is illustrated by prediction of the impact of changes in seed mass on protein crystallization. This new method offers a straightforward and low-cost alternative to traditional methods for the analysis and scale-up of protein crystallization data.     

Kluyveromyces lactis β-galactosidase crystallization using full-factorial experimental design

Kluyveromyces lactis β-galactosidase is an enzyme with numerous applications in the environmental, food and biotechnological industries. Despite of its biotechnological interest, its three-dimensional structure has not yet been determined. The growth of suitable crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures since their growth often depends on the combination of many different factors. Testing the influence on crystallization of even only a small number of these factors requires many experimental set-ups and large amounts of protein. In the present work, a full-factorial design has been used in order to find conditions for obtaining good-quality crystals of K. lactis β-galactosidase. With this full-factorial method protein crystals have been obtained.     

Current trends in α-helical membrane protein crystallization: An update

α-Helical membrane proteins (MPs) are the targets for many pharmaceutical drugs and play important roles in human physiology. In recent years, significant progress has been made in determining their atomic structure using X-ray crystallography. However, a major bottleneck in MP crystallography still remains, namely, the identification of conditions that give crystals that are suitable for structural determination. In 2008, we undertook an analysis of the crystallization conditions for 121 α-helical MPs to design a rationalized sparse matrix crystallization screen, MemGold. We now report an updated analysis that includes a further 133 conditions. The results reveal the current trends in α-helical MP crystallization with notable differences since 2008. The updated information has been used to design new crystallization and additive screens that should prove useful for both initial crystallization scouting and subsequent crystal optimization.     

Synergy within structural biology of single crystal optical spectroscopy and X-ray crystallography

Advances in the adaptation of optical spectroscopy to monitor photo-induced or enzyme-catalyzed reactions in the crystalline state have enabled X-ray crystal structures to be accurately linked with spectroscopically defined intermediates. This, in turn, has led to a deeper understanding of the role protein structural changes play in function. The integration of optical spectroscopy with X-ray crystallography is growing and now extends beyond linking crystal structure to reaction intermediate. Recent examples of this synergy include applications in protein crystallization, X-ray data acquisition, radiation damage, and acquisition of phase information important for structure determination.     

Rational protein crystallization by mutational surface engineering

Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their replacement with smaller amino acids lead to effective preparation of X-ray quality crystals of proteins otherwise recalcitrant to crystallization. Furthermore, this technique can also be used to obtain crystals of superior quality as compared to those grown for the wild-type protein, sometimes increasing the effective resolution by as much as 1 A or more. Several recent examples of this new methodology suggest that the method has the potential to become a routine tool in protein crystallography.     

Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.     

The use of recombinant methods and molecular engineering in protein crystallization

Recombinant techniques are routinely used for the preparation of protein samples for structural studies including X-ray crystallography. Among other benefits, these methods allow for a vast increase in the amount of obtained protein as compared to purification from source tissues, ease of purification when fusion proteins containing affinity tags are used, introduction of SeMet for phasing, and the opportunity to modify the protein to enhance its crystallizability. Protein engineering may involve removal of flexible regions including termini and interior loops, as well as replacement of residues that affect solubility. Moreover, modification of the protein surface to induce crystal growth may include rational engineering of surface patches that can readily mediate crystal contacts. The latter approach can be used to obtain proteins of crystals recalcitrant to crystallization or to obtain well-diffracting crystals in lieu of wild-type crystals yielding data to limited resolution. This review discusses recent advances in the field and describes a number of examples of diverse protein engineering techniques used in crystallographic investigations.     

High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.     

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.     

Automated technologies and novel techniques to accelerate protein crystallography for structural genomics

The sequence infrastructure that has arisen through large-scale genomic projects dedicated to protein analysis, has provided a wealth of information and brought together scientists and institutions from all over the world. As a consequence, the development of novel technologies and methodologies in proteomics research is helping to unravel the biochemical and physiological mechanisms of complex multivariate diseases at both a functional and molecular level. In the late sixties, when X-ray crystallography had just been established, the idea of determining protein structure on an almost universal basis was akin to an impossible dream or a miracle. Yet only forty years after, automated protein structure determination platforms have been established. The widespread use of robotics in protein crystallography has had a huge impact at every stage of the pipeline from protein cloning, over-expression, purification, crystallization, data collection, structure solution, refinement, validation and data management- all of which have become more or less automated with minimal human intervention necessary. Here, recent advances in protein crystal structure analysis in the context of structural genomics will be discussed. In addition, this review aims to give an overview of recent developments in high throughput instrumentation, and technologies and strategies to accelerate protein structure/function analysis.     

Automated crystal mounting and data collection for protein crystallography

To increase the efficiency of diffraction data collection for protein crystallographic studies, an automated system designed to store frozen protein crystals, mount them sequentially, align them to the X-ray beam, collect complete data sets, and return the crystals to storage has been developed. Advances in X-ray data collection technology including more brilliant X-ray sources, improved focusing optics, and faster-readout detectors have reduced diffraction data acquisition times from days to hours at a typical protein crystallography laboratory [1,2]. In addition, the number of high-brilliance synchrotron X-ray beam lines dedicated to macromolecular crystallography has increased significantly, and data collection times at these facilities can be routinely less than an hour per crystal. Because the number of protein crystals that may be collected in a 24 hr period has substantially increased, unattended X-ray data acquisition, including automated crystal mounting and alignment, is a desirable goal for protein crystallography. The ability to complete X-ray data collection more efficiently should impact a number of fields, including the emerging structural genomics field [3], structure-directed drug design, and the newly developed screening by X-ray crystallography [4], as well as small molecule applications.     

Surface topography of the p3 and p6 annexin V crystal forms determined by atomic force microscopy

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. The structure of the soluble form of annexin V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography.     

Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily

Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

X-ray crystallography of chemical compounds

AimsAccurate knowledge of molecular structure is a prerequisite for rational drug design. This review examines the role of X-ray crystallography in providing the required structural information and advances in the field of X-ray crystallography that enhance or expand its role.Main methodsX-ray crystallography of new drugs candidates and intermediates can provide valuable information of new syntheses and parameters for quantitative structure activity relationships (QSAR).Key findingsCrystallographic studies play a vital role in many disciplines including materials science, chemistry, pharmacology, and molecular biology. X-ray crystallography is the most comprehensive technique available to determine molecular structure. A requirement for the high accuracy of crystallographic structures is that a ‘good crystal’ must be found, and this is often the rate-limiting step. In the past three decades developments in detectors, increases in computer power, and powerful graphics capabilities have contributed to a dramatic increase in the number of materials characterized by X-ray crystallography. More recently the advent of high-throughput crystallization techniques has enhanced our ability to produce that one good crystal required for crystallographic analysis.SignificanceContinuing advances in all phases of a crystallographic study have expanded the ranges of samples which can be analyzes by X-ray crystallography to include larger molecules, smaller or weakly diffracting crystals, and twinned crystals.     

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.     

In vivo protein crystallization opens new routes in structural biology

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.