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- PDB-5wjw: Cryo-EM structure of B. subtilis flagellar filaments H84R -

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Basic information

Entry
Database: PDB / ID: 5wjw
TitleCryo-EM structure of B. subtilis flagellar filaments H84R
ComponentsFlagellin
KeywordsPROTEIN FIBRIL / bacteria flagella / helical polymers / cryo-EM
Function / homologyFlagellin, C-terminal domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region / bacterial-type flagellum / structural molecule activity / extracellular region / Flagellin
Function and homology information
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / negative staining / cryo EM / Resolution: 4.4 Å
AuthorsWang, F. / Burrage, A.M. / Orlova, A. / Kearns, D.B. / Egelman, E.H.
Funding support United States, 2items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Flagellin
B: Flagellin
C: Flagellin
D: Flagellin
E: Flagellin
F: Flagellin
G: Flagellin
H: Flagellin
I: Flagellin
J: Flagellin
K: Flagellin
L: Flagellin
M: Flagellin
N: Flagellin
O: Flagellin
P: Flagellin
Q: Flagellin
R: Flagellin
S: Flagellin
T: Flagellin
U: Flagellin
V: Flagellin
W: Flagellin
X: Flagellin
Y: Flagellin
Z: Flagellin
a: Flagellin
b: Flagellin
c: Flagellin
d: Flagellin
e: Flagellin
f: Flagellin
g: Flagellin
h: Flagellin
i: Flagellin
j: Flagellin
k: Flagellin
l: Flagellin
m: Flagellin
n: Flagellin
o: Flagellin


Theoretical massNumber of molelcules
Total (without water)1,339,89441
Polymers1,339,89441
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, helical filament was observed by negative staining and Cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area291330 Å2
ΔGint-1075 kcal/mol
Surface area426020 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 20 / Rise per n subunits: 4.64 Å / Rotation per n subunits: 65.81 °)

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Components

#1: Protein ...
Flagellin


Mass: 32680.332 Da / Num. of mol.: 41 / Mutation: H84R,T209C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: B4417_3365 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A162QQD4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Bacillus subtilis flagella filament / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 6.8 / Details: Imidazole buffer
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES
EM stainingType: NEGATIVE / Material: negative stain
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 3 sec. / Electron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)
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).
Image scansMovie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: dev_2439: / Classification: refinement
EM software
IDNameCategory
1EMAN2particle selection
2EPUimage acquisition
4CTFFIND3CTF correction
7Rosettamodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11SPIDERinitial Euler assignment
12SPIDERfinal Euler assignment
13SPIDERclassification
14SPIDER3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 65.81 ° / Axial rise/subunit: 4.64 Å / Axial symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: OTHER / Num. of particles: 58771 / Algorithm: BACK PROJECTION / Details: model-map FSC 0.38 cut-off / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
RefinementHighest resolution: 4.4 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006185566
ELECTRON MICROSCOPYf_angle_d1.148335011
ELECTRON MICROSCOPYf_dihedral_angle_d7.67373308
ELECTRON MICROSCOPYf_chiral_restr0.04614965
ELECTRON MICROSCOPYf_plane_restr0.00331447

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