Towards an integrative structural biology approach: combining Cryo-TEM,X-ray crystallography,and NMR

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.     

Automation of NMR structure determination of proteins

The automation of protein structure determination using NMR is coming of age. The tedious processes of resonance assignment, followed by assignment of NOE (nuclear Overhauser enhancement) interactions (now intertwined with structure calculation), assembly of input files for structure calculation, intermediate analyses of incorrect assignments and bad input data, and finally structure validation are all being automated with sophisticated software tools. The robustness of the different approaches continues to deal with problems of completeness and uniqueness; nevertheless, the future is very bright for automation of NMR structure generation to approach the levels found in X-ray crystallography. Currently, near completely automated structure determination is possible for small proteins, and the prospect for medium-sized and large proteins is good.     

蛋白质晶体学技术的发展与展望

从50年前英国科学家解析出第一个蛋白质晶体结构以来,蛋白质晶体学历经数个里程碑式的发展,已经成为一门成熟的高科技学科,是结构生物学的主要研究手段。近年来结构生物学发展迅速并和其他学科相互渗透交叉,特别是受到结构基因组学等热点学科的极大带动。作为结构生物学的基本手段和技术,蛋白质晶体学从解析简单的蛋白质三维结构延伸到解决各类生物大分子及复合物结构,并更加注重研究结构与功能之间的相互关系,派生出诸如基于结构的药物设计等应用性很强的分支。生物技术及计算机技术的飞速发展,尤其是高通量技术在生物学领域的应用,为蛋白质晶体学带来了全新的概念和更加广阔的前景。文章将主要介绍蛋白质晶体学技术的一些历史发展以及对未来的展望。     

Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination

Counterdiffusion crystallization in capillary is a very simple, cost-effective, and practical procedure for obtaining protein crystals suitable for X-ray data analysis. Its principles have been derived using well-known concepts coupling the ideas of precipitation and diffusion mass transport in a restricted geometry. The counterdiffusion process has been used to simultaneously screen for optimal conditions for protein crystal growth, incorporate strong anomalous scattering atoms, and mix in cryogenic solutions in a single capillary tube. The crystals obtained in the capillary have been used in situ for X-ray analysis. The implementation of this technique linked to the advancement of current crystallography software leads to a powerful structure determination method consolidating crystal growth, X-ray data collection, and ab initio phase determination into one without crystal manipulation. We review the historical progress of counterdiffusion crystallization, its application to X-ray crystallography, and ongoing tool development for high-throughput protein structure determination.     

High-throughput crystallography to enhance drug discovery

Protein crystallography has traditionally been regarded as a resource-intensive, time-consuming technique that, with some notable exceptions, has not made a significant impact on drug discovery. However, inspired by successes in the genome-sequencing initiatives, recent years have seen major changes in X-ray crystallography methodologies and the concept of high-throughput crystallography has emerged. Advances have been made in all phases of the process, including improved molecular biology, protein expression, crystallization and structure determination. This transformation has allowed X-ray crystallography to impact more broadly in the drug-discovery process, extending its utility from structure-based lead optimisation to novel fragment-based lead generation approaches.     

Combination of NMR spectroscopy and X-ray crystallography offers unique advantages for elucidation of the structural basis of protein complex assembly

NMR spectroscopy and X-ray crystallography are two premium methods for determining the atomic structures of macro-biomolecular complexes. Each method has unique strengths and weaknesses. While the two techniques are highly complementary, they have generally been used separately to address the structure and functions of biomolecular complexes. In this review, we emphasize that the combination of NMR spectroscopy and X-ray crystallography offers unique power for elucidating the structures of complicated protein assemblies. We demonstrate, using several recent examples from our own laboratory, that the exquisite sensitivity of NMR spectroscopy in detecting the conformational properties of individual atoms in proteins and their complexes, without any prior knowledge of conformation, is highly valuable for obtaining the high quality crystals necessary for structure determination by X-ray crystallography. Thus NMR spectroscopy, in addition to answering many unique structural biology questions that can be addressed specifically by that technique, can be exceedingly powerful in modern structural biology when combined with other techniques including X-ray crystallography and cryo-electron microscopy.     

Analytical ultracentrifugation in structural biology

Researchers in the field of structural biology, especially X-ray crystallography and protein nuclear magnetic resonance, are interested in knowing as much as possible about the state of their target protein in solution. Not only is this knowledge relevant to studies of biological function, it also facilitates determination of a protein structure using homogeneous monodisperse protein samples. A researcher faced with a new protein to study will have many questions even after that protein has been purified. Analytical ultracentrifugation (AUC) can provide all of this information readily from a small sample in a non-destructive way, without the need for labeling, enabling structure determination experiments without any wasting time and material on uncharacterized samples. In this article, I use examples to illustrate how AUC can contribute to protein structural analysis. Integrating information from a variety of biophysical experimental methods, such as X-ray crystallography, small angle X-ray scattering, electrospray ionization-mass spectrometry, AUC allows a more complete understanding of the structure and function of biomacromolecules.     

Femtosecond nanocrystallography using X-ray lasers for membrane protein structure determination

The invention of free electron X-ray lasers has opened a new era for membrane protein structure determination with the recent first proof-of-principle of the new concept of femtosecond nanocrystallography. Structure determination is based on thousands of diffraction snapshots that are collected on a fully hydrated stream of nanocrystals. This review provides a summary of the method and describes how femtosecond X-ray crystallography overcomes the radiation-damage problem in X-ray crystallography, avoids the need for growth and freezing of large single crystals while offering a new method for direct digital phase determination by making use of the fully coherent nature of the X-ray beam. We briefly review the possibilities for time-resolved crystallography, and the potential for making 'molecular movies' of membrane proteins at work.     

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.     

Dissecting the structural features of β-arrestins as multifunctional proteins

β-arrestins bind active G protein-coupled receptors (GPCRs) and play a crucial role in receptor desensitization and internalization. The classical paradigm of arrestin function has been expanded with the identification of many non-receptor-binding partners, which indicated the multifunctional role of β-arrestins in cellular functions. To elucidate the molecular mechanism of β-arrestin-mediated signaling, the structural features of β-arrestins were investigated using X-ray crystallography and cryogenic electron microscopy (cryo-EM). However, the intrinsic conformational flexibility of β-arrestins hampers the elucidation of structural interactions between β-arrestins and their binding partners using conventional structure determination tools. Therefore, structural information obtained using complementary structure analysis techniques would be necessary in combination with X-ray crystallography and cryo-EM data. In this review, we describe how β-arrestins interact with their binding partners from a structural point of view, as elucidated by both traditional methods (X-ray crystallography and cryo-EM) and complementary structure analysis techniques.     

The crystal structure of a complex of cycloheptaamylose with 2,5-diiobobenzoic acid

The molecular structure of the 1:1 complex of cycloheptaamylose with 2,5-diiodobenzoic acid has been determined by X-ray crystallography. The iodine atoms of the guest molecular are disordered and were not used in the structure determination. The cycloheptaamylose molecules form channels in the crystal by means of head to head and tail to tail association using the two-fold crystallographic axis.     

Progress in the analysis of membrane protein structure and function

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.     

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.     

Protein expression systems for structural genomics and proteomics

One of the key steps of structural genomics and proteomics is high-throughput expression of many target proteins. Gene cloning, especially by ligation-independent cloning techniques, and recombinant protein expression using microbial hosts such as Escherichia coli and the yeast Pichia pastoris are well optimized and further robotized. Cell-free protein synthesis systems have been developed for large-scale production of protein samples for NMR (stable-isotope labeling) and X-ray crystallography (selenomethionine substitution). Protein folding is still a major bottleneck in protein expression. Cell-based and cell-free methods for screening of suitable samples for structure determination have been developed for achieving a high success rate.     

生物高分辨电子显微学进展

生物高分辨电子显微学是近年来发展起来的一种可与X射线晶体学相媲美的测定生物大分子高分辨结构的方法．它克服了一些限制X射线晶体学应用的困难,可以直接对非晶体状态的生物大分子或仅能形成二维晶体的蛋白进行结构测定．这一技术主要包括高分辨电子显微象的获得与电子显微象解析．文章就这一技术应用中的一些问题：自然结构的保持、辐射损伤、低衬度、低信噪比等进行了讨论．     

Towards automated screening of two-dimensional crystals

Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible. We, and others, have developed working prototypes for several of these tools and tested and evaluated them in a simple screen of 24 crystallization conditions. While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach.     

Protein production in Escherichia coli for structural studies by X-ray crystallography

The arrival of genomic sequences to the database has provided a seemingly unlimited supply of targets for protein structure determination and the possibility of solving the structure of an entire proteome. Based on our experience with the proteomes of Pyrobaculum aerophilum and Mycobacterium tuberculosis, we have developed a simple strategy for the production of proteins for structural studies by X-ray crystallography. Our scheme demonstrates a strong protein target commitment and includes the expression of genes from these organisms in Escherichia coli. These proteins are expressed with affinity tags and purified for characterization and crystallization. We have identified protein solubility and crystallization as the two major bottlenecks in the process toward the determination of protein structures by X-ray diffraction. Strategies to overcome these bottlenecks are discussed.     

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.     

Microscopic assessment of membrane protein structure and function

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.