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- EMDB-3882: Thermus thermophilus PilF ATPase (apoprotein form) -

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Basic information

Entry
Database: EMDB / ID: EMD-3882
TitleThermus thermophilus PilF ATPase (apoprotein form)
Map dataThermus thermophilus PilF ATPase (apoprotein form)
Sample
  • Complex: Thermus thermophilus PilF ATPase (AMPPNP-bound form)
    • Protein or peptide: Type IV pilus assembly protein PilF
    • Protein or peptide: Type IV pilus assembly protein PilF
    • 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
DepositionSep 21, 2017-
Header (metadata) releaseAug 1, 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: 1.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ejf
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3882.png

Downloads & links

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Map

FileDownload / File: emd_3882.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermus thermophilus PilF ATPase (apoprotein form)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.6 Å/pix.
x 192 pix.
= 307.2 Å		1.6 Å/pix.
x 192 pix.
= 307.2 Å		1.6 Å/pix.
x 192 pix.
= 307.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.9 / Movie #1: 1.9
Minimum - Maximum-4.042804 - 6.4494243
Average (Standard dev.)0.032942884 (±0.3669116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 307.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.61.61.6
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z307.200307.200307.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-4.0436.4490.033

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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
    • Protein or peptide: Type IV pilus assembly protein PilF
    • 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
Details: Domain is part of the larger protein but connectivity to other domains cannot be confirmed.
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 16.249751 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
LPRAKPLGEI LVELGLARPE DVEEALQKQR RGGGRLEDTL VQSGKLRPEA LAQAVATQLG YPYVDPEEDP PDPGAPLLLP EDLCRRYGV FPHRLEGNRL VLLMKDPRNI LALDDVRLAL KRKGLNYEVA PAVATEAAIT KLIERFY

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Macromolecule #2: Type IV pilus assembly protein PilF

MacromoleculeName: Type IV pilus assembly protein PilF / type: protein_or_peptide / ID: 2
Details: Domain is part of the larger protein but connectivity to other domains cannot be confirmed.
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 15.440708 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QKDLKLGELL LQKGWISREA LEEALVEQEK TGDLLGRILV RKGLPEEALY RALAEQKGLE FLESTEGIVP DPSAALLLLR SDALRYGAV PIGFQNGEVE VVLSDPRHKE AVAQLLNRPA RFYLALPQAW EELFRRAY

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Macromolecule #3: Type IV pilus assembly protein PilF

MacromoleculeName: Type IV pilus assembly protein PilF / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 45.203906 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SAAQKFVKQV IREAFLQDAS DIHIEPRQND VQVRLRIDGA LRPYSTLPKG ALNAVISVVK IMGGLNIAEK RLPQDGRVRY REGAIDVDL RLSTLPTVYG EKAVMRLLKK ASDIPEIEDL GFAPGVFERF KEVISKPYGI FLITGPTGSG KSFTTFSILK R IATPDKNT ...String:
SAAQKFVKQV IREAFLQDAS DIHIEPRQND VQVRLRIDGA LRPYSTLPKG ALNAVISVVK IMGGLNIAEK RLPQDGRVRY REGAIDVDL RLSTLPTVYG EKAVMRLLKK ASDIPEIEDL GFAPGVFERF KEVISKPYGI FLITGPTGSG KSFTTFSILK R IATPDKNT QTIEDPVEYE IPGINQTQVN PQAGLTFARA LRAFLRQDPD IIMVGEIRDS ETAKIATEAA LTGHLVIATL HT NDAAQAI TRLDEMGVEP FNISAALIGV LSQRLVRRVC EHCKVEVKPD PETLRRLGLS EAEIQGARLY KGMGCERCGG TGY KGRYAI HELLVVDDEI RHAIVAGKSA TEIKEIARRK GMKTLREDGL YKALQGITTL EEVLARTIEA AAELALVPRG SSAH HHHHH 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
Image recordingFilm or detector model: GATAN K2 BASE (4k x 4k) / Average electron dose: 45.0 e/Å2
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
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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 45000
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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