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- EMDB-3023: Structure of the type IV pilus machinery from Thermus thermophilu... -

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Basic information

Entry
Database: EMDB / ID: EMD-3023
TitleStructure of the type IV pilus machinery from Thermus thermophilus in the open state
Map dataSubtomogram average (from NAD filtered tomograms) of the Thermus thermophilus type IV pilus machinery in the open state.
Sample
  • Sample: Type IV pilus machinery in the open state (filtered)
  • Protein or peptide: Type IV pilus machinery
KeywordsElectron cryo-tomography / subtomogram averaging / type IV pilus / DNA transporter / bacterial secretion / T. thermophilus
Biological speciesThermus thermophilus HB27 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 45.0 Å
AuthorsGold VAM / Salzer R / Averhoff B / Kuehlbrandt W
CitationJournal: Elife / Year: 2015
Title: Structure of a type IV pilus machinery in the open and closed state.
Authors: Vicki A M Gold / Ralf Salzer / Beate Averhoff / Werner Kühlbrandt /
Abstract: Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion ...Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ~30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.
History
DepositionMay 21, 2015-
Header (metadata) releaseJun 10, 2015-
Map releaseJun 10, 2015-
UpdateJun 10, 2015-
Current statusJun 10, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 150.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 150.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3023.tif

Downloads & links

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Map

FileDownload / File: emd_3023.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average (from NAD filtered tomograms) of the Thermus thermophilus type IV pilus machinery in the open state.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.42 Å/pix.
x 110 pix.
= 925.87 Å		8.42 Å/pix.
x 60 pix.
= 505.02 Å		8.42 Å/pix.
x 60 pix.
= 505.02 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 8.417 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 150.900000000000006 / Movie #1: 150.9
Minimum - Maximum145.999984740000002 - 157.0
Average (Standard dev.)149.636398320000012 (±0.90361208)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-46-47-9
Dimensions6060110
Spacing6060110
CellA: 505.02 Å / B: 505.02 Å / C: 925.87 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.4178.4178.417
M x/y/z6060110
origin x/y/z0.0000.0000.000
length x/y/z505.020505.020925.870
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-47-46-9
NC/NR/NS6060110
D min/max/mean146.000157.000149.636

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Supplemental data

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Sample components

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Entire : Type IV pilus machinery in the open state (filtered)

EntireName: Type IV pilus machinery in the open state (filtered)
Components
  • Sample: Type IV pilus machinery in the open state (filtered)
  • Protein or peptide: Type IV pilus machinery

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Supramolecule #1000: Type IV pilus machinery in the open state (filtered)

SupramoleculeName: Type IV pilus machinery in the open state (filtered) / type: sample / ID: 1000
Oligomeric state: The type IV pilus machinery is a hetero-oligomer, formed of at least 10 different proteins
Number unique components: 1

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Macromolecule #1: Type IV pilus machinery

MacromoleculeName: Type IV pilus machinery / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus HB27 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: 20 mM Tris, 100 mM EDTA
GridDetails: 300 mesh copper grid with quantifoil support film (R2/2), glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 20 % / Instrument: HOMEMADE PLUNGER
Method: Cell solutions were mixed 1:1 (v/v) with 10 nm ProteinA-gold particles. 3 microlitres of sample were applied to grids and blotted on one side for ~5s before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected on the K2 camera at magnification used for imaging
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateSep 26, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 140 e/Å2
Details: Each image in every tilt series is a drift corrected sum of 3-5 frames
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 11640 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 33000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsC12 symmetry was applied to the masked central PilQ core by rotation of each subvolume by 30 degrees (360 degrees/12) prior to alignment search.
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 45.0 Å / Resolution method: OTHER / Software - Name: IMOD, (ETOMO, &, PEET), Spider
Details: Resolution estimate was calculated using a mask to exclude the membrane and peptidoglycan
Number subtomograms used: 312

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