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- EMDB-4194: Thermus thermophilus PilF ATPase (AMPPNP-bound form) -

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Basic information

Entry
Database: EMDB / ID: EMD-4194
TitleThermus thermophilus PilF ATPase (AMPPNP-bound form)
Map dataThermus thermophilus PilF ATPase (AMPPNP-bound form)
Sample
  • Complex: Thermus thermophilus PilF ATPase (AMPPNP-bound form)
    • Protein or peptide: Type IV pilus assembly protein PilF
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 HB8 (bacteria) / Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsDerrick JP / Collins RF
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
DepositionDec 13, 2017-
Header (metadata) releaseJan 17, 2018-
Map releaseAug 1, 2018-
UpdateOct 24, 2018-
Current statusOct 24, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.45
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6f8l
  • Surface level: 1.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4194.png

Downloads & links

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Map

FileDownload / File: emd_4194.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermus thermophilus PilF ATPase (AMPPNP-bound form)
Voxel sizeX=Y=Z: 1.78 Å
Density
Contour LevelBy AUTHOR: 1.45 / Movie #1: 1.45
Minimum - Maximum-4.813376 - 5.397968
Average (Standard dev.)0.021497991 (±0.28989422)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 341.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z341.760341.760341.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-4.8135.3980.021

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Supplemental data

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Sample components

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Entire : Thermus thermophilus PilF ATPase (AMPPNP-bound form)

EntireName: Thermus thermophilus PilF ATPase (AMPPNP-bound form)
Components
  • Complex: Thermus thermophilus PilF ATPase (AMPPNP-bound form)
    • Protein or peptide: Type IV pilus assembly protein PilF

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Supramolecule #1: Thermus thermophilus PilF ATPase (AMPPNP-bound form)

SupramoleculeName: Thermus thermophilus PilF ATPase (AMPPNP-bound form) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Type IV pilus assembly protein PilF

MacromoleculeName: Type IV pilus assembly protein PilF / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 101.021289 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSVLTIGDKR LGAALLDAGL LTDEELQRAL ERHREVGGSL AEVLVDMGLL SERRIAQTIE DRFGIPLVEL HRVEIPPKVK ALLPAEKAK ELKAIPFALD EEAGVVRVAF LNPLDTLSLE EVEDLTGLVV EPYQTTKSAF LYALAKHYPE LGLPVPPPPS G EGQKDLKL ...String:
MSVLTIGDKR LGAALLDAGL LTDEELQRAL ERHREVGGSL AEVLVDMGLL SERRIAQTIE DRFGIPLVEL HRVEIPPKVK ALLPAEKAK ELKAIPFALD EEAGVVRVAF LNPLDTLSLE EVEDLTGLVV EPYQTTKSAF LYALAKHYPE LGLPVPPPPS G EGQKDLKL GELLLQKGWI SREALEEALV EQEKTGDLLG RILVRKGLPE EALYRALAEQ KGLEFLESTE GIVPDPSAAL LL LRSDALR YGAVPIGFQN GEVEVVLSDP RHKEAVAQLL NRPARFYLAL PQAWEELFRR AYPQKNRLGE VLVQEGKLSR EAL KEALEV QKGLPRAKPL GEILVELGLA RPEDVEEALQ KQRRGGGRLE DTLVQSGKLR PEALAQAVAT QLGYPYVDPE EDPP DPGAP LLLPEDLCRR YGVFPHRLEG NRLVLLMKDP RNILALDDVR LALKRKGLNY EVAPAVATEA AITKLIERFY GKAEL SEIA KEFAKKQAEE EVPSPLELDE SAAQKFVKQV IREAFLQDAS DIHIEPRQND VQVRLRIDGA LRPYSTLPKG ALNAVI SVV KIMGGLNIAE KRLPQDGRVR YREGAIDVDL RLSTLPTVYG EKAVMRLLKK ASDIPEIEDL GFAPGVFERF KEVISKP YG IFLITGPTGS GKSFTTFSIL KRIATPDKNT QTIEDPVEYE IPGINQTQVN PQAGLTFARA LRAFLRQDPD IIMVGEIR D SETAKIATEA ALTGHLVIAT LHTNDAAQAI TRLDEMGVEP FNISAALIGV LSQRLVRRVC EHCKVEVKPD PETLRRLGL SEAEIQGARL YKGMGCERCG GTGYKGRYAI HELLVVDDEI RHAIVAGKSA TEIKEIARRK GMKTLREDGL YKALQGITTL EEVLARTIE AAAELALVPR GSSAHHHHHH HHHH

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2 and 5 % glycerol (v/v)
VitrificationCryogen name: ETHANE
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.5 µm / Calibrated defocus min: 1.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: k2 / Energy filter - Lower energy threshold: 20 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 BASE (4k x 4k) / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: EMAN, RELION)
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION / Number images used: 450000

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