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- EMDB-8847: Cryo-EM structure of B. subtilis flagellar filaments N226Y -

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Basic information

Entry
Database: EMDB / ID: EMD-8847
TitleCryo-EM structure of B. subtilis flagellar filaments N226Y
Map dataCryo-EM structure of B. subtilis flagellar filaments N226Y
Sample
  • Complex: Bacillus subtilis flagella filament
    • Protein or peptide: Flagellin
Keywordsbacteria flagella / helical polymers / cryo-EM / PROTEIN FIBRIL
Function / homology
Function and homology information


bacterial-type flagellum / structural molecule activity / extracellular region
Similarity search - Function
Flagellin, C-terminal domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region
Similarity search - Domain/homology
Flagellin / Flagellin
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 3.8 Å
AuthorsWang F / Burrage AM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122510 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM093030 United States
CitationJournal: Nat Commun / Year: 2017
Title: A structural model of flagellar filament switching across multiple bacterial species.
Authors: Fengbin Wang / Andrew M Burrage / Sandra Postel / Reece E Clark / Albina Orlova / Eric J Sundberg / Daniel B Kearns / Edward H Egelman /
Abstract: The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we ...The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B. subtilis, P. aeruginosa and Salmonella enterica. Using the detailed information about the molecular interactions in two filament states, we successfully predict point mutations that shift the equilibrium between those two states. Further, we observe the dimerization of P. aeruginosa outer domains without any perturbation of the conserved interior of the filament. Our results give new insights into how the flagellin sequence has been "tuned" over evolution.Bacterial flagellar filaments are composed almost entirely of a single protein-flagellin-which can switch between different supercoiled states in a highly cooperative manner. Here the authors present near-atomic resolution cryo-EM structures of nine flagellar filaments, and begin to shed light on the molecular basis of filament switching.
History
DepositionJul 24, 2017-
Header (metadata) releaseAug 30, 2017-
Map releaseOct 25, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wjt
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5wjt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8847.png

Downloads & links

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Map

FileDownload / File: emd_8847.map.gz / Format: CCP4 / Size: 250 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of B. subtilis flagellar filaments N226Y
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 1000 pix.
= 1050. Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.02 / Movie #1: 0.06
Minimum - Maximum-0.09178648 - 0.21849495
Average (Standard dev.)0.0027166572 (±0.016780777)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-500
Dimensions2562561000
Spacing2562561000
CellA: 268.8 Å / B: 268.8 Å / C: 1050.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z2562561000
origin x/y/z0.0000.0000.000
length x/y/z268.800268.8001050.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-500
NC/NR/NS2562561000
D min/max/mean-0.0920.2180.003

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Supplemental data

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Sample components

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Entire : Bacillus subtilis flagella filament

EntireName: Bacillus subtilis flagella filament
Components
  • Complex: Bacillus subtilis flagella filament
    • Protein or peptide: Flagellin

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Supramolecule #1: Bacillus subtilis flagella filament

SupramoleculeName: Bacillus subtilis flagella filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: Flagellin

MacromoleculeName: Flagellin / type: protein_or_peptide / ID: 1 / Number of copies: 41 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 32.710355 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MRINHNIAAL NTLNRLSSNN SASQKNMEKL SSGLRINRAG DDAAGLAISE KMRGQIRGLE MASKNSQDGI SLIQTAEGAL TETHAILQR VRELVVQAGN TGTQDKATDL QSIQDEISAL TDEIDGISNR TEFNGKKLLD GTYKVDTATP ANQKNLVFQI G ANATQQIS ...String:
MRINHNIAAL NTLNRLSSNN SASQKNMEKL SSGLRINRAG DDAAGLAISE KMRGQIRGLE MASKNSQDGI SLIQTAEGAL TETHAILQR VRELVVQAGN TGTQDKATDL QSIQDEISAL TDEIDGISNR TEFNGKKLLD GTYKVDTATP ANQKNLVFQI G ANATQQIS VNIEDMGADA LGIKEADGSI AALHSVNDLD VTKFADNAAD CADIGFDAQL KVVDEAIYQV SSQRAKLGAV QN RLEHTIN NLSASGENLT AAESRIRDVD MAKEMSEFTK NNILSQASQA MLAQANQQPQ NVLQLLR

UniProtKB: Flagellin

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 6.8 / Details: Imidazole buffer
StainingType: NEGATIVE / Material: negative stain
GridPretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 2.0 sec. / Average electron dose: 20.0 e/Å2
Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the ...Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the estimation of the CTF as well as for boxing filaments. Only the first two parts were used for the reconstruction (~5 electrons per Angstrom^2).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 4.64 Å
Applied symmetry - Helical parameters - Δ&Phi: 65.83 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: SPIDER / Details: model-map FSC 0.38 cut-off / Number images used: 72005
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-5wjt:
Cryo-EM structure of B. subtilis flagellar filaments N226Y

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