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Yorodumi- EMDB-3022: Structure of the type IV pilus machinery from Thermus thermophilu... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3022 | |||||||||
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Title | Structure of the type IV pilus machinery from Thermus thermophilus in the closed state | |||||||||
Map data | Subtomogram average (from unfiltered tomograms) of the Thermus thermophilus type IV pilus machinery in the closed state. | |||||||||
Sample |
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Keywords | Electron cryo-tomography / subtomogram averaging / type IV pilus / DNA transporter / bacterial secretion / T. thermophilus | |||||||||
Biological species | Thermus thermophilus HB27 (bacteria) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 35.0 Å | |||||||||
Authors | Gold VAM / Salzer R / Averhoff B / Kuehlbrandt W | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3022.map.gz | 1.1 MB | EMDB map data format | |
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Header (meta data) | emd-3022-v30.xml emd-3022.xml | 9.2 KB 9.2 KB | Display Display | EMDB header |
Images | emd_3022.tif | 755.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3022 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3022 | HTTPS FTP |
-Validation report
Summary document | emd_3022_validation.pdf.gz | 201.5 KB | Display | EMDB validaton report |
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Full document | emd_3022_full_validation.pdf.gz | 200.5 KB | Display | |
Data in XML | emd_3022_validation.xml.gz | 4.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3022 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3022 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3022.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Subtomogram average (from unfiltered tomograms) of the Thermus thermophilus type IV pilus machinery in the closed state. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.417 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Type IV pilus machinery in the closed state (unfiltered)
Entire | Name: Type IV pilus machinery in the closed state (unfiltered) |
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Components |
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-Supramolecule #1000: Type IV pilus machinery in the closed state (unfiltered)
Supramolecule | Name: Type IV pilus machinery in the closed state (unfiltered) 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
Macromolecule | Name: Type IV pilus machinery / type: protein_or_peptide / ID: 1 / Recombinant expression: No |
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Source (natural) | Organism: Thermus thermophilus HB27 (bacteria) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 / Details: 20 mM Tris, 100 mM EDTA |
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Grid | Details: 300 mesh copper grid with quantifoil support film (R2/2), glow discharged |
Vitrification | Cryogen 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 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected on the K2 camera at magnification used for imaging |
Specialist optics | Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | Sep 27, 2014 |
Image recording | Category: 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated 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 stage | Specimen 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 |
-Image processing
Details | C12 symmetry was applied to the masked central PilQ core by rotation of each subvolume by 30 degrees (360 degrees/12) prior to alignment search. |
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Final reconstruction | Applied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 35.0 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: 3984 |