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.

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

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 TGFβ binding protein-4, and fibulin-4 and -5.

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 β2 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.

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 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.

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.

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.

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.

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

Unlabelled: 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.

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.

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).

Structure of hepatitis B surface antigen from subviral tubes determined by electron cryomicroscopy.

Hepatitis B virus consists of an icosahedral core containing the double-stranded DNA genome, enveloped by a membrane with embedded surface proteins. The crystal structure of the core protein has been solved but little information about the structure of the surface proteins has so far been available. There are three sizes of surface protein, small (S), medium (M) and large (L), which form disulfide-bonded homo- and heterodimers.

A structural model for maturation of the hepatitis B virus core.

Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions.

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM.

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice.

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface.

Automatic particle selection: results of a comparative study.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area.

Particle finding in electron micrographs using a fast local correlation algorithm.

A versatile tool for selecting particles from electron micrographs, intended for single particle analysis and three-dimensional reconstruction, is presented. It is based on a local real-space correlation method. Real-space correlations calculated over a local area are suitable for finding small objects or patterns in a larger field.