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.     

Putting the pieces together: integrative modeling platform software for structure determination of macromolecular assemblies

A set of software tools for building and distributing models of macromolecular assemblies uses an integrative structure modeling approach, which casts the building of models as a computational optimization problem where information is encoded into a scoring function used to evaluate candidate models.     

Visualization software for molecular assemblies

Software for viewing three-dimensional models and maps of viruses, ribosomes, filaments, and other molecular assemblies is advancing on many fronts. New developments include molecular representations that offer better control over level of detail, lighting that improves the perception of depth, and two-dimensional projections that simplify data interpretation. Programmable graphics processors offer quality, speed, and visual effects not previously possible, while 3D printers, haptic interaction devices, and auto-stereo displays show promise in more naturally engaging our senses. Visualization methods are developed by diverse groups of researchers with differing goals: experimental biologists, database developers, computer scientists, and package developers. We survey recent developments and problems faced by the developer community in bringing innovative visualization methods into widespread use.     

A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm

We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.     

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.     

Cryo-EM of macromolecular assemblies at near-atomic resolution

With single-particle electron cryomicroscopy (cryo-EM), it is possible to visualize large, macromolecular assemblies in near-native states. Although subnanometer resolutions have been routinely achieved for many specimens, state of the art cryo-EM has pushed to near-atomic (3.3-4.6 ?) resolutions. At these resolutions, it is now possible to construct reliable atomic models directly from the cryo-EM density map. In this study, we describe our recently developed protocols for performing the three-dimensional reconstruction and modeling of Mm-cpn, a group II chaperonin, determined to 4.3 ? resolution. This protocol, utilizing the software tools EMAN, Gorgon and Coot, can be adapted for use with nearly all specimens imaged with cryo-EM that target beyond 5 ? resolution. Additionally, the feature recognition and computational modeling tools can be applied to any near-atomic resolution density maps, including those from X-ray crystallography.     

CROW for large scale macromolecular simulations

CROW (Columns and Rows Of Workstations - http://www.sicmm.org/crow/) is a parallel computer cluster based on the Beowulf (http://www.beowulf.org/) idea, modified to support a larger number of processors. Its architecture is based on point-to-point network architecture, which does not require the use of any network switching equipment in the system. Thus, the cost is lower, and there is no degradation in network performance even for a larger number of processors.     

Alignment of 3D structures of macromolecular assemblies

MOTIVATION: A number of macromolecular assemblies are being reconstructed in 3D from electron micrographs. The analysis yields a 3D matrix representing the protein density map. In reconstruction processes and in comparing the results of different experiments, it is often necessary to obtain all models oriented the same way in three dimensions. The problem is not trivial since there exist no 3D counterpart of correlation analysis used for 2D images. It is usually solved by time consuming trial and error algorithms. RESULTS: 3D density distributions can be brought to a 'canonical' orientation. The tensor of inertia of the distribution is determined and its eigenvectors are oriented along the coordinate axes. The method is fast and essentially free of reference. It is suitable for structures whose inertial axes do not completely degenerate as they do in icosahedral viruses or if symmetry is cubic. Applications are presented for asymmetric objects and for molecules possessing symmetry axes higher than twofold. IMPLEMENTATION: The implementation simply requires the accumulation of the inertial tensor and its diagonalisation. Volume data rotation has been already illustrated in this journal by the authors.     

SVA: software for annotating and visualizing sequenced human genomes

SUMMARY: Here we present Sequence Variant Analyzer (SVA), a software tool that assigns a predicted biological function to variants identified in next-generation sequencing studies and provides a browser to visualize the variants in their genomic contexts. SVA also provides for flexible interaction with software implementing variant association tests allowing users to consider both the bioinformatic annotation of identified variants and the strength of their associations with studied traits. We illustrate the annotation features of SVA using two simple examples of sequenced genomes that harbor Mendelian mutations. Availability and implementation: Freely available on the web at http://www.svaproject.org.     

A novel mirror-symmetry analysis approach for the study of macromolecular assemblies imaged by electron microscopy

Multivariate statistical symmetry analysis is widely employed in single-particle electron-microscopy studies for the detection of symmetry components within a set of noisy two-dimensional images. So far, this technique has been used to retrieve information from the analysis of end-on view oriented particles only. Here, we propose a method to detect symmetry components from side- and tilted-view oriented particles. This method is validated using a number of in silico generated as well as real datasets, can be used to analyze stoichiometrically heterogeneous datasets, and is useful for separating particle datasets with respect to their symmetry components. Additionally, translational components in lock-washer ring configurations can be detected. Most relevantly, this method represents a powerful tool for the characterisation of distinct symmetry components within multi-layered protein assemblies, and any putative symmetry mismatch between layers. Such configurations have often been postulated, though rarely observed directly, and are thought to have a crucial role in conferring dynamicity to molecular machineries like nucleic acid packaging motors, ClpAP/ClpXP proteases, flagellar motors and the F1/F0 ATPase.     

The Phenix software for automated determination of macromolecular structures

X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.     

WebFEATURE: An interactive web tool for identifying and visualizing functional sites on macromolecular structures

WebFEATURE (http://feature.stanford.edu/webfeature/) is a web-accessible structural analysis tool that allows users to scan query structures for functional sites in both proteins and nucleic acids. WebFEATURE is the public interface to the scanning algorithm of the FEATURE package, a supervised learning algorithm for creating and identifying 3D, physicochemical motifs in molecular structures. Given an input structure or Protein Data Bank identifier (PDB ID), and a statistical model of a functional site, WebFEATURE will return rank-scored 'hits' in 3D space that identify regions in the structure where similar distributions of physicochemical properties occur relative to the site model. Users can visualize and interactively manipulate scored hits and the query structure in web browsers that support the Chime plug-in. Alternatively, results can be downloaded and visualized through other freely available molecular modeling tools, like RasMol, PyMOL and Chimera. A major application of WebFEATURE is in rapid annotation of function to structures in the context of structural genomics.     

Sun1 forms immobile macromolecular assemblies at the nuclear envelope

SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Sun1 proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun1 segments are essential for maintenance of Sun1 immobility at the NE. Our data in particular underline the self-association properties of the C-terminal coiled-coil Sun1 segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM. Our data support a model in which SUN-protein-containing multi-variate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments.     

Rigid-cluster models of conformational transitions in macromolecular machines and assemblies

We present a rigid-body-based technique (called rigid-cluster elastic network interpolation) to generate feasible transition pathways between two distinct conformations of a macromolecular assembly. Many biological molecules and assemblies consist of domains which act more or less as rigid bodies during large conformational changes. These collective motions are thought to be strongly related with the functions of a system. This fact encourages us to simply model a macromolecule or assembly as a set of rigid bodies which are interconnected with distance constraints. In previous articles, we developed coarse-grained elastic network interpolation (ENI) in which, for example, only Calpha atoms are selected as representatives in each residue of a protein. We interpolate distance differences of two conformations in ENI by using a simple quadratic cost function, and the feasible conformations are generated without steric conflicts. Rigid-cluster interpolation is an extension of the ENI method with rigid-clusters replacing point masses. Now the intermediate conformations in an anharmonic pathway can be determined by the translational and rotational displacements of large clusters in such a way that distance constraints are observed. We present the derivation of the rigid-cluster model and apply it to a variety of macromolecular assemblies. Rigid-cluster ENI is then modified for a hybrid model represented by a mixture of rigid clusters and point masses. Simulation results show that both rigid-cluster and hybrid ENI methods generate sterically feasible pathways of large systems in a very short time. For example, the HK97 virus capsid is an icosahedral symmetric assembly composed of 60 identical asymmetric units. Its original Hessian matrix size for a Calpha coarse-grained model is >(300,000)(2). However, it reduces to (84)(2) when we apply the rigid-cluster model with icosahedral symmetry constraints. The computational cost of the interpolation no longer scales heavily with the size of structures; instead, it depends strongly on the minimal number of rigid clusters into which the system can be decomposed.     

From short peptides to nanofibers to macromolecular assemblies in biomedicine

In the last few years, a variety of self-assembling short peptides that consist exclusively of simple amino acids have been designed and modified. These peptides exhibit self-assembling dynamic behaviors. At the molecular structural level, they form α-helical, β-sheet and β-hairpins structures in water. These structures further undergo spontaneous assembly to form nanofibers which aggregate into supramolecular scaffolds that entrap large volumes of water. Furthermore, nanostructures and supramolecular structures that self-organized from these short peptides also have a broad spectrum of biotechnological applications. They are useful as biological materials for 2D and 3D tissue cell cultures, regenerative and reparative medicine, tissue engineering as well as injectable drug delivery matrices that gel in situ. We have endeavored to do a comprehensive review of short peptides that form nanofibrous hydrogels. In particular, we have focused on recent advances in peptide assembly motifs and applications.     

Single particle analysis of filamentous and highly elongated macromolecular assemblies

The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of approximately 20 degrees was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of approximately 25 A. If no out-of-plane views are available, an alternative approach is to use a prior 3D model as a reference for the initial angle assignment. Tests with the thin filament model indicated that reasonably accurate angular assignment can be made using a reference containing actin, but lacking the regulatory proteins tropomyosin and troponin. We also found that an adaptation of the exact filtered back projection method is required to allow the correct weighting of projection images in which the particle has a very large axial ratio. This adaptation resulted in significant improvements in the reconstruction.