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- EMDB-5631: Cryo-electron tomography of in situ flagellar motor of Borrelia b... -

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Basic information

Entry
Database: EMDB / ID: EMD-5631
TitleCryo-electron tomography of in situ flagellar motor of Borrelia burgdorferi FlhO mutant
Map dataFlagellar motor of a FlhO mutant in B. burgdorferi
Sample
  • Sample: In situ flagellar motor from B. burgdorferi FlhO mutant cells
  • Organelle or cellular component: flagellar motor
KeywordsFlagellar motor / assembly / Type III secretion / Lyme disease spirochete / molecular machine
Biological speciesBorrelia burgdorferi (Lyme disease spirochete)
Methodsubtomogram averaging / cryo EM / Resolution: 39.0 Å
AuthorsZhao X / Zhang K / Boquoi T / Hu B / Motaleb MA / Miller KA / James ME / Charon NW / Manson MD / Norris SJ ...Zhao X / Zhang K / Boquoi T / Hu B / Motaleb MA / Miller KA / James ME / Charon NW / Manson MD / Norris SJ / Li C / Liu J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi.
Authors: Xiaowei Zhao / Kai Zhang / Tristan Boquoi / Bo Hu / M A Motaleb / Kelly A Miller / Milinda E James / Nyles W Charon / Michael D Manson / Steven J Norris / Chunhao Li / Jun Liu /
Abstract: Periplasmic flagella are essential for the distinctive morphology, motility, and infectious life cycle of the Lyme disease spirochete Borrelia burgdorferi. In this study, we genetically trapped ...Periplasmic flagella are essential for the distinctive morphology, motility, and infectious life cycle of the Lyme disease spirochete Borrelia burgdorferi. In this study, we genetically trapped intermediates in flagellar assembly and determined the 3D structures of the intermediates to 4-nm resolution by cryoelectron tomography. We provide structural evidence that secretion of rod substrates triggers remodeling of the central channel in the flagellar secretion apparatus from a closed to an open conformation. This open channel then serves as both a gateway and a template for flagellar rod assembly. The individual proteins assemble sequentially to form a modular rod. The hook cap initiates hook assembly on completion of the rod, and the filament cap facilitates filament assembly after formation of the mature hook. Cryoelectron tomography and mutational analysis thus combine synergistically to provide a unique structural blueprint of the assembly process of this intricate molecular machine in intact cells.
History
DepositionApr 5, 2013-
Header (metadata) releaseApr 17, 2013-
Map releaseAug 14, 2013-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5631.png

Downloads & links

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Map

FileDownload / File: emd_5631.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFlagellar motor of a FlhO mutant in B. burgdorferi
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.7 Å/pix.
x 256 pix.
= 1459.2 Å		5.7 Å/pix.
x 256 pix.
= 1459.2 Å		5.7 Å/pix.
x 256 pix.
= 1459.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-14.16023159 - 13.52649879
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-9-9-9
Dimensions256256256
Spacing256256256
CellA=B=C: 1459.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.75.75.7
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z1459.2001459.2001459.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-9-9-9
NC/NR/NS256256256
D min/max/mean-14.16013.526-0.000

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Supplemental data

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Sample components

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Entire : In situ flagellar motor from B. burgdorferi FlhO mutant cells

EntireName: In situ flagellar motor from B. burgdorferi FlhO mutant cells
Components
  • Sample: In situ flagellar motor from B. burgdorferi FlhO mutant cells
  • Organelle or cellular component: flagellar motor

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Supramolecule #1000: In situ flagellar motor from B. burgdorferi FlhO mutant cells

SupramoleculeName: In situ flagellar motor from B. burgdorferi FlhO mutant cells
type: sample / ID: 1000 / Number unique components: 20

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Supramolecule #1: flagellar motor

SupramoleculeName: flagellar motor / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Borrelia burgdorferi (Lyme disease spirochete) / Strain: B31 / synonym: Lyme disease spirochete

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferDetails: BSK-II liquid medium supplemented with 6% rabbit serum
GridDetails: freshly glow-discharged holey carbon grids
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Method: Bacterial culture was deposited onto freshly glow-discharged, holey carbon grids for 1 min. Grids were blotted with filter paper and then rapidly frozen in liquid ethane, using a homemade ...Method: Bacterial culture was deposited onto freshly glow-discharged, holey carbon grids for 1 min. Grids were blotted with filter paper and then rapidly frozen in liquid ethane, using a homemade gravity-driven plunger apparatus.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 170 K
DateJul 1, 2012
Image recordingNumber real images: 12963 / Average electron dose: 100 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe subvolumes of the flagellar motors were extracted computationally from the tomograms and were further aligned in 3D.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 39.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD, Raptor, Protomo / Number subtomograms used: 999
Final 3D classificationNumber classes: 4

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