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- EMDB-11060: Flagellar motor of Shewanella putrefaciens in situ, flhG deletion -

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Basic information

Entry
Database: EMDB / ID: EMD-11060
TitleFlagellar motor of Shewanella putrefaciens in situ, flhG deletion
Map dataFlagellar motor of Shewanella putrefaciens in situ, flhG deletion
Sample
  • Cell: Flagellar motor of Shewanella putrefaciens in situ, wildtype
Biological speciesShewanella putrefaciens CN-32 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 79.0 Å
AuthorsBlagotinsek V / Schwan M / Steinchen W / Mrusek D / Hook J / Rossmann FM / Freibert SA / Kressler D / Beeby M / Thormann KM / Bange G
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: Proc Natl Acad Sci U S A / Year: 2020
Title: An ATP-dependent partner switch links flagellar C-ring assembly with gene expression.
Authors: Vitan Blagotinsek / Meike Schwan / Wieland Steinchen / Devid Mrusek / John C Hook / Florian Rossmann / Sven A Freibert / Hanna Kratzat / Guillaume Murat / Dieter Kressler / Roland Beckmann / ...Authors: Vitan Blagotinsek / Meike Schwan / Wieland Steinchen / Devid Mrusek / John C Hook / Florian Rossmann / Sven A Freibert / Hanna Kratzat / Guillaume Murat / Dieter Kressler / Roland Beckmann / Morgan Beeby / Kai M Thormann / Gert Bange /
Abstract: Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its ...Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species , we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.
History
DepositionMay 21, 2020-
Header (metadata) releaseOct 21, 2020-
Map releaseOct 21, 2020-
UpdateOct 21, 2020-
Current statusOct 21, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.26
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11060.png

Downloads & links

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Map

FileDownload / File: emd_11060.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFlagellar motor of Shewanella putrefaciens in situ, flhG deletion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.28 Å/pix.
x 150 pix.
= 1242. Å		8.28 Å/pix.
x 150 pix.
= 1242. Å		8.28 Å/pix.
x 150 pix.
= 1242. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 8.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.26 / Movie #1: 0.26
Minimum - Maximum-0.5246716 - 0.66255856
Average (Standard dev.)0.06086719 (±0.21940218)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-75-75-75
Dimensions150150150
Spacing150150150
CellA=B=C: 1242.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.288.288.28
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z1242.0001242.0001242.000
α/β/γ90.00090.00090.000
start NX/NY/NZ9482110
NX/NY/NZ11313776
MAP C/R/S123
start NC/NR/NS-75-75-75
NC/NR/NS150150150
D min/max/mean-0.5250.6630.061

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Supplemental data

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Sample components

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Entire : Flagellar motor of Shewanella putrefaciens in situ, wildtype

EntireName: Flagellar motor of Shewanella putrefaciens in situ, wildtype
Components
  • Cell: Flagellar motor of Shewanella putrefaciens in situ, wildtype

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Supramolecule #1: Flagellar motor of Shewanella putrefaciens in situ, wildtype

SupramoleculeName: Flagellar motor of Shewanella putrefaciens in situ, wildtype
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Shewanella putrefaciens CN-32 (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: LB 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: 2 / 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: 25000
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 reconstructionApplied symmetry - Point group: C100 (100 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 79.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET (ver. 1.10.1) / Number subtomograms used: 349
ExtractionNumber tomograms: 118 / Number images used: 349 / Reference model: sperical / Method: manual / Software - Name: PEET (ver. 1.10.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: PEET (ver. 1.10.1)

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