Biology subjects

Grigorieff, N.

Publications and source records attributed to Grigorieff, N..

30 records · Page 2Linked to original sources

bioRxiv2021

Cryo-TEM simulations of amorphous radiation-sensitive samples using multislice wave propagation.

Image simulation plays a central role in the development and practice of high-resolution electron microscopy, including transmission electron microscopy of frozen-hydrated specimens (cryo-EM). Simulating images with contrast that matches the contrast observed in experimental images remains challenging, especially for amorphous samples. Current state-of-the-art simulators apply post hoc scaling to approximate empirical solvent contrast, attenuated image intensity due to specimen thickness, and amplitude contrast. This practice fails for images that require spatially variable scaling, e.g., simulations of a crowded or cellular environment. Modeling both the signal and the noise accurately is necessary to simulate images of biological specimens with contrast that is correct on an absolute scale. The "Frozen-Plasmon" method is introduced which explicitly models spatially variable inelastic scattering processes in cryo-EM specimens. This approach produces amplitude contrast that depends on the atomic composition of the specimen, reproduces the total inelastic mean free path as observed experimentally, and allows for the incorporation of radiation damage in the simulation. These improvements are quantified using the matched-filter concept to compare simulation and experiment. The Frozen-Plasmon method, in combination with a new mathematical formulation for accurately sampling the tabulated atomic scattering potentials onto a Cartesian grid, is implemented in the open-source software package cisTEM.

biophysics↗

bioRxiv2020

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

biochemistry↗

bioRxiv2019

Structure and assembly of calcium homeostasis modulator proteins

Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. Examples include connexin, innexin, and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), which permeate ions and ATP in a voltage-dependent manner to control neuronal excitability, taste signaling, and pathologies of depression and Alzheimers disease. Despite such critical biological roles, the structures and patterns of oligomeric assembly remain unclear. Here, we reveal the first structures of two CALHMs, CALHM1 and CALHM2, by single particle cryo-electron microscopy, which show novel assembly of the four transmembrane helices into channels of 8-mers and 11-mers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore-size, lipid accommodation, and channel activity.

biophysics↗

bioRxiv2019

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated and polyadenylated mRNAs and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 [A] resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with the RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5-nucleotide.

molecular biology↗

bioRxiv2019

In situ structure of rotavirus VP1 RNA-dependent RNA polymerase

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the \"double-layer particle\" or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (\"triple-layer particles\" or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

molecular biology↗

bioRxiv2019

High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations

Noroviruses are a leading cause of food-borne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6-4.1 [A]) cryo-electron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7 and GI.1 human norovirus outbreak strain virus-like particles (VLPs). Although norovirus VLPs have been thought to exist in a single-sized assembly, our structures reveal polymorphism between and within genogroups with small, medium and large particle sizes observed. We developed a new asymmetric reconstruction method and resolved a metal ion adjacent to the co-receptor binding site, which affected the structural stability of the shell. Our structures serve as valuable templates for facilitating vaccine formulations.

biophysics↗

bioRxiv2018

Analysis of Local Variability and Allostery in Macromolecular Assemblies using Cryo-EM and Focused Classification

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 allostery, 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.\n\nHighlightsO_LIDescription of different image classification strategies in single-particle cryo-EM\nC_LIO_LIQuantitative evaluation of two classification methods using simulated data\nC_LIO_LIApplication of the two classification methods to an experimental dataset\nC_LI

biophysics↗

bioRxiv2018

Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microcopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We developed a novel hybrid-method called \"TomographY-Guided 3D REconstruction of Subcellular Structures\" (TYGRESS) that combines cryo-ET with SP-cryo-EM to achieve close-to-nanometer resolution of complexes inside crowded environments. Using TYGRESS, we determined the native 3D structures of the intact ciliary axoneme with up to 12 [A] resolution. These results reveal many structures and details that were not visible by cryo-ET. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging, bringing us a step closer to (pseudo-)atomic models of cells.\n\nOne Sentence SummaryA hybrid cryo-electron microscopy method reveals subcellular structures at unprecedented resolution.

cell biology↗

bioRxiv2018

Cryo-EM structure of an amyloid fibril from systemic amyloidosis

Systemic AA amyloidosis is a worldwide occurring disease of humans and animals that arises from the misfolding of serum amyloid A protein. To provide insights into the molecular basis of this disease we used electron cryo-microscopy and determined the structure of an ex vivo amyloid fibril purified from AA amyloidotic mice at 3.0 [A] resolution. The fibril consists of C-terminally truncated serum amyloid A protein arranged into a compactly folded all-{beta} conformation. The structure identifies the protein N-terminus as central for the assembly of this fibril and provides a mechanism for its prion-like replication. Our data further explain how amino acid substitutions within the tightly packed fibril core can lead to amyloid resistance in vivo.

biophysics↗

bioRxiv2018

Mechanistic Origins of Dynamic Instability in Filaments from the Phage Tubulin, PhuZ

A bacteriophage-encoded tubulin homologue, PhuZ, harnesses dynamic instability to position genomes of KZ-like bacteriophage at the midline of their Pseudomonas hosts, facilitating phage infectivity. While much has been learned about molecular origins of microtubule dynamics, how GTP binding and hydrolysis control dynamics in the divergent 3-stranded PhuZ filaments is not understood. Here we present cryo-EM reconstructions of the PhuZ filamentin a pre-hydrolysis (3.5[A]) and three post-hydrolysis states (4.2 [A], 7.3 [A] and 8.1 [A] resolutions), likely representing distinct depolymerization stages. Core polymerization-induced structural changes reveal similarities to {beta}-tubulin, suggesting broad conservation within the tubulin family. By contrast, GTP hydrolysis is sensed quite differently and is communicated by the divergent PhuZ C-terminus to the lateral interface, leading to PhuZ polymer destabilization. This provides a contrasting molecular description of how nucleotide state can be harnessed by the tubulin fold to regulate filament assembly, metastability and disassembly.

cell biology↗

bioRxiv2018

cisTEM: User-friendly software for single-particle image processing

We have developed new open-source software called cisTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cisTEM 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. cisTEM is available for download from cistem.org.

biophysics↗

bioRxiv2016

Mechanism of ribosome rescue by ArfA and RF2

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-[A] 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, hitherto unobserved in 70S termination complexes. 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.

biochemistry↗