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62 Publications

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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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    Grigorieff Lab
    06/02/16 | Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.
    Tajima N, Karakas E, Grant T, Simorowski N, Diaz-Avalos R, Grigorieff N, Furukawa H
    Nature. 2016 Jun 2;534(7605):63-8. doi: 10.1038/nature17679

    The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

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    Grigorieff Lab
    05/25/16 | Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction.
    Laxmikanthan G, Xu C, Brilot AF, Warren D, Steele L, Seah N, Tong W, Grigorieff N, Landy A, Van Duyne GD
    eLife. 2016 May 25;5:. doi: 10.7554/eLife.14313

    The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.

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    Grigorieff Lab
    05/16/16 | Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril.
    Schmidt A, Annamalai K, Schmidt M, Grigorieff N, Fändrich M
    Proceedings of the National Academy of Sciences of the United States of America. 2016 May 16;113(22):6200-5. doi: 10.1073/pnas.1522282113

    Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles.

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    Grigorieff Lab
    05/09/16 | Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.
    Abeyrathne PD, Koh CS, Grant T, Grigorieff N, Korostelev AA
    eLife. 2016 May 9;5:. doi: 10.7554/eLife.14874

    Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.

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    Grigorieff Lab
    11/01/15 | Automatic estimation and correction of anisotropic magnification distortion in electron microscopes.
    Grant T, Grigorieff N
    Journal of Structural Biology. 2015 Nov;192(2):204-8. doi: 10.1016/j.jsb.2015.08.006

    We demonstrate a significant anisotropic magnification distortion, found on an FEI Titan Krios microscope and affecting magnifications commonly used for data acquisition on a Gatan K2 Summit detector. We describe a program (mag_distortion_estimate) to automatically estimate anisotropic magnification distortion from a set of images of a standard gold shadowed diffraction grating. We also describe a program (mag_distortion_correct) to correct for the estimated distortion in collected images. We demonstrate that the distortion present on the Titan Krios microscope limits the resolution of a set of rotavirus VP6 images to ∼7 Å, which increases to ∼3 Å following estimation and correction of the distortion. We also use a 70S ribosome sample to demonstrate that in addition to affecting resolution, magnification distortion can also interfere with the classification of heterogeneous data.

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    Grigorieff Lab
    10/06/15 | Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.
    Zhou A, Rohou A, Schep DG, Bason JV, Montgomery MG, Walker JE, Grigorieff N, Rubinstein JL
    eLife. 2015 Oct 06;4:. doi: 10.7554/eLife.10180

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

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    Grigorieff Lab
    09/22/15 | Peptide dimer structure in an Aβ(1-42) fibril visualized with cryo-EM.
    Schmidt M, Rohou A, Lasker K, Yadav JK, Schiene-Fischer C, Fändrich M, Grigorieff N
    Proceedings of the National Academy of Sciences of the United States of America. 2015 Sep 22;112(38):11858-63. doi: 10.1073/pnas.1503455112

    Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1-42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.

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    Grigorieff Lab
    08/28/15 | Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0.
    Chiu P, Li X, Li Z, Beckett B, Brilot AF, Grigorieff N, Agard DA, Cheng Y, Walz T
    Journal of Structural Biology. 2015 Aug 28:. doi: 10.1016/j.jsb.2015.08.015

    The K2 Summit camera was initially the only commercially available direct electron detection camera that was optimized for high-speed counting of primary electrons and was also the only one that implemented centroiding so that the resolution of the camera can be extended beyond the Nyquist limit set by the physical pixel size. In this study, we used well-characterized two-dimensional crystals of the membrane protein aquaporin-0 to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases.

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    Grigorieff Lab
    08/13/15 | CTFFIND4: Fast and accurate defocus estimation from electron micrographs.
    Rohou A, Grigorieff N
    Journal of Structural Biology. 2015 Aug 13;192(2):216-21. doi: 10.1016/j.jsb.2015.08.008

    CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

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