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- PDB-6nrv: Cryo-EM reconstruction of CFA/I pili -

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Basic information

Entry
Database: PDB / ID: 6nrv
TitleCryo-EM reconstruction of CFA/I pili
ComponentsCFA/I fimbrial subunit B
KeywordsPROTEIN FIBRIL / Enterotoxigenic Escherichia coli (ETEC) / CFA/I pili / helical reconstruction
Function / homologyCFA/I fimbrial subunit E, pilin domain / Fimbrial major subunit, CS1-type / CS1 type fimbrial major subunit / pilus / Immunoglobulin-like / Sandwich / Mainly Beta / CFA/I fimbrial subunit B
Function and homology information
Biological speciesESCHERICHIA COLI ETEC O78:H12 (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsZheng, W. / Andersson, M. / Bullitt, E. / Egelman, E.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122510 United States
CitationJournal: IUCrJ / Year: 2019
Title: Cryo-EM structure of the CFA/I pilus rod.
Authors: Weili Zheng / Magnus Andersson / Narges Mortezaei / Esther Bullitt / Edward Egelman /
Abstract: Enterotoxigenic (ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization ...Enterotoxigenic (ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of class 5 pili. The helical quaternary structure of CFA/I can be unwound under tensile force and it has been shown that this mechanical property helps bacteria to withstand shear forces from fluid motion. We report in this work the CFA/I pilus structure at 4.3 Å resolution from electron cryomicroscopy (cryo-EM) data, and report details of the donor strand complementation. The CfaB pilins modeled into the cryo-EM map allow us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and chaperone-usher pili. In addition, from the model built using the EM structure we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament's structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and a point-mutated CFA/I with a propensity for unwinding. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target adhesion pili needed for initial attachment and sustained adhesion of ETEC.
History
DepositionJan 24, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Database references / Category: citation / citation_author
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: CFA/I fimbrial subunit B
B: CFA/I fimbrial subunit B
C: CFA/I fimbrial subunit B
D: CFA/I fimbrial subunit B
E: CFA/I fimbrial subunit B
F: CFA/I fimbrial subunit B
G: CFA/I fimbrial subunit B
H: CFA/I fimbrial subunit B
I: CFA/I fimbrial subunit B
J: CFA/I fimbrial subunit B
K: CFA/I fimbrial subunit B
L: CFA/I fimbrial subunit B
M: CFA/I fimbrial subunit B


Theoretical massNumber of molelcules
Total (without water)194,59513
Polymers194,59513
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CFA/I fimbrial subunit B / CFA/I antigen / CFA/I pilin / Colonization factor antigen I subunit B


Mass: 14968.839 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Source: (natural) ESCHERICHIA COLI ETEC O78:H12 (E. coli) / Strain: E7473 / References: UniProt: P0CK93

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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: Enterotoxigenic Escherichia coli (ETEC) CFA/I pili / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli ETEC H10407 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_2919: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN2particle selection
2EPUimage acquisition
4CTFFIND3CTF correction
5SPIDERCTF correction
8RosettaEMmodel fitting
10SPIDERinitial Euler assignment
13SPIDER3D reconstruction
14Cootmodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 113.3 ° / Axial rise/subunit: 8.6 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 117011
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97295 / Symmetry type: HELICAL
Atomic model buildingPDB-ID: 3F85

3f85


Accession code: 3F85 / Source name: PDB / Type: experimental model

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