|Entry||Database: PDB / ID: 5nbz|
|Title||Wzz dodecamer fitted by MDFF to the Wzz experimental map from cryo-EM|
|Keywords||MEMBRANE PROTEIN / Wzz Polysaccharide Chain Length membrane protein Cryo-electron microscopy Single particle analysis / Membrane protein|
|Specimen source||Staphylococcus aureus / / bacteria|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 9 Å resolution|
|Authors||Ford, R.C. / Kargas, V. / Collins, R.F. / Whitfield, C. / Clarke, B.R. / Siebert, A. / Bond, P.J. / Clare, D.K.|
|Citation||Journal: Structure / Year: 2017|
Title: Full-length, Oligomeric Structure of Wzz Determined by Cryoelectron Microscopy Reveals Insights into Membrane-Bound States.
Authors: Richard F Collins / Vasileios Kargas / Brad R Clarke / C Alistair Siebert / Daniel K Clare / Peter J Bond / Chris Whitfield / Robert C Ford
Abstract: Wzz is an integral inner membrane protein involved in regulating the length of lipopolysaccharide O-antigen glycans and essential for the virulence of many Gram-negative pathogens. In all Wzz ...Wzz is an integral inner membrane protein involved in regulating the length of lipopolysaccharide O-antigen glycans and essential for the virulence of many Gram-negative pathogens. In all Wzz homologs, the large periplasmic domain is proposed to be anchored by two transmembrane helices, but no information is available for the transmembrane and cytosolic domains. Here we have studied purified oligomeric Wzz complexes using cryoelectron microscopy and resolved the transmembrane regions within a semi-continuous detergent micelle. The transmembrane helices of each monomer display a right-handed super-helical twist, and do not interact with the neighboring transmembrane domains. Modeling, flexible fitting and multiscale simulation approaches were used to study the full-length complex and to provide explanations for the influence of the lipid bilayer on its oligomeric status. Based on structural and in silico observations, we propose a new mechanism for O-antigen chain-length regulation that invokes synergy of Wzz and its polymerase partner, Wzy.
Copyright: 2017 Elsevier Ltd. All rights reserved.
SummaryFull reportAbout validation report
|Date||Deposition: Mar 2, 2017 / Release: Apr 5, 2017|
|Structure viewer||Molecule: |
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Mass: 26760.129 Da / Num. of mol.: 12 / Source: (gene. exp.) Staphylococcus aureus / / bacteria / Production host: Escherichia coli
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: Dodecameric complex / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.36 deg. / Units: MEGADALTONS / Experimental value: NO|
|Source (natural)||Organism: Staphylococcus aureus|
|Source (recombinant)||Organism: Escherichia coli|
|Buffer solution||Details: 20mM Tris pH 7.5, 150mM NaCl, 0.025% dodecyl maltoside.|
|Specimen||Conc.: 0.3 mg/ml|
Details: Dodecamers with C12 symmetry with a small fraction of head-to-head D12 complexes.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Grid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 kelvins / Details: 1 x 4sec blot|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 9 Å / Resolution method: OTHER / Number of particles: 22000 / Details: ResMap / Symmetry type: POINT|
|Atomic model building||Ref protocol: FLEXIBLE FIT / Ref space: REAL|
|Atomic model building||PDB-ID: 4E29 / Pdb chain ID: A / Pdb chain residue range: 55-291|
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