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91 Publications

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    Gonen Lab
    05/12/13 | Crystal structure of a nitrate/nitrite exchanger.
    Zheng H, Wisedchaisri G, Gonen T
    Nature. 2013 May 12;497(7451):647-51. doi: 10.1038/nature12139

    Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

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    Gonen Lab
    04/02/13 | Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.
    Nannenga BL, Iadanza MG, Vollmar BS, Gonen T
    Current Protocols in Protein Science . 2013 Apr 2;Chapter 17:Unit 17.15. doi: 10.1002/0471140864.ps1715s72

    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.

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    Gonen Lab
    02/01/13 | HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.
    Filbin ME, Vollmar BS, Shi D, Gonen T, Kieft JS
    Nature Structural & Molecular Biology. 2013 Feb;20(2):150-8. doi: 10.1038/nsmb.2465

    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’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’s structure and function can be altered by bound RNAs, including those derived from cellular invaders.

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    Gonen Lab
    01/01/13 | High throughput methods for electron crystallography.
    Stoke D, Ubarretxena-Belandia I, Gonen T, Engel A
    Methods in Molecular Biology. 2013;955:273-96. doi: 10.1007/978-1-62703-176-9_15

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

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    Gonen Lab
    01/01/13 | Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement.
    Wisedchaisri G, Gonen T
    Methods in Molecular Biology. 2013;955:243-72. doi: 10.1007/978-1-62703-176-9_14

    Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.

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    Gonen Lab
    01/01/13 | The collection of high-resolution electron diffraction data.
    Gonen T
    Methods in Molecular Biology. 2013;955:153-169. doi: 10.1007/978-1-62703-176-9_9

    A number of atomic-resolution structures of membrane proteins (better than 3Å resolution) have been determined recently by electron crystallography. While this technique was established more than 40 years ago, it is still in its infancy with regard to the two-dimensional (2D) crystallization, data collection, data analysis, and protein structure determination. In terms of data collection, electron crystallography encompasses both image acquisition and electron diffraction data collection. Other chapters in this volume outline protocols for image collection and analysis. This chapter, however, outlines detailed protocols for data collection by electron diffraction. These include microscope setup, electron diffraction data collection, and troubleshooting.

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    Gonen Lab
    08/20/12 | The Ndc80 kinetochore complex directly modulates microtubule dynamics.
    Umbreit NT, Gestaut DR, Tien JF, Vollmar BS, Gonen T, Asbury CL, Davis TN
    Proceedings of the National Academy of Sciences of the United States of America. 2012 Aug 20;109(40):16113-8. doi: 10.1073/pnas.1209615109

    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.

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    Gonen Lab
    08/12/12 | The structure of purified kinetochores reveals multiple microtubule-attachment sites.
    Gonen S, Akiyoshi B, Iadanza MG, Shi D, Duggan N, Biggins S, Gonen T
    Nature Structural & Molecular Biology. 2012 Aug 12;19(9):925-9. doi: 10.1038/nsmb.2358

    Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an  126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.

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    Gonen Lab
    08/01/12 | Recent progress in membrane protein structures and investigation methods.
    Gonen T, Waksman G
    Current Opinion in Structural Biology. 2012 Aug;22(4):467-8. doi: 10.1016/
    Gonen Lab
    06/01/12 | Computational design of self-assembling protein nanomaterials with atomic level accuracy.
    King NP, Sheffler W, Sawaya MR, Vollmar BS, Sumida JP, André I, Gonen T, Yeates TO, Baker D
    Science. 2012 Jun 1;336(6085):1171-4. doi: 10.1126/science.1219364

    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.

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