@article {5803, title = {HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.}, journal = {Nature Structural \& Molecular Biology}, volume = {20}, year = {2013}, month = {2013 Feb}, pages = {150-8}, abstract = {

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit{\textquoteright}s decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome{\textquoteright}s structure and function can be altered by bound RNAs, including those derived from cellular invaders.

}, issn = {1545-9985}, doi = {10.1038/nsmb.2465}, author = {Filbin, Megan E and Vollmar, Breanna S and Shi, Dan and Gonen, Tamir and Kieft, Jeffrey S} } @article {5959, title = {Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.}, journal = {Current Protocols in Protein Science }, volume = {Chapter 17}, year = {2013}, month = {2013 Apr 2}, pages = {Unit 17.15}, abstract = {

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

}, issn = {1934-3663}, doi = {10.1002/0471140864.ps1715s72}, author = {Nannenga, Brent L and Iadanza, Matthew G and Vollmar, Breanna S and Gonen, Tamir} } @article {4920, title = {Computational design of self-assembling protein nanomaterials with atomic level accuracy.}, journal = {Science}, volume = {336}, year = {2012}, month = {2012 Jun 1}, pages = {1171-4}, abstract = {

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

}, issn = {1095-9203}, doi = {10.1126/science.1219364}, author = {King, Neil P and Sheffler, William and Sawaya, Michael R and Vollmar, Breanna S and Sumida, John P and Andr{\'e}, Ingemar and Gonen, Tamir and Yeates, Todd O and Baker, David} } @article {5153, title = {The Ndc80 kinetochore complex directly modulates microtubule dynamics.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Aug 20}, pages = {16113-8}, abstract = {

The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.

}, issn = {1091-6490}, doi = {10.1073/pnas.1209615109}, author = {Umbreit, Neil T and Gestaut, Daniel R and Tien, Jerry F and Vollmar, Breanna S and Gonen, Tamir and Asbury, Charles L and Davis, Trisha N} } @article {3629, title = {N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Apr 19}, pages = {6409-14}, abstract = {

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

}, keywords = {alpha-Crystallin B Chain, Animals, Biomolecular, Humans, Missense, Models, Molecular, Mutation, Nuclear Magnetic Resonance, Protein Multimerization, Protein Structure, Quaternary, Tertiary}, issn = {1091-6490}, doi = {10.1073/pnas.1014656108}, author = {Jehle, Stefan and Vollmar, Breanna S and Bardiaux, Benjamin and Dove, Katja K and Rajagopal, Ponni and Gonen, Tamir and Oschkinat, Hartmut and Klevit, Rachel E} }