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- PDB-6ejf: Thermus thermophilus PilF ATPase (apoprotein form) -

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Basic information

Entry
Database: PDB / ID: 6ejf
TitleThermus thermophilus PilF ATPase (apoprotein form)
Components(Type IV pilus assembly protein PilF) x 3
KeywordsMOTOR PROTEIN / type IV pilus / type II secretion / macromolecular machine
Function / homology
Function and homology information


ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Type II secretion system protein GspE, N-terminal / MshEN domain / Type II secretion system protein GspE, N-terminal superfamily / 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
Type IV pilus assembly ATPase PilB
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8 Å
AuthorsDerrick, J.P. / Collins, R.F.
CitationJournal: Sci Rep / Year: 2018
Title: Structural cycle of the Thermus thermophilus PilF ATPase: the powering of type IVa pilus assembly.
Authors: Richard Collins / Vijaykumar Karuppiah / C Alistair Siebert / Rana Dajani / Angela Thistlethwaite / Jeremy P Derrick /
Abstract: Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an ...Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an inner membrane complex of biogenesis proteins which, in turn, bind to nascent pilin subunits and mediate fiber assembly. Here we report the structural characterization of the PilF TFP assembly ATPase from Thermus thermophilus. The crystal structure of a recombinant C-terminal fragment of PilF revealed bound, unhydrolysed ATP, although the full length complex was enzymatically active. 3D reconstructions were carried out by single particle cryoelectron microscopy for full length apoprotein PilF and in complex with AMPPNP. The structure forms an hourglass-like shape, with the ATPase domains in one half and the N1 domains in the second half which, we propose, interact with the other pilus biogenesis components. Molecular models for both forms were generated: binding of AMPPNP causes an upward shift of the N1 domains towards the ATPase domains of ~8 Å. We advocate a model in which ATP hydrolysis is linked to displacement of the N1 domains which is associated with lifting pilin subunits out of the inner membrane, and provide the activation energy needed to form the pilus fiber.
History
DepositionSep 21, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
G: Type IV pilus assembly protein PilF
J: Type IV pilus assembly protein PilF
K: Type IV pilus assembly protein PilF
L: Type IV pilus assembly protein PilF
H: Type IV pilus assembly protein PilF
I: Type IV pilus assembly protein PilF
M: Type IV pilus assembly protein PilF
N: Type IV pilus assembly protein PilF
O: Type IV pilus assembly protein PilF
P: Type IV pilus assembly protein PilF
Q: Type IV pilus assembly protein PilF
R: Type IV pilus assembly protein PilF
A: Type IV pilus assembly protein PilF
B: Type IV pilus assembly protein PilF
C: Type IV pilus assembly protein PilF
D: Type IV pilus assembly protein PilF
E: Type IV pilus assembly protein PilF
F: Type IV pilus assembly protein PilF


Theoretical massNumber of molelcules
Total (without water)461,36618
Polymers461,36618
Non-polymers00
Water0
1


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

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Components

#1: Protein
Type IV pilus assembly protein PilF


Mass: 16249.751 Da / Num. of mol.: 6 / Fragment: UNP residues 330-475
Source method: isolated from a genetically manipulated source
Details: Domain is part of the larger protein but connectivity to other domains cannot be confirmed.
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: TTHA0364 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5SLC9
#2: Protein
Type IV pilus assembly protein PilF


Mass: 15440.708 Da / Num. of mol.: 6 / Fragment: UNP residues 163-299
Source method: isolated from a genetically manipulated source
Details: Domain is part of the larger protein but connectivity to other domains cannot be confirmed.
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: TTHA0364 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5SLC9
#3: Protein
Type IV pilus assembly protein PilF


Mass: 45203.906 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: TTHA0364 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5SLC9

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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: Thermus thermophilus PilF ATPase (AMPPNP-bound form) / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2 and 5 % glycerol (v/v)
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 4500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 45000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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