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- EMDB-2222: Structure of the PilF DNA transformation ATPase (AMPPNP bound form) -

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Basic information

Entry
Database: EMDB / ID: EMD-2222
TitleStructure of the PilF DNA transformation ATPase (AMPPNP bound form)
Map dataPilF DNA Transformation ATPase (apoprotein form)
Sample
  • Sample: PilF ATPase from Thermus thermophilus
  • Protein or peptide: ATPase
KeywordsATPase / DNA transformation
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsCollins RF / Hassan D / Karuppiah V / Thistlethwaite A / Derrick JP
CitationJournal: Biochem J / Year: 2013
Title: Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus.
Authors: Richard F Collins / Darin Hassan / Vijaykumar Karuppiah / Angela Thistlethwaite / Jeremy P Derrick /
Abstract: Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus ...Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus thermophilus) is required for high transformation efficiency, but its mechanism of action is unknown. In the present study, we show that TtPilF is able to bind to both DNA and RNA. The structure of TtPilF was determined by cryoelectron microscopy in the presence and absence of the ATP analogue p[NH]ppA (adenosine 5'-[β,γ-imido]triphosphate), at 10 and 12 Å (1 Å=0.1 nm) resolutions respectively. It consists of two distinct N- and C-terminal regions, separated by a short stem-like structure. Binding of p[NH]ppA induces structural changes in the C-terminal domains, which are transmitted via the stem to the N-terminal domains. Molecular models were generated for the apoenzyme and p[NH]ppA-bound states in the C-terminal regions by docking of a model based on a crystal structure from a closely related enzyme. Analysis of DNA binding by electron microscopy, using gold labelling, localized the binding site to the N-terminal domains. The results suggest a model in which DNA uptake by TtPilF is powered by ATP hydrolysis, causing conformational changes in the C-terminal domains, which are transmitted via the stem to take up DNA into the cell.
History
DepositionOct 17, 2012-
Header (metadata) releaseNov 21, 2012-
Map releaseJan 9, 2013-
UpdateFeb 27, 2013-
Current statusFeb 27, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.83
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.83
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2222.png

Downloads & links

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Map

FileDownload / File: emd_2222.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPilF DNA Transformation ATPase (apoprotein form)
Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 0.83 / Movie #1: 0.83
Minimum - Maximum-0.36359513 - 1.84904575
Average (Standard dev.)0.03646536 (±0.18055071)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z384.000384.000384.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.3641.8490.036

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Supplemental data

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Sample components

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Entire : PilF ATPase from Thermus thermophilus

EntireName: PilF ATPase from Thermus thermophilus
Components
  • Sample: PilF ATPase from Thermus thermophilus
  • Protein or peptide: ATPase

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Supramolecule #1000: PilF ATPase from Thermus thermophilus

SupramoleculeName: PilF ATPase from Thermus thermophilus / type: sample / ID: 1000 / Details: Monodisperse sample / Oligomeric state: homohexamer / Number unique components: 1
Molecular weightTheoretical: 586 KDa

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Macromolecule #1: ATPase

MacromoleculeName: ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 586 KDa

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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: 6.5 / Details: 50mM Mes pH 6.5, 200mM NaCl and 10mM MgCl2
GridDetails: Freshly glow discharged 2-2 Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV
Method: continuously blotted for 4-5 seconds in a 90% humidity chamber before plunge freezing into liquid ethane at 22 degrees.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder: Gatan MSC / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 85 K
DateNov 1, 2011
Image recordingDigitization - Sampling interval: 3.5 µm / Number real images: 97 / Average electron dose: 20 e/Å2 / Details: Data were recorded on a Gatan 4Kx4K CCD / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: EMAN2 - each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 37000
DetailsSemi-automated picking as implemented within EMAN2

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