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https://www.readbyqxmd.com/read/28323617/cryoem-structures-of-membrane-pore-and-prepore-complex-reveal-cytolytic-mechanism-of-pneumolysin
#1
Katharina van Pee, Alexander Neuhaus, Edoardo D'Imprima, Deryck J Mills, Werner Kühlbrandt, Özkan Yildiz
Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain D3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel...
March 21, 2017: ELife
https://www.readbyqxmd.com/read/28300075/a-human-antibody-against-zika-virus-crosslinks-the-e-protein-to-prevent-infection
#2
S Saif Hasan, Andrew Miller, Gopal Sapparapu, Estefania Fernandez, Thomas Klose, Feng Long, Andrei Fokine, Jason C Porta, Wen Jiang, Michael S Diamond, James E Crowe, Richard J Kuhn, Michael G Rossmann
The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117...
March 16, 2017: Nature Communications
https://www.readbyqxmd.com/read/28287545/method-to-visualize-and-analyze-membrane-interacting-proteins-by-transmission-electron-microscopy
#3
Ramakrishnan B Kumar, Lin Zhu, Hans Hebert, Caroline Jegerschöld
Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc...
March 5, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28283532/molecular-architecture-of-the-n-type-atpase-rotor-ring-from-burkholderia-pseudomallei
#4
Sarah Schulz, Martin Wilkes, Deryck J Mills, Werner Kühlbrandt, Thomas Meier
The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N1No-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution...
March 10, 2017: EMBO Reports
https://www.readbyqxmd.com/read/28254381/vitrification-after-multiple-rounds-of-sample-application-and-blotting-improves-particle-density-on-cryo-electron-microscopy-grids
#5
Joost Snijder, Andrew J Borst, Annie Dosey, Alexandra C Walls, Anika Burrell, Vijay S Reddy, Justin M Kollman, David Veesler
Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane...
February 22, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/28250125/conformational-states-of-a-soluble-uncleaved-hiv-1-envelope-trimer
#6
Yuhang Liu, Junhua Pan, Yongfei Cai, Nikolaus Grigorieff, Stephen C Harrison, Bing Chen
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...
March 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28192420/atomic-resolution-structures-from-fragmented-protein-crystals-with-the-cryoem-method-microed
#7
M Jason de la Cruz, Johan Hattne, Dan Shi, Paul Seidler, Jose Rodriguez, Francis E Reyes, Michael R Sawaya, Duilio Cascio, Simon C Weiss, Sun Kyung Kim, Cynthia S Hinck, Andrew P Hinck, Guillermo Calero, David Eisenberg, Tamir Gonen
Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography...
February 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28181439/cryoem-structure-refinement-by-integrating-nmr-chemical-shifts-with-molecular-dynamics-simulations
#8
Juan R Perilla, Gongpu Zhao, Manman Lu, Jiying Ning, Guangjin Hou, In-Ja L Byeon, Angela M Gronenborn, Tatyana Polenova, Peijun Zhang
Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging...
February 9, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28165000/high-affinity-anchoring-of-the-decoration-protein-pb10-onto-the-bacteriophage-t5-capsid
#9
Emeline Vernhes, Madalena Renouard, Bernard Gilquin, Philippe Cuniasse, Dominique Durand, Patrick England, Sylviane Hoos, Alexis Huet, James F Conway, Anatoly Glukhov, Vladimir Ksenzenko, Eric Jacquet, Naïma Nhiri, Sophie Zinn-Justin, Pascale Boulanger
Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent...
February 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28148799/computational-prediction-of-the-heterodimeric-and-higher-order-structure-of-gpe1-gpe2-envelope-glycoproteins-encoded-by-the-hepatitis-c-virus-hcv
#10
Holly Freedman, Michael R Logan, Darren Hockman, Julia Koehler Leman, John Lok Man Law, Michael Houghton
Despite the recent success of newly developed direct acting antivirals against Hepatitis C, the disease continues to be a global health threat due to lack of diagnosis of most carriers and the high cost of treatment. The heterodimer formed by the glycoproteins E1 and E2 within the hepatitis C virus (HCV) lipid envelope is a potential vaccine candidate and antiviral target. While the structure of E1/E2 has not yet been resolved, partial crystal structures of the E1 and E2 ectodomains have been determined. The unresolved parts of the structure are within the realm of what can be modeled with current computational modeling tools...
February 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28056362/structures-of-biomolecular-complexes-by-combination-of-nmr-and-cryoem-methods
#11
REVIEW
Philippe Cuniasse, Paulo Tavares, Elena V Orlova, Sophie Zinn-Justin
CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid- or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics...
January 2, 2017: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/28045370/atomic-structures-of-fibrillar-segments-of-hiapp-suggest-tightly-mated-%C3%AE-sheets-are-important-for-cytotoxicity
#12
Pascal Krotee, Jose A Rodriguez, Michael R Sawaya, Duilio Cascio, Francis E Reyes, Dan Shi, Johan Hattne, Brent L Nannenga, Marie E Oskarsson, Stephan Philipp, Sarah Griner, Lin Jiang, Charles G Glabe, Gunilla T Westermark, Tamir Gonen, David S Eisenberg
hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils...
January 3, 2017: ELife
https://www.readbyqxmd.com/read/28004660/corrigendum-cryoem-structure-of-the-methanospirillum-hungatei-archaellum-reveals-structural-features-distinct-from-the-bacterial-flagellum-and-type-iv-pilus
#13
Nicole Poweleit, Peng Ge, Hong H Nguyen, Rachel R Ogorzalek Loo, Robert P Gunsalus, Z Hong Zhou
No abstract text is available yet for this article.
December 22, 2016: Nature Microbiology
https://www.readbyqxmd.com/read/27992877/in-situ-structures-of-the-genome-and-genome-delivery-apparatus-in-a-single-stranded-rna-virus
#14
Xinghong Dai, Zhihai Li, Mason Lai, Sara Shu, Yushen Du, Z Hong Zhou, Ren Sun
Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly...
December 19, 2016: Nature
https://www.readbyqxmd.com/read/27958264/in-vitro-protease-cleavage-and-computer-simulations-reveal-the-hiv-1-capsid-maturation-pathway
#15
Jiying Ning, Gonca Erdemci-Tandogan, Ernest L Yufenyuy, Jef Wagner, Benjamin A Himes, Gongpu Zhao, Christopher Aiken, Roya Zandi, Peijun Zhang
HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel...
December 13, 2016: Nature Communications
https://www.readbyqxmd.com/read/27922015/cryoem-structure-of-the-methanospirillum-hungatei-archaellum-reveals-structural-features-distinct-from-the-bacterial-flagellum-and-type-iv-pili
#16
Nicole Poweleit, Peng Ge, Hong H Nguyen, Rachel R Ogorzalek Loo, Robert P Gunsalus, Z Hong Zhou
Archaea use flagella known as archaella-distinct both in protein composition and structure from bacterial flagella-to drive cell motility, but the structural basis of this function is unknown. Here, we report an atomic model of the archaella, based on the cryo electron microscopy (cryoEM) structure of the Methanospirillum hungatei archaellum at 3.4 Å resolution. Each archaellum contains ∼61,500 archaellin subunits organized into a curved helix with a diameter of 10 nm and average length of 10,000 nm...
December 5, 2016: Nature Microbiology
https://www.readbyqxmd.com/read/27882950/neutralization-mechanism-of-a-highly-potent-antibody-against-zika-virus
#17
Shuijun Zhang, Victor A Kostyuchenko, Thiam-Seng Ng, Xin-Ni Lim, Justin S G Ooi, Sebastian Lambert, Ter Yong Tan, Douglas G Widman, Jian Shi, Ralph S Baric, Shee-Mei Lok
The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4...
November 24, 2016: Nature Communications
https://www.readbyqxmd.com/read/27852863/cryo-electron-microscopy-structures-of-expanded-poliovirus-with-vhhs-sample-the-conformational-repertoire-of-the-expanded-state
#18
Mike Strauss, Lise Schotte, Krishanthi S Karunatilaka, David J Filman, James M Hogle
By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion...
February 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/27834216/schizosaccharomyces-pombe-kinesin-5-switches-direction-using-a-steric-blocking-mechanism
#19
Mishan Britto, Adeline Goulet, Syeda Rizvi, Ottilie von Loeffelholz, Carolyn A Moores, Robert A Cross
Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking...
November 22, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27752045/structures-and-stabilization-of-kinetoplastid-specific-split-rrnas-revealed-by-comparing-leishmanial-and-human-ribosomes
#20
Xing Zhang, Mason Lai, Winston Chang, Iris Yu, Ke Ding, Jan Mrazek, Hwee L Ng, Otto O Yang, Dmitri A Maslov, Z Hong Zhou
The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments...
October 18, 2016: Nature Communications
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