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Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

Author(s): Tobias Herrmann [sup.1] [sup.2] , Raúl Torres [sup.3] , Eric N. Salgado [sup.3] [sup.7] , Cristina Berciu [sup.4] [sup.8] , Daniel Stoddard [sup.4] [sup.5] , Daniela Nicastro [sup.4] [sup.5] [...], A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell.sup.1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4.sup.2,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses. Electron cryomicroscopy and cryotomography studies reveal that rotaviruses attach to a target cell through the outer-layer protein VP4, which--following cleavage--rearranges to enable perforation of the membrane and delivery of the viral genome into the host cell.

Author(s): David Tomasek [sup.1] [sup.2] , Shaun Rawson [sup.3] , James Lee [sup.1] [sup.2] [sup.5] , Joseph S. Wzorek [sup.2] [sup.6] , Stephen C. Harrison [sup.3] [sup.4] , Zongli Li [...], Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a [beta]-barrel structure.sup.1,2. [beta]-Barrels are sheets of [beta]-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane.sup.3-5. One subunit of the machines is itself a [beta]-barrel protein that has a central role in folding other [beta]-barrels. In Gram-negative bacteria, the [beta]-barrel assembly machine (BAM) consists of the [beta]-barrel protein BamA, and four lipoproteins.sup.5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP).sup.9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid [beta]-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies.sup.10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete. Cryo-electron microscopy structures of a folding intermediate on the BAM complex of Escherichia coli reveal how interactions between the BamA catalyst and substrate permit stable association during folding, followed by rapid turnover.

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry. Author summary: To infect a cell, non-enveloped viruses must have a mechanism to transfer their genomes across a membrane. The double-strand RNA (dsRNA) viruses deliver into the cytosol not just their genomes but an intact subviral particle (diameter ~70 nm). Rhesus rotavirus (RRV) particles attach through a spike-like outer-layer protein, VP4 (cleaved to VP5* and VP8*), then wrap themselves in membrane at the cell surface, producing a small, cytosolic vesicle surrounding each virion, from which the subviral particles escape directly. Loss of Ca2+ from the entering virion always precedes escape. In previous work we found that a conformational change in VP5*/VP8* projects the C-terminal "foot" domain of VP5* outward. We now show, by combining cryo-EM and single-particle optical microscopy of RRV particles interacting with liposomes, that the outward-projected foot penetrates the lipid bilayer, permeabilizing it to Ca2+. The new experiments thus recapitulate the initial step in particle escape and show that the conformational change must occur while the spike protein is membrane-attached, to couple the VP5* conformational change with membrane insertion. These findings bring us closer to a full molecular description how a large, subviral particle (about 70 nm diameter) can be delivered into a target host cell. [ABSTRACT FROM AUTHOR]