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- PDB-5nbz: Wzz dodecamer fitted by MDFF to the Wzz experimental map from cryo-EM -

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Entry
Database: PDB / ID: 5nbz
TitleWzz dodecamer fitted by MDFF to the Wzz experimental map from cryo-EM
ComponentsWzzB
KeywordsMEMBRANE PROTEIN / Wzz Polysaccharide Chain Length membrane protein Cryo-electron microscopy Single particle analysis / Membrane protein
Specimen sourceStaphylococcus aureus / / bacteria
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 9 Å resolution
AuthorsFord, R.C. / Kargas, V. / Collins, R.F. / Whitfield, C. / Clarke, B.R. / Siebert, A. / Bond, P.J. / Clare, D.K.
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 2, 2017 / Release: Apr 5, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 5, 2017Structure modelrepositoryInitial release
1.1Jun 28, 2017Structure modelDatabase referencescitation_citation.title

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Assembly

Deposited unit
A: WzzB
B: WzzB
C: WzzB
D: WzzB
E: WzzB
F: WzzB
G: WzzB
H: WzzB
I: WzzB
J: WzzB
K: WzzB
L: WzzB


Theoretical massNumber of molelcules
Total (without water)321,12212
Polyers321,12212
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)41770
ΔGint (kcal/M)-125
Surface area (Å2)142600
MethodPISA

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Components

#1: Protein/peptide
WzzB


Mass: 26760.129 Da / Num. of mol.: 12 / Source: (gene. exp.) Staphylococcus aureus / / bacteria / Production host: Escherichia coli

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dodecameric complex / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.36 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus
Source (recombinant)Organism: Escherichia coli
Buffer solutionDetails: 20mM Tris pH 7.5, 150mM NaCl, 0.025% dodecyl maltoside.
pH: 7.5
SpecimenConc.: 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 supportGrid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 kelvins / Details: 1 x 4sec blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 9 Å / Resolution method: OTHER / Number of particles: 22000 / Details: ResMap / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 4E29 / Pdb chain ID: A / Pdb chain residue range: 55-291

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