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10 Publications

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    Gonen Lab
    10/24/14 | High thermodynamic stability of parametrically designed helical bundles.
    Huang P, Oberdorfer G, Xu C, Pei XY, Nannenga BL, Rogers JM, DiMaio F, Gonen T, Luisi B, Baker D
    Science. 2014 Oct 24;346(6208):481-5. doi: 10.1126/science.1257481

    We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.

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    Gonen Lab
    10/17/14 | Structure of catalase determined by MicroED.
    Nannenga BL, Shi D, Hattne J, Reyes FE, Gonen T
    eLife. 2014 Oct 17;3:e03600. doi: 10.7554/eLife.03600

    MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

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    Gonen Lab
    09/01/14 | High-resolution structure determination by continuous-rotation data collection in MicroED.
    Nannenga BL, Shi D, Leslie AG, Gonen T
    Nature Methods. 2014 Sep;11(9):927-30. doi: 10.1038/nmeth.3043

    MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

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    Gonen Lab
    08/13/14 | A type VI secretion-related pathway in bacteroidetes mediates interbacterial antagonism.
    Russell AB, Wexler AG, Harding BN, Whitney JC, Bohn AJ, Goo YA, Tran BQ, Barry NA, Zheng H, Peterson SB, Chou S, Gonen T, Goodlett DR, Goodman AL, Mougous JD
    Cell Host Microbe. 2014 Aug 13;16(2):227-36. doi: 10.1016/j.chom.2014.07.007

    Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

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    Gonen Lab
    08/04/14 | Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.
    Wisedchaisri G, Park M, Iadanza MG, Zheng H, Gonen T
    Nature Communication. 2014 - Aug;5:4521. doi: 10.1038/ncomms5521

    The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

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    Gonen Lab
    08/01/14 | Editorial overview: Membranes: recent methods in the study of membrane protein structure.
    Gonen T, Waksman G
    Current Opinion in Structural Biology. 2014 Aug;27:iv-v. doi: 10.1016/j.sbi.2014.09.002
    Gonen Lab
    06/05/14 | Accurate design of co-assembling multi-component protein nanomaterials.
    King NP, Bale JB, Sheffler W, McNamara DE, Gonen S, Gonen T, Yeates TO, Baker D
    Nature. 2014 Jun 5;510(7503):103-8. doi: 10.1038/nature13404

    The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.

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    Gonen Lab
    06/01/14 | A suite of software for processing MicroED data of extremely small protein crystals.
    Iadanza MG, Gonen T
    Journal of Applied Crystallography. 2014 Jun 1;47(Pt 3):1140-45. doi: 10.1107/S1600576714008073

    Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.

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    Gonen Lab
    04/04/14 | Protein structure determination by MicroED.
    Nannenga BL, Gonen T
    Current Opinion in Structural Biology. 2014 Apr 4;27C:24-31. doi: 10.1016/j.sbi.2014.03.004

    In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9A resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.

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    Gonen Lab
    03/30/14 | Amphotericin forms an extramembranous and fungicidal sterol sponge.
    Anderson TM, Clay MC, Cioffi AG, Diaz KA, Hisao GS, Tuttle MD, Nieuwkoop AJ, Comellas G, Maryum N, Wang S, Uno BE, Wildeman EL, Gonen T, Rienstra CM, Burke MD
    Nature Chemical Biology. 2014 Mar 30;10(5):400-6. doi: 10.1038/nchembio.1496

    For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells.

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