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.     

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.     

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.     

Biomolecular analysis of elastic fibre molecules

Elastic fibres are macromolecular extracellular matrix assemblies that endow dynamic connective tissues such as arteries, lungs and skin with the property of elastic recoil. Here, we describe how we have purified elastic fibre molecules and then analysed them using a range of biochemical and biomolecular approaches. Such approaches have provided powerful insights into the complex hierarchical processes of extracellular matrix assembly. We outline molecular interaction and kinetics assays using Biacore, biophysical approaches such as multi-angle laser light scattering and analytical ultracentrifugation which provide information on molecular and macromolecular shape and mass in solution, the visualisation of molecules and assemblies using microscopy approaches such as atomic force microscopy and environmental scanning electron microscopy, and compositional analysis of macromolecular complexes using mass spectrometry. Data from these in vitro analytical approaches can be combined to develop powerful new models of elastic fibre assembly.     

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.     

The neurobiologist's guide to structural biology: a primer on why macromolecular structure matters and how to evaluate structural data

Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD).     

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.     

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.     

The catalytic core of an archaeal 2-oxoacid dehydrogenase multienzyme complex is a 42-mer protein assembly

The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 ?. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.     

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.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

Emerging applications of small angle solution scattering in structural biology

Small angle solution X‐ray and neutron scattering recently resurfaced as powerful tools to address an array of biological problems including folding, intrinsic disorder, conformational transitions, macromolecular crowding, and self or hetero‐assembling of biomacromolecules. In addition, small angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy, and other structural methods to aid in the structure determinations of multidomain or multicomponent proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation, or X‐ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for characterizing the organizations of two‐component systems such as a nucleoprotein or a lipid‐protein assembly. Time‐resolved small and wide‐angle solution scattering to study biological processes in real time, and the use of localized heavy‐atom labeling and anomalous solution scattering for applications as FRET‐like molecular rulers, are amongst promising newer developments. Despite the challenges in data analysis and interpretation, these X‐ray/neutron solution scattering based approaches hold great promise for understanding a wide variety of complex processes prevalent in the biological milieu.     

Gene expression within a dynamic nuclear landscape

Molecular imaging in living cells or organisms now allows us to observe macromolecular assemblies with a time resolution sufficient to address cause-and-effect relationships on specific molecules. These emerging technologies have gained much interest from the scientific community since they have been able to reveal novel concepts in cell biology, thereby changing our vision of the cell. One main paradigm is that cells stochastically vary, thus implying that population analysis may be misleading. In fact, cells should be analyzed within time-resolved single-cell experiments rather than being compared to other cells within a population. Technological imaging developments as well as the stochastic events present in gene expression have been reviewed. Here, we discuss how the structural organization of the nucleus is revealed using noninvasive single-cell approaches, which ultimately lead to the resolution required for the analysis of highly controlled molecular processes taking place within live cells. We also describe the efforts being made towards physiological approaches within the context of living organisms.     

X-ray scattering combined with coordinate-based analyses for applications in natural and artificial photosynthesis

Advances in X-ray light sources and detectors have created opportunities for advancing our understanding of structure and structural dynamics for supramolecular assemblies in solution by combining X-ray scattering measurement with coordinate-based modeling methods. In this review the foundations for X-ray scattering are discussed and illustrated with selected examples demonstrating the ability to correlate solution X-ray scattering measurements to molecular structure, conformation, and dynamics. These approaches are anticipated to have a broad range of applications in natural and artificial photosynthesis by offering possibilities for structure resolution for dynamic supramolecular assemblies in solution that can not be fully addressed with crystallographic techniques, and for resolving fundamental mechanisms for solar energy conversion by mapping out structure in light-excited reaction states.     

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.     

Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps

The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10 ? resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions.     

How to avoid premature decay of your macromolecular crystal: a quick soak for long life

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.     

A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.     

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.     

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.