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56 Janelia Publications

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    Gonen Lab
    06/01/18 | Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state.
    Lei H, Ma J, Sanchez Martinez S, Gonen T
    Nature Structural & Molecular Biology. 2018 Jun;25(6):522-527. doi: 10.1038/s41594-018-0072-2

    Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.

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    Gonen Lab
    05/03/18 | MicroED structure of the NaK ion channel reveals a Na partition process into the selectivity filter.
    Liu S, Gonen T
    Communications Biology. 2018;1:38. doi: 10.1038/s42003-018-0040-8

    Sodium (Na) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na ion at the entrance of the channel pore. A process of dilation coupled with Na movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.

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    Gonen Lab
    04/18/18 | Analysis of global and site-specific radiation damage in cryo-EM.
    Hattne J, Shi D, Glynn C, Zee C, Gallagher-Jones M, Martynowycz MW, Rodriguez JA, Gonen T
    Structure (London, England : 1993). 2018 Apr 18;26(5):759-66. doi: 10.1016/j.str.2018.03.021

    Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

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    Gonen Lab
    03/14/18 | Integrative structure and functional anatomy of a nuclear pore complex.
    Kim SJ, Fernandez-Martinez J, Nudelman I, Shi Y, Zhang W, Raveh B, Herricks T, Slaughter BD, Hogan JA, Upla P, Chemmama IE, Pellarin R, Echeverria I, Shivaraju M, Chaudhury AS, Wang J, Williams R, Unruh JR, Greenberg CH, Jacobs EY, Yu Z, de la Cruz MJ, Mironska R, Stokes DL, Aitchison JD, Jarrold MF, Gerton JL, Ludtke SJ, Akey CW, Chait BT, Sali A, Rout MP
    Nature. 2018 Mar 14;555(7697):475-82. doi: 10.1038/nature26003

    Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

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    Gonen Lab
    03/12/18 | Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2.
    Guenther EL, Ge P, Trinh H, Sawaya MR, Cascio D, Boyer DR, Gonen T, Zhou ZH, Eisenberg DS
    Nature Structural & Molecular Biology. 2018 Mar 12:. doi: 10.1038/s41594-018-0045-5

    Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that theDLIIKGISVHIsegment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.

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    Gonen Lab
    03/01/18 | From electron crystallography of 2D crystals to MicroED of 3D crystals.
    Martynowycz MW, Gonen T
    Current Opinion in Colloid & Interface Science . 2018 Mar;34:9-16. doi: 10.1016/j.cocis.2018.01.010

    Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

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    Gonen Lab
    02/01/18 | Structure-based inhibitors of tau aggregation.
    Seidler PM, Boyer DR, Rodriguez JA, Sawaya MR, Cascio D, Murray K, Gonen T, Eisenberg DS
    Nature Chemistry. 2018 Feb;10(2):170-176. doi: 10.1038/nchem.2889

    Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

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    Gonen Lab
    01/15/18 | Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp.
    Gallagher-Jones M, Glynn C, Boyer DR, Martynowycz MW, Hernandez E, Miao J, Zee C, Novikova IV, Goldschmidt L, McFarlane HT, Helguera GF, Evans JE, Sawaya MR, Cascio D, Eisenberg DS, Gonen T, Rodriguez JA
    Nature Structural & Molecular Biology. 2018 Jan 15:. doi: 10.1038/s41594-017-0018-0

    The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.

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    Gonen Lab
    12/27/17 | Common fibrillar spines of amyloid-β and human Islet Amyloid Polypeptide revealed by Micro Electron Diffraction and inhibitors developed using structure-based design.
    Krotee P, Griner SL, Sawaya MR, Cascio D, Rodriguez JA, Shi D, Philipp S, Murray K, Saelices L, Lee J, Seidler P, Glabe CG, Jiang L, Gonen T, Eisenberg DS
    The Journal of Biological Chemistry. 2017 Dec 27:. doi: 10.1074/jbc.M117.806109

    Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues, and are associated with Alzheimer's disease (AD) and Type-II Diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method Micro-Electron Diffraction (MicroED) we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ 24-34 WT and hIAPP 19-29 S20G, with 64% sequence similarity. We observe a high degree of structural similarity between their backbone atoms (0.96 Å RMSD). Moreover, fibrils of these segments induce amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment show cross-efficacy for full-length Aβ and hIAPP and reduce cytotoxicity of both proteins, though by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ 24-34 WT and hIAPP 19-29 S20G offers a molecular model for cross-seeding between Aβ and hIAPP.

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    Gonen Lab
    11/20/17 | Structure-based inhibitors of tau aggregation.
    Seidler PM, Boyer DR, Rodriguez JA, Sawaya MR, Cascio D, Murray K, Gonen T, Eisenberg DS
    Nature Chemistry. 2017 Nov 20:. doi: 10.1038/nchem.2889

    Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

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