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- EMDB-47437: Consensus map of the CpaF compact structure with two ATPs and two... -

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Basic information

Entry
Database: EMDB / ID: EMD-47437
TitleConsensus map of the CpaF compact structure with two ATPs and two ADPs (Under-saturated ATP/ADP dataset)
Map data
Sample
  • Complex: Compact state of CpaF with two ATPs and four ADPs
    • Protein or peptide: CpaF
KeywordsATPase / MOTOR PROTEIN
Function / homology: / Type II/IV secretion system protein / Type II/IV secretion system protein / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / CpaF
Function and homology information
Biological speciesCaulobacter vibrioides (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYen IY / Howell PL
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-169053 Canada
CitationJournal: Nat Commun / Year: 2025
Title: Conformational changes in the motor ATPase CpaF facilitate a rotary mechanism of Tad pilus assembly.
Authors: Ian Y Yen / Gregory B Whitfield / John L Rubinstein / Lori L Burrows / Yves V Brun / P Lynne Howell /
Abstract: The type IV pilus family uses PilT/VirB11-like ATPases to rapidly assemble and disassemble pilin subunits. Among these, the tight adherence (Tad) pilus performs both functions using a single ...The type IV pilus family uses PilT/VirB11-like ATPases to rapidly assemble and disassemble pilin subunits. Among these, the tight adherence (Tad) pilus performs both functions using a single bifunctional ATPase, CpaF. Here, we determine three conformationally distinct structures of CpaF hexamers with varying nucleotide occupancies by cryo-electron microscopy. Analysis of these structures suggest ATP binding and hydrolysis expand and rotate the hexamer pore clockwise while subsequent ADP release contracts the ATPase. Truncation of the intrinsically disordered region of CpaF in Caulobacter crescentus equally reduces pilus extension and retraction events observed using fluorescence microscopy, but does not reduce ATPase activity. AlphaFold3 modeling suggests that CpaF and other motors of the type IV filament superfamily employ conserved secondary structural features to engage their respective platform proteins. From these data, we propose that CpaF uses a clockwise, rotary mechanism of catalysis to assemble a right-handed, helical Tad pilus, a process broadly applicable to other single motor systems.
History
DepositionOct 21, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47437.png

Downloads & links

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Map

FileDownload / File: emd_47437.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 288 pix.
= 296.64 Å		1.03 Å/pix.
x 288 pix.
= 296.64 Å		1.03 Å/pix.
x 288 pix.
= 296.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.226
Minimum - Maximum-0.506627 - 1.3228207
Average (Standard dev.)-0.00068255013 (±0.036925506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 296.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_47437_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_47437_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Compact state of CpaF with two ATPs and four ADPs

EntireName: Compact state of CpaF with two ATPs and four ADPs
Components
  • Complex: Compact state of CpaF with two ATPs and four ADPs
    • Protein or peptide: CpaF

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Supramolecule #1: Compact state of CpaF with two ATPs and four ADPs

SupramoleculeName: Compact state of CpaF with two ATPs and four ADPs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Hexameric assembly with each chain of CpaF missing the first 79 residues
Source (natural)Organism: Caulobacter vibrioides (bacteria) / Strain: NA1000
Molecular weightTheoretical: 278 KDa

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Macromolecule #1: CpaF

MacromoleculeName: CpaF / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides (bacteria) / Strain: NA1000
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DYYHATKTTI FNALLNTIDL SQLAQLDLKQ AGEEIRDIVA ELVAIKNVSM SVAEQEHLVQ DIINDVLGYG PLEPLLARDD IADIMVNGAH RVFIEVGGKV QLTNVRFRDN LQLMNICQRI VSQVGRRVDE SSPICDARLP DGSRVNVIAP PLALDGPTLT IRKFKKDKLT ...String:
DYYHATKTTI FNALLNTIDL SQLAQLDLKQ AGEEIRDIVA ELVAIKNVSM SVAEQEHLVQ DIINDVLGYG PLEPLLARDD IADIMVNGAH RVFIEVGGKV QLTNVRFRDN LQLMNICQRI VSQVGRRVDE SSPICDARLP DGSRVNVIAP PLALDGPTLT IRKFKKDKLT MKNLVEFASI SPEGARVLGV IGACRCNLVI SGGTGSGKTT LLNTMTAFID PTERVVTCED AAELQLQQPH VVRLETRPPN LEGSGAVTMR DLVKNCLRMR PERIIVGEVR GPEAFDLLQA MNTGHDGSMG TLHANSPREA ISRIESMITM GGYGLPSKTI KEMIVGSVDV IIQAARLRDG SRRITHITEV VGLEGDVIVT QDLFVYEITG EDEHGKVVGK HRSTGIARPR FWDRARYYGL ERELAEALDA AE

UniProtKB: CpaF

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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

Concentration5 mg/mL
BufferpH: 8
GridModel: Homemade / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 360 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
DetailsCHAPS detergent at a final concentration of 0.05% (w/v) was added to the buffer prior to vitrification

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 10576 / Average electron dose: 36.7 e/Å2
Details: 7576 movies are untilted and 3000 movies are tilted at 30 degrees.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2078699 / Details: Combined from both datasets
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.2) / Number images used: 226031
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.2)
FSC plot (resolution estimation)

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