Optimal 3D angular sampling with applications to cryo-EM problems.

The goal of cryo-EM experiments in the biological sciences is to determine the atomic structure of a molecule and deduce insights into its functions and mechanisms. Despite improvements in instrumentation for data collection and new software algorithms, in most cases, individual atoms are not resolved. Model building of proteins, nucleic acids, or molecules in general, is feasible from the experimentally determined density maps at resolutions up to the range of 3-4 Angstroms. For lower-resolution maps or parts of maps, fitting smaller structures obtained by modelling or experimental techniques with higher resolution is a way to resolve the issue. In practice, we have an atomic structure, generate its density map at a given resolution, and translate/rotate the map within a region of interest in the experimental map, computing a measure-of-fit score with the corresponding areas of the experimental map. This procedure is computationally intensive since we work in 6D space. An optimal ordered list of rotations will reduce the angular error and help to find the best-fitting positions faster for a coarse global search or a local refinement. It can be used for adaptive approaches to stop fitting algorithms earlier once the desired accuracy has been achieved. We demonstrate how the performance of some fitting algorithms can be improved by grouping sets of rotations. We present an approach to generate more efficient 3D angular sampling, and provide the computer code to generate lists of optimal orientations for single and grouped rotations and the lists themselves.

Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory latent TGFß-binding site.

Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFß-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvß3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.

Elastic Fibre Proteins in Elastogenesis and Wound Healing.

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFß binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Theoretical and practical approaches to improve the performance of local correlation algorithms for volume data analysis and shape recognition.

In this paper, several approaches to be used to accelerate algorithms for fitting an atomic structure into a given 3D density map determined by cryo-EM are discussed. Rotation and translation of the atomic structure to find similarity scores are used and implemented with discrete Fourier transforms. Several rotations can be combined into groups to accelerate processing. The finite resolution of experimental and simulated maps allows a reduction in the number of rotations and translations needed in order to estimate similarity-score values.

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine against serogroup B meningococci have focused on the use of surface-exposed outer membrane proteins. Here we report the use of virus-like particles derived from the core protein of Hepatitis B Virus, HBc, to incorporate antigen domains derived from Factor H binding protein (FHbp) and the adhesin NadA. The extracellular domain of NadA was inserted into the major immunodominant region of HBc, and the C-terminal domain of FHbp at the C-terminus (CFHbp), creating a single polypeptide chain 3.7-fold larger than native HBc. Remarkably, cryoelectron microscopy revealed that the construct formed assemblies that were able to incorporate both antigens with minimal structural changes to native HBc. Electron density was weak for NadA and absent for CFHbp, partly attributable to domain flexibility. Following immunization of mice, three HBc fusions (CFHbp or NadA alone, NadA + CFHbp) were able to induce production of IgG1, IgG2a and IgG2b antibodies reactive against their respective antigens at dilutions in excess of 1:18,000. However, only HBc fusions containing NadA elicited the production of antibodies with serum bactericidal activity. It is hypothesized that this improved immune response is attributable to the adoption of a more native-like folding of crucial conformational epitopes of NadA within the chimeric VLP. This work demonstrates that HBc can incorporate insertions of large antigen domains but that maintenance of their three-dimensional structure is likely to be critical in obtaining a protective response.

The MUC5B mucin polymer is dominated by repeating structural motifs and its topology is regulated by calcium and pH.

The polymeric mucin MUC5B provides the structural and functional framework of respiratory mucus, conferring both viscoelastic and antimicrobial properties onto this vital protective barrier. Whilst it is established that MUC5B forms disulfide-linked linear polymers, how this relates to their packaging in secretory granules, and their molecular form in mucus remain to be fully elucidated. Moreover, the role of the central heavily O-glycosylated mucin domains in MUC5B conformation is incompletely described. Here we have completed a detailed structural analysis on native MUC5B polymers purified from saliva and subsequently investigated how MUC5B conformation is affected by changes in calcium concentration and pH, factors important for mucin intragranular packaging and post-secretory expansion. The results identify that MUC5B has a beaded structure repeating along the polymer axis and suggest that these repeating motifs arise from distinct glycosylation patterns. Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca2+, 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties.

The structural basis of translational control by eIF2 phosphorylation.

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.

Imaging Three-Dimensional Elemental Inhomogeneity in Pt-Ni Nanoparticles Using Spectroscopic Single Particle Reconstruction.

The properties of nanoparticles are known to critically depend on their local chemistry but characterizing three-dimensional (3D) elemental segregation at the nanometer scale is highly challenging. Scanning transmission electron microscope (STEM) tomographic imaging is one of the few techniques able to measure local chemistry for inorganic nanoparticles but conventional methodologies often fail due to the high electron dose imparted. Here, we demonstrate realization of a new spectroscopic single particle reconstruction approach built on a method developed by structural biologists. We apply this technique to the imaging of PtNi nanocatalysts and find new evidence of a complex inhomogeneous alloying with a Pt-rich core, a Ni-rich hollow octahedral intermediate shell and a Pt-rich rhombic dodecahedral skeleton framework with less Pt at ⟨100⟩ vertices. The ability to gain evidence of local surface enrichment that varies with the crystallographic orientation of facets and vertices is expected to provide significant insight toward the development of nanoparticles for sensing, medical imaging, and catalysis.

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils.

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. STATEMENT OF SIGNIFICANCE: Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.

Structural analysis of X-linked retinoschisis mutations reveals distinct classes which differentially effect retinoschisin function.

Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for the construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ∼4.2Šresolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.

Collaborative computational project for electron cryo-microscopy.

The Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) has recently been established. The aims of the project are threefold: to build a coherent cryoEM community which will provide support for individual scientists and will act as a focal point for liaising with other communities, to support practising scientists in their use of cryoEM software and finally to support software developers in producing and disseminating robust and user-friendly programs. The project is closely modelled on CCP4 for macromolecular crystallography, and areas of common interest such as model fitting, underlying software libraries and tools for building program packages are being exploited. Nevertheless, cryoEM includes a number of techniques covering a large range of resolutions and a distinct project is required. In this article, progress so far is reported and future plans are discussed.

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly.

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.

Collagen VI, conformation of A-domain arrays and microfibril architecture.

Collagen VI is a ubiquitous extracellular matrix protein that assembles into beaded microfibrils that form networks linking cells to the matrix. Collagen VI microfibrils are typically formed from a heterotrimer of the α1, α2, and α3 chains. The α3 chain is distinct as it contains an extended N terminus with up to 10 consecutive von Willebrand factor type A-domains (VWA). Here, we use solution small angle x-ray scattering (SAXS) and single particle analysis EM to determine the nanostructure of nine of these contiguous A-domains. Both techniques reveal a tight C-shape conformation for the A-domains. Furthermore, using biophysical approaches, we demonstrate that the N-terminal region undergoes a conformational change and a proportion forms dimers in the presence of Zn(2+). This is the first indication that divalent cations interact with collagen VI A-domains. A three-dimensional reconstruction of tissue-purified collagen VI microfibrils was generated using EM and single particle image analysis. The reconstruction showed the intricate architecture of the collagen VI globular regions, in particular the highly structurally conserved C-terminal region and variations in the appearance of the N-terminal region. The N-terminal domains project out from the globular beaded region like angled radial spokes. These could potentially provide interactive surfaces for other cell matrix molecules.