@article {65286, title = {Structure and assembly of calcium homeostasis modulator proteins.}, journal = {Nature Structural and Molecular Biology}, volume = {27}, year = {2020}, month = {2020 Jan 27}, pages = {150-9}, abstract = {

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. 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), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer{\textquoteright}s disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, 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.

}, issn = {1545-9985}, doi = {10.1038/s41594-019-0369-9}, author = {Syrjanen, Johanna L and Michalski, Kevin and Chou, Tsung-Han and Grant, Timothy and Rao, Shanlin and Simorowski, Noriko and Tucker, Stephen J and Grigorieff, Nikolaus and Furukawa, Hiro} } @article {64971, title = {High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, year = {2019}, month = {2019 Jun 25}, pages = {12828-32}, abstract = {

Noroviruses are a leading cause of foodborne 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- to 4.1-{\r A}) cryoelectron 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. Using asymmetric reconstruction, we were able to resolve a Zn2+\ metal ion adjacent to the coreceptor binding site, which affected the structural stability of the shell. Our structures serve as valuable templates for facilitating vaccine formulations.

}, doi = {10.1073/pnas.1903562116}, author = {Jung, James and Grant, Timothy and Thomas, Dennis R. and Diehnelt, Chris W. and Grigorieff, Nikolaus and Leemor, Joshua-Tor} } @article {65024, title = {In situ structure of rotavirus VP1 RNA-dependent RNA polymerase.}, journal = {Journal of Molecular Biology}, volume = {431}, year = {2019}, month = {2019 Jun 21}, pages = {3124-38}, abstract = {

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 {\textquotedblleft}double-layer particle{\textquotedblright} 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 ({\textquotedblleft}triple-layer particles{\textquotedblright} 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.

}, doi = {10.1016/j.jmb.2019.06.016}, author = {Jenni, Simon and Salgado, Eric N. and Herrmann, Tobias and Li, Zongli and Grant, Timothy and Grigorieff, Nikolaus and Trapani, Stefano and Estrozi, Leandro F. and Harrison, Stephen C.} } @article {49087, title = {cisTEM, User-friendly software for single-particle image processing.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Mar 07}, abstract = {

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.

}, issn = {2050-084X}, doi = {10.7554/eLife.35383}, author = {Grant, Timothy and Rohou, Alexis and Grigorieff, Nikolaus} } @article {49152, title = {Structural mechanism of functional modulation by gene splicing in NMDA receptors.}, journal = {Neuron}, volume = {98}, year = {2018}, month = {2018 Apr 10}, pages = {521-9}, abstract = {

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.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2018.03.034}, author = {Regan, Michael C and Grant, Timothy and McDaniel, Miranda J and Karakas, Erkan and Zhang, Jing and Traynelis, Stephen F and Grigorieff, Nikolaus and Furukawa, Hiro} } @article {48344, title = {Mechanism of ribosome rescue by ArfA and RF2.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Mar 16}, pages = {e23687}, abstract = {

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-{\r A} resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA{\textbullet}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.

}, issn = {2050-084X}, doi = {10.7554/eLife.23687}, author = {Demo, Gabriel and Svidritskiy, Egor and Madireddy, Rohini and Diaz-Avalos, Ruben and Grant, Timothy and Grigorieff, Nikolaus and Sousa, Duncan and Korostelev, Andrei A} } @article {47509, title = {Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.}, journal = {Nature}, volume = {534}, year = {2016}, month = {2016 Jun 2}, pages = {63-8}, abstract = {

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.

}, issn = {1476-4687}, doi = {10.1038/nature17679}, author = {Tajima, Nami and Karakas, Erkan and Grant, Timothy and Simorowski, Noriko and Diaz-Avalos, Ruben and Grigorieff, Nikolaus and Furukawa, Hiro} } @article {47465, title = {Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 May 9}, abstract = {

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{\textbullet}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.

}, issn = {2050-084X}, doi = {10.7554/eLife.14874}, author = {Abeyrathne, Priyanka D and Koh, Cha San and Grant, Timothy and Grigorieff, Nikolaus and Korostelev, Andrei A} } @article {47885, title = {Automatic estimation and correction of anisotropic magnification distortion in electron microscopes.}, journal = {Journal of Structural Biology}, volume = {192}, year = {2015}, month = {2015 Nov}, pages = {204-8}, abstract = {

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 {\r A}, which increases to \~{}3 {\r A} 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.

}, keywords = {Anisotropy, Cryoelectron Microscopy, Gold, Image Enhancement}, issn = {1095-8657}, doi = {10.1016/j.jsb.2015.08.006}, author = {Grant, Timothy and Grigorieff, Nikolaus} } @article {46815, title = {Measuring the optimal exposure for single particle cryo-EM using a 2.6 {\r A} reconstruction of rotavirus VP6.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 May 29}, pages = {10.7554}, abstract = {

Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 {\r A} resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/{\r A}(2), we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This {\textquoteright}high-exposure{\textquoteright} technique should benefit cryo-EM work on all types of samples, especially those of relatively low molecular mass.

}, issn = {2050-084X}, doi = {10.7554/eLife.06980}, author = {Grant, Timothy and Grigorieff, Nikolaus} } @article {48316, title = {Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy.}, journal = {Cell}, volume = {162}, year = {2015}, month = {2015 Jul 16}, pages = {314-27}, abstract = {

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 {\r A}, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

}, keywords = {Cryoelectron Microscopy, DNA-Directed RNA Polymerases, Models, Molecular, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Transcription, Genetic, Vesicular stomatitis Indiana virus, Viral Proteins}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.06.018}, author = {Liang, Bo and Li, Zongli and Jenni, Simon and Rahmeh, Amal A and Morin, Benjamin M and Grant, Timothy and Grigorieff, Nikolaus and Harrison, Stephen C and Whelan, Sean P J} }