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- EMDB-20503: Subtomogram average of a purified Leptospira biflexa -fcpB mutant... -

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Basic information

Entry
Database: EMDB / ID: EMD-20503
TitleSubtomogram average of a purified Leptospira biflexa -fcpB mutant flagellum
Map dataMap of the Leptospira biflexa purified -fcpB flagellum, determined through subtomogram averaging with emClarity.
Sample
  • Complex: Flagellum
Biological speciesLeptospira biflexa serovar Patoc (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 18.4 Å
AuthorsBrady MR / Gibson KH / Sindelar CV
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Front Cell Infect Microbiol / Year: 2018
Title: FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete .
Authors: Elsio A Wunder / Leyla Slamti / David N Suwondo / Kimberley H Gibson / Zhiguo Shang / Charles V Sindelar / Felipe Trajtenberg / Alejandro Buschiazzo / Albert I Ko / Mathieu Picardeau /
Abstract: The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have ...The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete . By screening a library of random transposon mutants in the spirochete , we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene for flagellar-coiling protein B. is conserved in all members of the genus, but not present in other organisms including other spirochetes. Complementation of the mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are -specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.
History
DepositionJul 22, 2019-
Header (metadata) releaseMar 4, 2020-
Map releaseMar 25, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.01
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20503.png

Downloads & links

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Map

FileDownload / File: emd_20503.map.gz / Format: CCP4 / Size: 19.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the Leptospira biflexa purified -fcpB flagellum, determined through subtomogram averaging with emClarity.
Voxel sizeX=Y=Z: 4.97 Å
Density
Contour LevelBy AUTHOR: 4.01 / Movie #1: 4.01
Minimum - Maximum-12.255934 - 18.001131
Average (Standard dev.)-0.0000000001 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions171171171
Spacing171171171
CellA=B=C: 849.86993 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.974.974.97
M x/y/z171171171
origin x/y/z0.0000.0000.000
length x/y/z849.870849.870849.870
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ172172172
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS171171171
D min/max/mean-12.25618.001-0.000

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Supplemental data

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Half map: Half mask of the Leptospira biflexa purified -fcpB...

Fileemd_20503_half_map_1.map
AnnotationHalf mask of the Leptospira biflexa purified -fcpB flagellum, determined through subtomogram averaging with emClarity.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half mask of the Leptospira biflexa purified -fcpB...

Fileemd_20503_half_map_2.map
AnnotationHalf mask of the Leptospira biflexa purified -fcpB flagellum, determined through subtomogram averaging with emClarity.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Flagellum

EntireName: Flagellum
Components
  • Complex: Flagellum

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Supramolecule #1: Flagellum

SupramoleculeName: Flagellum / type: complex / ID: 1 / Parent: 0
Details: Purified periplasmic flagella from Leptospira biflexa serovar Patoc -fcpB mutant
Source (natural)Organism: Leptospira biflexa serovar Patoc (bacteria) / Location in cell: Periplasmic space

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 6.8 / Component - Formula: H2O
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: CONTINUOUS / Support film - #0 - Film thickness: 3.0 nm / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: The sample was glow-discharged with ArO2 for 6 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK III / Details: Blot time of 6 seconds, blot offset -1mm.
DetailsFlagella were purified from Leptospira biflexa serovar Patoc according to the method described from Wunder et al. 2016 (Molecular Microbiology). After the final centrifugation, the flagella were resuspended in 0.5 mL H2O, and approximately 0.2% sodium azide was added as a preservative. Flagella were stored at 4 degrees C.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 1.5 sec. / Average electron dose: 8.35 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus min: 2.5 µm / Nominal magnification: 14500
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER 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: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 18.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity
Details: emClarity was used to generate a final structure from the 10 best tomograms (that contained 14 filaments).
Number subtomograms used: 10
ExtractionNumber tomograms: 24 / Number images used: 5917 / Software - Name: RELION
CTF correctionSoftware - Name: IMOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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