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34 Janelia Publications

Showing 11-20 of 34 results
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    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.

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    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.

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    Grigorieff Lab
    10/13/17 | Structure of RNA polymerase bound to ribosomal 30S subunit.
    Demo G, Rasouly A, Vasilyev N, Svetlov V, Loveland AB, Diaz-Avalos R, Grigorieff N, Nudler E, Korostelev AA
    eLife. 2017 Oct 13;6:. doi: 10.7554/eLife.28560

    In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S•RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.

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    Grigorieff Lab
    07/10/17 | Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1.
    Abeyrathne PD, Grigorieff N
    PloS One. 2017;12(7):e0180163. doi: 10.1371/journal.pone.0180163

    Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.

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    Grigorieff Lab
    05/12/17 | CryoEM structure of an influenza virus receptor-binding site antibody-antigen interface.
    Liu Y, Pan J, Jenni S, Raymond DD, Caradonna T, Do KT, Schmidt AG, Harrison SC, Grigorieff N
    Journal of Molecular Biology. 2017 May 12;429(12):1829-39. doi: 10.1016/j.jmb.2017.05.011

    Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes. Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain Fv (scFv) complexes, by studying a complex we failed to crystallize in pursuing an extended project of the human immune response to influenza vaccines. The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen:antibody interface, although small variation in the twist of the rod-like HA trimer limited the overall resolution to about 4.5Å. Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

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    Grigorieff Lab
    05/03/17 | Single-protein detection in crowded molecular environments in cryo-EM images.
    Rickgauer JP, Grigorieff N, Denk W
    eLife. 2017 May 03;6:. doi: 10.7554/eLife.25648

    We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and-in the presence of protein background-a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.

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    Grigorieff Lab
    03/16/17 | Mechanism of ribosome rescue by ArfA and RF2.
    Demo G, Svidritskiy E, Madireddy R, Diaz-Avalos R, Grant T, Grigorieff N, Sousa D, Korostelev AA
    eLife. 2017 Mar 16;6:e23687. doi: 10.7554/eLife.23687

    ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA "senses" the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

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    Grigorieff Lab
    03/01/17 | Conformational states of a soluble, uncleaved HIV-1 envelope trimer.
    Liu Y, Pan J, Cai Y, Grigorieff N, Harrison SC, Chen B
    Journal of Virology. 2017 Mar 01;91(10):e00175-17. doi: 10.1128/JVI.00175-17

    HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers - the uncleaved ectodomains of (gp160)3 - from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20Å) cryoEM (electron cryomicroscopy) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with truncated cytoplasmic tail (EnvΔCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env.IMPORTANCE Development of any HIV vaccine with a protein component (either prime or boost) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike, in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications including an engineered disulfide. This construct, difficult to produce in large quantities, has yet to induce better antibody responses than other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and of inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.

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    Grigorieff Lab
    12/09/16 | Structure of the transporter associated with antigen processing trapped by herpes simplex virus.
    Oldham ML, Grigorieff N, Chen J
    eLife. 2016 Dec 09;5:. doi: 10.7554/eLife.21829

    The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) transporter essential to cellular immunity against viral infection. Some persistent viruses have evolved strategies to inhibit TAP so that they may go undetected by the immune system. The herpes simplex virus for example evades immune surveillance by blocking peptide transport with a small viral protein ICP47. In this study, we determined the structure of human TAP bound to ICP47 by electron cryo-microscopy (cryo-EM) to 4.0 Å. The structure shows that ICP47 traps TAP in an inactive conformation distinct from the normal transport cycle. The specificity and potency of ICP47 inhibition result from contacts between the tip of the helical hairpin and the apex of the transmembrane cavity. This work provides a clear molecular description of immune evasion by a persistent virus. It also establishes the molecular structure of TAP to facilitate mechanistic studies of the antigen presentation process.

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    Grigorieff Lab
    06/07/16 | Frealign: an exploratory tool for single-particle cryo-EM.
    Grigorieff N
    Methods in Enzymology. 2016 Jun 07:. doi: 10.1016/bs.mie.2016.04.013

    Frealign is a software tool designed to process electron microscope images of single molecules and complexes to obtain reconstructions at the highest possible resolution. It provides a number of refinement parameters and options that allow users to tune their refinement to achieve specific goals, such as masking to classify selected regions within a particle, control over the refinement of specific alignment parameters to accommodate various data collection schemes, refinement of pseudosymmetric particles, and generation of initial maps. This chapter provides a general overview of Frealign functions and a more detailed guide to using Frealign in typical scenarios.

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