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- PDB-8rkl: TadA/CpaF nucleotide free -

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Basic information

Entry
Database: PDB / ID: 8rkl
TitleTadA/CpaF nucleotide free
ComponentsPilus assembly ATPase CpaF
KeywordsMOTOR PROTEIN / secretion / ATPase / Apo
Function / homology: / Type II/IV secretion system protein / Type II/IV secretion system protein / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / Pilus assembly ATPase CpaF
Function and homology information
Biological speciesCaulobacter vibrioides NA1000 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsHohl, M. / Low, H.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Bidirectional pilus processing in the Tad pilus system motor CpaF.
Authors: Michael Hohl / Emma J Banks / Max P Manley / Tung B K Le / Harry H Low /
Abstract: The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which ...The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.
History
DepositionDec 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pilus assembly ATPase CpaF
B: Pilus assembly ATPase CpaF
C: Pilus assembly ATPase CpaF
D: Pilus assembly ATPase CpaF
E: Pilus assembly ATPase CpaF
F: Pilus assembly ATPase CpaF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)279,74410
Polymers278,7916
Non-polymers9534
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Pilus assembly ATPase CpaF


Mass: 46465.199 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caulobacter vibrioides NA1000 (bacteria)
Gene: cpaF, CCNA_03037 / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: A0A0H3CDS2
#2: Chemical
ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES


Mass: 238.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: hexamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria)
Source (recombinant)Organism: Escherichia coli MC1061 (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_4933: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51697 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00518846
ELECTRON MICROSCOPYf_angle_d0.58325568
ELECTRON MICROSCOPYf_dihedral_angle_d11.466916
ELECTRON MICROSCOPYf_chiral_restr0.0453036
ELECTRON MICROSCOPYf_plane_restr0.0053390

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