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1.  Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores 
Cell  2016;167(4):1028-1040.e15.
Summary
Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.
Graphical Abstract
Highlights
•We report a crystal structure of human MIS12 complex, a crucial kinetochore component•The structure reveals how the MIS12 complex binds its kinetochore receptor CENP-C•We dissect how Aurora B kinase promotes the MIS12:CENP-C interaction•A combination of diverse structural methods reveals outer kinetochore organization
Structural analyses show how a human kinetochore subcomplex serves as an adaptor between centromeric nucleosomes and outer kinetochore components.
doi:10.1016/j.cell.2016.10.005
PMCID: PMC5101189  PMID: 27881301
CENP-C; KMN network; Mis12; PMF1; DSN1; NSL1; MIND; kinetochore; centromere; CCAN
2.  Structure of the L-protein of vesicular stomatitis virus from electron cryomicroscopy 
Cell  2015;162(2):314-327.
The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a noncanonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains [RNA-dependent RNA polymerase (RdRp), polyribonucleotidyl transferase (PRNTase), and methyl transferase] and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.
doi:10.1016/j.cell.2015.06.018
PMCID: PMC4557768  PMID: 26144317
RNA-dependent RNA polymerase; RNA capping; cryoEM single-particle analysis
3.  The crystal structure of dynamin 
Nature  2011;477(7366):561-566.
Dynamin-related proteins (DRPs) are multi-domain GTPases that function via oligomerization and GTP-dependent conformational changes to play central roles in regulating membrane structure across phylogenetic kingdoms. How DRPs harness self-assembly and GTP-dependent conformational changes to remodel membranes is not understood. Here we present the crystal structure of an assembly-deficient mammalian endocytic DRP, dynamin 1, lacking the proline-rich domain, in its nucleotide-free state. The dynamin 1 monomer is an extended structure with the GTPase domain and bundle signalling element positioned on top of a long helical stalk with the pleckstrin homology domain flexibly attached on its opposing end. Dynamin 1 dimer and higher order dimer multimers form via interfaces located in the stalk. Analysis of these interfaces provides insight into DRP family member specificity and regulation and provides a framework for understanding the biogenesis of higher order DRP structures and the mechanism of DRP-mediated membrane scission events.
doi:10.1038/nature10441
PMCID: PMC4075756  PMID: 21927001
4.  Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthase 
A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns.
The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model.
doi:10.1107/S0907444909000778
PMCID: PMC2631638  PMID: 19171964
imperfect pseudo-merohedral twinning; fungal fatty acid synthase
5.  Structure of the E. coli protein-conducting channel bound to a translating ribosome 
Nature  2005;438(7066):318-324.
Secreted and membrane proteins are translocated across/into cell membranes via a protein-conducting channel (PCC). We present a cryo-EM reconstruction of the E. coli PCC, SecYEG, complexed with the ribosome and a signal anchor containing nascent chain, showing mRNA, three tRNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEβ structure into the PCC EM densities favors a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. Based on our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.
doi:10.1038/nature04133
PMCID: PMC1351281  PMID: 16292303

Results 1-5 (5)