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- EMDB-19275: TadA/CpaF with AMPPNP -

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Basic information

Entry
Database: EMDB / ID: EMD-19275
TitleTadA/CpaF with AMPPNP
Map data
Sample
  • Complex: Hexameric complex of TadA
    • Protein or peptide: Pilus assembly ATPase CpaF
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Keywordssecretion / ATPase / MOTOR PROTEIN
Function / homology: / Type II/IV secretion system protein / Type II/IV secretion system protein / P-loop containing nucleoside triphosphate hydrolase / Pilus assembly ATPase CpaF
Function and homology information
Biological speciesCaulobacter vibrioides NA1000 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHohl M / Low H
Funding support United Kingdom, 1 items
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 24, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19275.png

Downloads & links

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Map

FileDownload / File: emd_19275.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-25.184898 - 46.518256999999998
Average (Standard dev.)0.000000000006466 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Hexameric complex of TadA

EntireName: Hexameric complex of TadA
Components
  • Complex: Hexameric complex of TadA
    • Protein or peptide: Pilus assembly ATPase CpaF
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Hexameric complex of TadA

SupramoleculeName: Hexameric complex of TadA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria)

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Macromolecule #1: Pilus assembly ATPase CpaF

MacromoleculeName: Pilus assembly ATPase CpaF / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides NA1000 (bacteria)
Molecular weightTheoretical: 46.465199 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: DYYHATKTTI FNALLNTIDL SQLAQLDLKQ AGEEIRDIVA ELVAIKNVSM SVAEQEHLVQ DIINDVLGYG PLEPLLARDD IADIMVNGA HRVFIEVGGK VQLTNVRFRD NLQLMNICQR IVSQVGRRVD ESSPICDARL PDGSRVNVIA PPLALDGPTL T IRKFKKDK ...String:
DYYHATKTTI FNALLNTIDL SQLAQLDLKQ AGEEIRDIVA ELVAIKNVSM SVAEQEHLVQ DIINDVLGYG PLEPLLARDD IADIMVNGA HRVFIEVGGK VQLTNVRFRD NLQLMNICQR IVSQVGRRVD ESSPICDARL PDGSRVNVIA PPLALDGPTL T IRKFKKDK LTMKNLVEFA SISPEGARVL GVIGACRCNL VISGGTGSGK TTLLNTMTAF IDPTERVVTC EDAAELQLQQ PH VVRLETR PPNLEGSGAV TMRDLVKNCL RMRPERIIVG EVRGPEAFDL LQAMNTGHDG SMGTLHANSP REAISRIESM ITM GGYGLP SKTIKEMIVG SVDVIIQAAR LRDGSRRITH ITEVVGLEGD VIVTQDLFVY EITGEDEHGK VVGKHRSTGI ARPR FWDRA RYYGLERELA EALDAAEA

UniProtKB: Pilus assembly ATPase CpaF

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID

MacromoleculeName: 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / type: ligand / ID: 3 / Number of copies: 4 / Formula: EPE
Molecular weightTheoretical: 238.305 Da
Chemical component information

ChemComp-EPE:
4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / pH buffer*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73200
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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