Cryo-EM structure in situ reveals a molecular switch that safeguards virus against genome loss

The portal protein is a key component of many double-stranded DNA viruses, governing capsid assembly and genome packaging. Twelve subunits of the portal protein define a tunnel, through which DNA is translocated into the capsid. It is unknown how the portal protein functions as a gatekeeper, preventing DNA slippage, whilst allowing its passage into the capsid, and how these processes are controlled. A cryo-EM structure of the portal protein of thermostable virus P23-45, determined in situ in its procapsid-bound state, indicates a mechanism that naturally safeguards the virus against genome loss. This occurs via an inversion of the conformation of the loops that define the constriction in the central tunnel, accompanied by a hydrophilic-hydrophobic switch. The structure also shows how translocation of DNA into the capsid could be modulated by a changing mode of protein-protein interactions between portal and capsid, across a symmetry-mismatched interface

Digital Image Processing of Electron Micrographs: The PIC System II

The PIC system, an integrated package of Fortran programs and subroutines designed to run on the Digital Equipment Corporation VAX family of computers, has been developed for analysis of electron micrographs with emphasis on the particular requirements for structural analysis of biological macromolecules. The substantially improved VAX version of PIC reported here has been developed from an earlier PDP-11 version which was, in turn, developed from a set of IBM 370 programs called MDPP. PIC now encompasses over 150 commands or processing operations that afford a comprehensive range of image processing operations including image restoration, enhancement, Fourier analysis, correlation averaging, and multivariate statistical analysis including clustering and classification. In particular, we describe our software for correction of imperfect lattices, as well as programs for correlation alignment and averaging of single particle images

Активизация инвестиционной деятельности коммерческими банками

В статье рассмотрены основные вопросы активизации инвестиционной деятельности коммерческими банками Украины.У статті розглядаються основні питання активізації інвестиційної діяльності комерційними банками України.The main questions of the investment activity by the commercial banks of Ukraine were descried in the article

Structure of the Herpes Simplex Virus Capsid: Peptide A862-H880 of the Major Capsid Protein Is Displayed on the Rim of the Capsomer Protrusions

AbstractThe herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on aT= 16 icosahedral lattice. The major capsid protein, VP5 (MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid protomers, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862–880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of thetrans-capsomeric channels. We conclude that these sites are occupied by peptide 862–880 in the mature HSV-1 capsid

Quasi-Normal Cornified Cell Envelopes in Loricrin Knockout Mice Imply the Existence of a Loricrin Backup System

The cornified cell envelope, a lipoprotein layer that assembles at the surface of terminally differentiated keratinocytes, is a resilient structure on account of covalent crosslinking of its constituent proteins, principally loricrin, which accounts for up to 60%-80% of total protein. Despite the importance of the cell envelope as a protective barrier, knocking out the loricrin gene in mice results in only mild syndromes. We have investigated the epidermis and forestomach epithelium of these mice by electron microscopy. In both tissues, corneocytes have normal-looking cell envelopes, despite the absence of loricrin, which was confirmed by immunolabeling, and the absence of the distinctive loricrin-containing keratohyalin granules (L-granules). Isolated cell envelopes were normal in thickness (≈15 nm) and mass per unit area (≈7.3 kDa per nm2); however, metal shadowing revealed an altered substructure on their cytoplasmic surface. Their amino acid compositions indicate altered protein compositions. Analysis of these data implies that the epidermal cell envelopes have elevated levels of the small proline-rich proteins, and cell envelopes of both kinds contain other protein(s) that, like loricrin, are rich in glycine and serine. These observations imply that, in the absence of loricrin, the mechanisms that govern cell envelope assembly function normally but employ different building-blocks

Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside