Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.     

Cryo-electron tomography: gaining insight into cellular processes by structural approaches

Visualization of cellular processes at a resolution of the individual protein should involve integrative and complementary approaches that can eventually draw realistic functional and cellular landscapes. Electron tomography of vitrified but otherwise unaltered cells emerges as a central method for three-dimensional reconstruction of cellular architecture at a resolution of 2-6 nm. While a combination of correlative light-based microscopy with cryo-electron tomography (cryo-ET) provides medium-resolution insight into pivotal cellular processes, fitting high-resolution structural approaches, for example, X-ray crystallography, into reconstructed macromolecular assemblies provides unprecedented information on native protein assemblies. Thus, cryo-ET bridges the resolution gap between cellular and structural biology. In this article, we focus on the study of eukaryotic cells and macromolecular complexes in a close-to-life-state. We discuss recent developments and structural findings enabling major strides to be made in understanding complex physiological functions.     

GPU-powered tools boost molecular visualization

Recent advances in experimental structure determination provide a wealth of structural data on huge macromolecular assemblies such as the ribosome or viral capsids, available in public databases. Further structural models arise from reconstructions using symmetry orders or fitting crystal structures into low-resolution maps obtained by electron-microscopy or small angle X-ray scattering experiments. Visual inspection of these huge structures remains an important way of unravelling some of their secrets. However, such visualization cannot conveniently be carried out using conventional rendering approaches, either due to performance limitations or due to lack of realism. Recent developments, in particular drawing benefit from the capabilities of Graphics Processing Units (GPUs), herald the next generation of molecular visualization solutions addressing these issues. In this article, we present advances in computer science and visualization that help biologists visualize, understand and manipulate large and complex molecular systems, introducing concepts that remain little-known in the bioinformatics field. Furthermore, we compile currently available software and methods enhancing the shape perception of such macromolecular assemblies, for example based on surface simplification or lighting ameliorations.     

Towards atomic resolution structural determination by single-particle cryo-electron microscopy

Recent advances in cryo-electron microscopy and single-particle reconstruction (collectively referred to as 'cryoEM') have made it possible to determine the three-dimensional (3D) structures of several macromolecular complexes at near-atomic resolution ( approximately 3.8-4.5A). These achievements were accomplished by overcoming the challenges in sample handling, instrumentation, image processing, and model building. At near-atomic resolution, many detailed structural features can be resolved, such as the turns and deep grooves of helices, strand separation in beta sheets, and densities for loops and bulky amino acid side chains. Such structural data of the cytoplasmic polyhedrosis virus (CPV), the Epsilon 15 bacteriophage and the GroEL complex have provided valuable constraints for atomic model building using integrative tools, thus significantly enhancing the value of the cryoEM structures. The CPV structure revealed a drastic conformational change from a helix to a beta hairpin associated with RNA packaging and replication, coupling of RNA processing and release, and the long sought-after polyhedrin-binding domain. These latest advances in single-particle cryoEM provide exciting opportunities for the 3D structural determination of viruses and macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR) methods.     

Strategies for the covalent conjugation of a bifunctional chelating agent to albumin: synthesis and characterization of potential MRI contrast agents

With the purpose to develop macromolecular magnetic resonance imaging contrast agents, we herein report three different synthetic approaches to the covalent attachment of bifunctional chelating agents to human serum albumin followed by coordination to contrast enhancing gadolinium(III). Applied methods cover active ester-mediated conjugation, linkage through glutaryl spacer, as well as the connection by the employment of glutaraldehyde. The content of gadolinium(III) was evaluated by inductively-coupled-plasma mass-spectrometry (ICP-MS) measurements and indicated reproducible amounts of conjugated contrast enhancing material. Small angle X-ray scattering (SAXS) experiments provided the size and altered shape of the gadolinium loaded proteins in comparison to unmodified albumin. Finally, the magnetic resonance properties of the protein conjugates were evaluated. The results indicated suitability of the gadolinium(III) loaded protein conjugates for use as macromolecular contrast agents in magnetic resonance imaging (MRI).     

Defining and solving the essential protein-protein interactions in HIV infection

The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV - host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV.     

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.     

The structural basis for the regulation of tissue transglutaminase by calcium ions.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.     

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.     

Inference of macromolecular assemblies from crystalline state

We discuss basic physical-chemical principles underlying the formation of stable macromolecular complexes, which in many cases are likely to be the biological units performing a certain physiological function. We also consider available theoretical approaches to the calculation of macromolecular affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. We develop a new method, based on chemical thermodynamics, for automatic detection of macromolecular assemblies in the Protein Data Bank (PDB) entries that are the results of X-ray diffraction experiments. As found, biological units may be recovered at 80-90% success rate, which makes X-ray crystallography an important source of experimental data on macromolecular complexes and protein-protein interactions. The method is implemented as a public WWW service.     

Experimental approaches for solution X-ray scattering and fiber diffraction

X-ray scattering and diffraction from non-crystalline systems have gained renewed interest in recent years, as focus shifts from the structural chemistry information gained by high-resolution studies to the context of structural physiology at larger length scales. Such techniques permit the study of isolated macromolecules as well as highly organized macromolecular assemblies as a whole under near-physiological conditions. Time-resolved approaches, made possible by advanced synchrotron instrumentation, add a crucial dimension to many of these investigations. This article reviews experimental approaches in non-crystalline X-ray scattering and diffraction that may be used to illuminate important scientific questions such as protein/nucleic acid folding and structure-function relationships in large macromolecular assemblies.     

Trapping reaction intermediates in macromolecular crystals for structural analyses

The development of "time-resolved" crystallographic methods, including trapping of reaction intermediates and rapid data collection, allows the comparative study of discrete structural species formed during a macromolecular reaction, such as enzymatic catalysis, ribozyme cleavage, or a protein photocycle. The primary technical details that must be addressed in such studies are the reaction initiation, the accumulation of a specific reaction species throughout the crystal, the lifetime of that species and of the crystal under the experimental conditions, and the method used to collect X-ray data. Methods of reaction initiation range from substrate diffusion, which is appropriate for the visualization of very long-lived intermediates, to photolysis, which is appropriate for the accumulation of rate-limited species with half-lives ranging from milliseconds to nanoseconds. This review discusses various methods for initiating turnover in crystals and trapping rate-limiting species for structural studies.     

Bridging structural biology and genomics: assessing protein interaction data with known complexes

Currently, there is a major effort to map protein-protein interactions on a genome-wide scale. The utility of the resulting interaction networks will depend on the reliability of the experimental methods and the coverage of the approaches. Known macromolecular complexes provide a defined and objective set of protein interactions with which to compare biochemical and genetic data for validation. Here, we show that a significant fraction of the protein-protein interactions in genome-wide datasets, as well as many of the individual interactions reported in the literature, are inconsistent with the known 3D structures of three recent complexes (RNA polymerase II, Arp2/3 and the proteasome). Furthermore, comparison among genome-wide datasets, and between them and a larger (but less well resolved) group of 174 complexes, also shows marked inconsistencies. Finally, individual interaction datasets, being inherently noisy, are best used when integrated together, and we show how simple Bayesian approaches can combine them, significantly decreasing error rate.     

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.     

Annealing of normal and mutant wheat starches. LM, SEM, DSC, and SAXS studies

Structure and thermodynamic properties of native and annealed wheat starches with different amylose content (from 1.5% to 39.5%) have been studied by high-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray diffraction (SAXS), light (LM), and scanning electron microscopy (SEM). Starch morphology, the values of the melting cooperative unit, the thickness of crystalline lamellae and the size of amylopectin clusters as well as thermodynamic parameters characterizing surface of the face side in starch crystals were determined. Some suppositions based on different physical approaches are used for a discussion of the results concerning structural reorganization of starches on different levels of macromolecular organization.     

The structural basis of XRCC1-mediated DNA repair

Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined.