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.     

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。     

Overestimated accuracy of circular dichroism in determining protein secondary structure

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein’s secondary structure.     

Cover Image,Volume 88, Issue 9

There have been several studies suggesting that protein structures solved by NMR spectroscopy and X-ray crystallography show significant differences. To understand the origin of these differences, we assembled a database of high-quality protein structures solved by both methods. We also find significant differences between NMR and crystal structures—in the root-mean-square deviations of the C _α atomic positions, identities of core amino acids, backbone, and side-chain dihedral angles, and packing fraction of core residues. In contrast to prior studies, we identify the physical basis for these differences by modeling protein cores as jammed packings of amino acid-shaped particles. We find that we can tune the jammed packing fraction by varying the degree of thermalization used to generate the packings. For an athermal protocol, we find that the average jammed packing fraction is identical to that observed in the cores of protein structures solved by X-ray crystallography. In contrast, highly thermalized packing-generation protocols yield jammed packing fractions that are even higher than those observed in NMR structures. These results indicate that thermalized systems can pack more densely than athermal systems, which suggests a physical basis for the structural differences between protein structures solved by NMR and X-ray crystallography.     

结构生物学研究在中国

1970年代初期,中国科学工作者测定了亚洲地区第一个蛋白质晶体结构——猪胰岛素三方二锌晶体结构,成为中国结构生物学历史发展的起点.进入新世纪,该学科领域已进入国际前沿,展现出快速发展态势,正在迎来发展新时期.本篇评述包含"历史发展","现代化实验设施建设"和"深入生命世界,走进国际前沿——近年代表性研究成果集萃"三个主题节段,以较全视野反映结构生物学研究在中国的发展历程.     

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.     

Neutron Laue macromolecular crystallography

Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.     

Membrane transporter research in times of countless structures

Structural biology has advanced our understanding of membrane proteins like no other scientific discipline in the past two decades and the number of high resolution membrane transporter structures solved by X-ray crystallography has increased exponentially over this time period. Currently, single particle cryo-EM is in full swing due to a recent resolution revolution and permits for structural insights of proteins that were refractory to crystallization. It is foreseeable that multiple structures of many human transporters will be solved in the coming five years. Nevertheless, many scientifically important questions remain unanswered despite of available structures, as is illustrated in this article at the example of multidrug efflux pumps and ABC transporters. Structure-function studies likely continue to be a supporting pillar of membrane transporter research. However, there is a trend towards the “integrated structural biologist”, whose research focusses on a biological question and who closely collaborates with other research groups specialized in spectroscopy techniques or molecular dynamics simulation. Future membrane protein research requires joint efforts from specialists of various disciplines to finally work towards a molecular understanding of membrane transport in the context of the living cell. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

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.     

Relative stability of protein structures determined by X-ray crystallography or NMR spectroscopy: a molecular dynamics simulation study

The relative stability of protein structures determined by either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy has been investigated by using molecular dynamics simulation techniques. Published structures of 34 proteins containing between 50 and 100 residues have been evaluated. The proteins selected represent a mixture of secondary structure types including all alpha, all beta, and alpha/beta. The proteins selected do not contain cysteine-cysteine bridges. In addition, any crystallographic waters, metal ions, cofactors, or bound ligands were removed before the systems were simulated. The stability of the structures was evaluated by simulating, under identical conditions, each of the proteins for at least 5 ns in explicit solvent. It is found that not only do NMR-derived structures have, on average, higher internal strain than structures determined by X-ray crystallography but that a significant proportion of the structures are unstable and rapidly diverge in simulations.     

The structural basis of allosteric regulation in proteins

Allosteric regulation of protein function occurs when the regulatory trigger, such as the binding of a small-molecule effector or inhibitor, takes place some distance from the protein’s, or protein complex’s, active site. This distance can be a few Å, or tens of Å. Many proteins are regulated in this way and exhibit a variety of allosteric mechanisms. Here we review how analyses of experimentally determined models of protein 3D structures, using either X-ray crystallography or NMR spectroscopy, have revealed some of the mechanisms involved.     

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used "negative stains". Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low dose imaging techniques to avoiding radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM.     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.     

Recent developments in structural proteomics for protein structure determination

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.     

Conformational correlation and coupled motion between residue A21 and B25 side chain observed in crystal structures of insulin mutants at position A21

The C-terminal residue of the insulin A chain is invariant and kept as asparagine in all known insulin molecules from hagfish through birds to mammals. To get information on the role of this conserved residue, which is still unclear, the three-dimensional structures of four human insulin mutants, A21 Asn-->Gly, A21 Asn-->Asp, A21 Asn-->Ala, and A21 Asn-->Gln DesB30, were determined by X-ray crystallography. The four mutants crystallize separately into two kinds (rhombohedral and cubic) of crystals. In the refined structures, conformational correlation and coupled motion between the A chain C-terminal residue A21 and the B25 side chain was observed, in contrast to the nearly unchanged general structures as compared with the native insulin structures in their respective crystals. A detailed analysis suggests that residue A21 can affect insulin receptor binding by interaction with the B25 side chain and the B chain C-terminal segment to assist the B25 side chain rearranging into the 'active' conformation.     

Comparison of Composer and ORCHESTRAR

Although the number of known protein structures is increasing, the number of protein sequences without determined structures is still much larger. Three-dimensional (3D) protein structure information helps in the understanding of functional mechanisms, but solving structures by X-ray crystallography or NMR is often a lengthy and difficult process. A relatively fast way of determining a protein's 3D structure is to construct a computer model using homologous sequence and structure information. Much work has gone into algorithms that comprise the ORCHESTRAR homology modeling program in the SYBYL software package. This novel homology modeling tool combines algorithms for modeling conserved cores, variable regions, and side chains. The paradigm of using existing knowledge from multiple templates and the underlying protein environment knowledgebase is used in all of these algorithms, and will become even more powerful as the number of experimentally derived protein structures increases. To determine how ORCHESTRAR compares to Composer (a broadly used, but an older tool), homology models of 18 proteins were constructed using each program so that a detailed comparison of each step in the modeling process could be carried out. Proteins modeled include kinases, dihydrofolate reductase, HIV protease, and factor Xa. In almost all cases ORCHESTRAR produces models with lower root-mean-squared deviation (RMSD) values when compared with structures determined by X-ray crystallography or NMR. Moreover, ORCHESTRAR produced a homology model for three target sequences where Composer failed to produce any. Data for RMSD comparisons between structurally conserved cores, structurally variable regions, side-chain conformations are presented, as well as analyses of active site and protein-protein interface configurations.     

Complementary approaches to structure determination of icosahedral viruses

Few biological macromolecular complexes exhibit the combination of massive size and hierarchical, symmetrical architecture embodied in icosahedral viruses. X-ray crystallography, electron cryomicroscopy and small-angle X-ray scattering provide complementary approaches to studying these remarkable structures. Through a combined approach, progress has been made towards providing detailed structures of highly complex and very large viruses, and towards imaging the dynamic structural changes performed by viruses at key stages in their life cycles.     

<Emphasis Type="Italic">Rapper</Emphasis><Emphasis Type="Bold">tk</Emphasis>: a versatile engine for discrete restraint-based conformational sampling of macromolecules

Background  

Macromolecular structures are modeled by conformational optimization within experimental and knowledge-based restraints. Discrete restraint-based sampling generates high-quality structures within these restraints and facilitates further refinement in a continuous all-atom energy landscape. This approach has been used successfully for protein loop modeling, comparative modeling and electron density fitting in X-ray crystallography.