Main Menu (Mobile)- Block

Main Menu - Block

janelia7_blocks-janelia7_fake_breadcrumb | block
Koyama Lab / Publications
custom | custom

Filter

facetapi-Q2b17qCsTdECvJIqZJgYMaGsr8vANl1n | block

Associated Lab

facetapi-PV5lg7xuz68EAY8eakJzrcmwtdGEnxR0 | block
facetapi-021SKYQnqXW6ODq5W5dPAFEDBaEJubhN | block
general_search_page-panel_pane_1 | views_panes

6 Publications

Showing 1-6 of 6 results
Your Criteria:
    Grigorieff Lab
    11/29/18 | Analysis of discrete local variability and structural covariance in macromolecular assemblies using Cryo-EM and focused classification.
    Zhang C, Cantara W, Jeon Y, Musier-Forsyth K, Grigorieff N, Lyumkis D
    Ultramicroscopy. 2018 Nov 29;203:170. doi: 10.1016/j.ultramic.2018.11.016

    Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM image processing packages, to focus classify on specific regions of a particle and detect potential covariance. The strategies are based on masking the region of interest using either a 2-D mask applied to reference projections and particle images, or a 3-D mask applied to the 3-D volume. We show that focused classification approaches can be used to study structural covariance, a concept that is likely to gain more importance as datasets grow in size, allowing the distinction of more structural states and smaller differences between states. Finally, we apply the approaches to an experimental dataset containing the HIV-1 Transactivation Response (TAR) element RNA fused into the large bacterial ribosomal subunit to deconvolve structural mobility within localized regions of interest, and to a dataset containing assembly intermediates of the large subunit to measure structural covariance.

    View Publication Page
    Grigorieff Lab
    08/24/18 | Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.
    Vemu A, Szczesna E, Zehr EA, Spector JO, Grigorieff N, Deaconescu AM, Roll-Mecak A
    Science (New York, N.Y.). 2018 Aug 24;361(6404):. doi: 10.1126/science.aau1504

    Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

    View Publication Page
    Grigorieff Lab
    04/30/18 | Atomic resolution cryo-EM structure of β-galactosidase.
    Bartesaghi A, Aguerrebere C, Falconieri V, Banerjee S, Earl LA, Zhu X, Grigorieff N, Milne JL, Sapiro G, Wu X, Subramaniam S
    Structure (London, England : 1993). 2018 Apr 30;26(6):848. doi: 10.1016/j.str.2018.04.004

    The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for β-galactosidase bound to the inhibitor phenylethyl β-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.

    View Publication Page
    Grigorieff Lab
    04/10/18 | Structural mechanism of functional modulation by gene splicing in NMDA receptors.
    Regan MC, Grant T, McDaniel MJ, Karakas E, Zhang J, Traynelis SF, Grigorieff N, Furukawa H
    Neuron. 2018 Apr 10;98(3):521-9. doi: 10.1016/j.neuron.2018.03.034

    Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the exon-5-encoded motif at the amino-terminal domain (ATD) of the GluN1 subunit are known to display robustly altered deactivation rates and pH sensitivity, but the underlying mechanism for this functional modification is largely unknown. Here, we show through cryoelectron microscopy (cryo-EM) that the presence of the exon 5 motif in GluN1 alters the local architecture of heterotetrameric GluN1-GluN2 NMDA receptors and creates contacts with the ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, which are absent in NMDA receptors lacking the exon 5 motif. The unique interactions established by the exon 5 motif are essential to the stability of the ATD/LBD and LBD/LBD interfaces that are critically involved in controlling proton sensitivity and deactivation.

    View Publication Page
    Grigorieff Lab
    03/07/18 | cisTEM, User-friendly software for single-particle image processing.
    Grant T, Rohou A, Grigorieff N
    eLife. 2018 Mar 07;7:. doi: 10.7554/eLife.35383

    We have developed new open-source software calledTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging.TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments.TEM is available for download from cistem.org.

    View Publication Page
    Grigorieff Lab
    02/16/18 | Physical basis of amyloid fibril polymorphism.
    Close W, Neumann M, Schmidt A, Hora M, Annamalai K, Schmidt M, Reif B, Schmidt V, Grigorieff N, Fändrich M
    Nature Communications. 2018 Feb 16;9(1):699. doi: 10.1038/s41467-018-03164-5

    Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide-peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.

    View Publication Page