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- EMDB-10945: In situ structure of the Caulobacter crescentus flagellar motor a... -

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Basic information

Entry
Database: EMDB / ID: EMD-10945
TitleIn situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog
Map dataFlagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised
Sample
  • Cell: Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised
Biological speciesCaulobacter vibrioides CB15 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 82.0 Å
AuthorsRossmann FM / Hug I / Sangermani M / Jenal U / Beeby M
Funding support Germany, United Kingdom, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)research fellowship 385257318 Germany
Biotechnology and Biological Sciences Research Council (BBSRC)BB/L023091/1 United Kingdom
CitationJournal: Mol Microbiol / Year: 2020
Title: In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog.
Authors: Florian M Rossmann / Isabelle Hug / Matteo Sangermani / Urs Jenal / Morgan Beeby /
Abstract: Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its ...Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.
History
DepositionApr 28, 2020-
Header (metadata) releaseJun 17, 2020-
Map releaseJun 17, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.461
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.461
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10945.png

Downloads & links

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Map

FileDownload / File: emd_10945.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFlagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised
Voxel sizeX=Y=Z: 7.00206 Å
Density
Contour LevelBy AUTHOR: 0.461 / Movie #1: 0.461
Minimum - Maximum-0.8858997 - 1.2116416
Average (Standard dev.)0.0007194363 (±0.19360326)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125-125-125
Dimensions250250250
Spacing250250250
CellA=B=C: 1750.515 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.002067.002067.00206
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z1750.5151750.5151750.515
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-125-125-125
NC/NR/NS250250250
D min/max/mean-0.8861.2120.001

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Supplemental data

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Sample components

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Entire : Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs d...

EntireName: Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised
Components
  • Cell: Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised

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Supramolecule #1: Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs d...

SupramoleculeName: Flagellar motor of Caulobactor crescentus CB 15, in situ, cheYs deletion strain, C11 symmetrised
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Caulobacter vibrioides CB15 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7 / Component - Name: peptone yeast extract; PYE medium
GridModel: UltrAuFoil / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: blot time 5 s, blot force 3, wait time 60s, drain time 0.5s,.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 7 / Average exposure time: 1.5 sec. / Average electron dose: 3.15 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C11 (11 fold cyclic) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 82.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET (ver. 1.10.1) / Number subtomograms used: 112
ExtractionNumber tomograms: 341 / Number images used: 278 / Reference model: sperical / Method: manual / Software - Name: PEET (ver. 1.10.1)
Final 3D classificationSoftware - Name: PEET (ver. 1.10.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: PEET (ver. 1.10.1)

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