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- PDB-6ykr: Structure of a protonation mimic of unplugged C. jejuni MotAB -

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Basic information

Entry
Database: PDB / ID: 6ykr
TitleStructure of a protonation mimic of unplugged C. jejuni MotAB
Components
  • Chemotaxis protein MotA, putative
  • Chemotaxis protein MotB, putative
KeywordsMEMBRANE PROTEIN / Bacterial flagellar motor / stator unit / locomotion / proton transport / ion transport
Function / homologyFlagellar motor protein MotA, conserved site / MotA/TolQ/ExbB proton channel / OmpA-like domain / Motility protein B, N-terminal domain / OmpA-like domain superfamily / integral component of membrane / Chemotaxis protein MotA, putative / Chemotaxis protein MotB, putative
Function and homology information
Biological speciesCampylobacter jejuni subsp. jejuni serotype O:23/36 (Campylobacter)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsSantiveri, M. / Roa-Eguiara, A. / Taylor, N.M.I.
Funding support Denmark, 2items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Danish Council for Independent Research8123-00002B Denmark
CitationJournal: Cell / Year: 2020
Title: Structure and Function of Stator Units of the Bacterial Flagellar Motor.
Authors: Mònica Santiveri / Aritz Roa-Eguiara / Caroline Kühne / Navish Wadhwa / Haidai Hu / Howard C Berg / Marc Erhardt / Nicholas M I Taylor /
Abstract: Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate ...Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Chemotaxis protein MotA, putative
B: Chemotaxis protein MotA, putative
C: Chemotaxis protein MotA, putative
D: Chemotaxis protein MotA, putative
E: Chemotaxis protein MotA, putative
F: Chemotaxis protein MotB, putative
G: Chemotaxis protein MotB, putative


Theoretical massNumber of molelcules
Total (without water)200,7047
Polymers200,7047
Non-polymers00
Water1086
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26050 Å2
ΔGint-236 kcal/mol
Surface area52770 Å2
MethodPISA

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Components

#1: Protein
Chemotaxis protein MotA, putative /


Mass: 28195.816 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176) (Campylobacter)
Strain: 81-176 / Gene: CJJ81176_0359 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H3PAV1
#2: Protein Chemotaxis protein MotB, putative /


Mass: 29862.643 Da / Num. of mol.: 2
Mutation: Single mutation D22N and deletion of aminoacids 41 to 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176) (Campylobacter)
Strain: 81-176 / Gene: CJJ81176_0358 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H3PBX6
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Stator unit MotAB(Delta41-60, D22N) / Type: COMPLEX
Details: The stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Single mutation D22N simulates the protonated state of the channel. This stator unit is unplugged (deletion of ...Details: The stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Single mutation D22N simulates the protonated state of the channel. This stator unit is unplugged (deletion of aminoacids 41 to 60 of MotB).
Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Campylobacter jejuni subsp. jejuni 81-176 (Campylobacter)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43(DE3)
Buffer solutionpH: 8
SpecimenConc.: 0.68 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 43.44 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM softwareName: RELION / Version: 3 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 456384 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 39.06 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004510372
ELECTRON MICROSCOPYf_angle_d0.835714014
ELECTRON MICROSCOPYf_chiral_restr0.0511640
ELECTRON MICROSCOPYf_plane_restr0.00711763
ELECTRON MICROSCOPYf_dihedral_angle_d11.47013766

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