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- PDB-6olm: CryoEM structure of PilT4 from Geobacter metallireducens with add... -

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Basic information

Entry
Database: PDB / ID: 6olm
TitleCryoEM structure of PilT4 from Geobacter metallireducens with added ATP: C6cccccc conformation
ComponentsTwitching motility pilus retraction ATPase
KeywordsMOTOR PROTEIN / ATPase / T4P / type iv pilus / motor
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Pilus retraction protein PilT/PilU / Bacterial type II secretion system protein E signature. / : / Type II/IV secretion system protein / Type II/IV secretion system protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Twitching motility pilus retraction ATPase
Similarity search - Component
Biological speciesGeobacter metallireducens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsMcCallum, M. / Howell, P.L.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Commun / Year: 2019
Title: Multiple conformations facilitate PilT function in the type IV pilus.
Authors: Matthew McCallum / Samir Benlekbir / Sheryl Nguyen / Stephanie Tammam / John L Rubinstein / Lori L Burrows / P Lynne Howell /
Abstract: Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by ...Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C symmetry; however, most of these ATPases crystallize with either C or C symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C C, and C conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.
History
DepositionApr 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 15, 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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Assembly

Deposited unit
A: Twitching motility pilus retraction ATPase
B: Twitching motility pilus retraction ATPase
C: Twitching motility pilus retraction ATPase
D: Twitching motility pilus retraction ATPase
E: Twitching motility pilus retraction ATPase
F: Twitching motility pilus retraction ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)260,34312
Polymers257,3006
Non-polymers3,0436
Water00
1


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

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Components

#1: Protein
Twitching motility pilus retraction ATPase


Mass: 42883.379 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter metallireducens (strain GS-15 / ATCC 53774 / DSM 7210) (bacteria)
Strain: GS-15 / ATCC 53774 / DSM 7210 / Gene: pilT-4, Gmet_1394 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / References: UniProt: Q39VU6
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*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: PilT Hexamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Geobacter metallireducens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET28a
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Homemade
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 35.7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2particle selection
4cryoSPARC2CTF correction
7UCSF Chimera8.6.8model fitting
9cryoSPARC2initial Euler assignment
10cryoSPARC2final Euler assignment
11cryoSPARC2classification
12cryoSPARC23D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43823 / Symmetry type: POINT
Atomic model buildingB value: 68.8 / Protocol: FLEXIBLE FIT
Details: Initial fitting in chimera, flexible fitting in Phenix-refine
Atomic model buildingPDB-ID: 60JX

60jx


Accession code: 60JX / Source name: PDB / Type: experimental model

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