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- EMDB-21559: Single particle cryoEM structure of V. cholerae Type IV competenc... -

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Basic information

Entry
Database: EMDB / ID: EMD-21559
TitleSingle particle cryoEM structure of V. cholerae Type IV competence pilus secretin PilQ
Map data
SampleVibrio cholerae Type IV competence pilus secretin PilQ
  • Type IV pilus secretin PilQ family protein
Function / homology
Function and homology information


protein secretion / cell outer membrane
GspD/PilQ family / Type II secretion system protein GspD, conserved site / Type II/III secretion system / NolW-like / Secretin/TonB, short N-terminal domain / Type IV pilus secretin PilQ / NolW-like superfamily
Type IV pilus secretin PilQ family protein
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWeaver SJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128674 United States
CitationJournal: Nat Commun / Year: 2020
Title: CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae.
Authors: Sara J Weaver / Davi R Ortega / Matthew H Sazinsky / Triana N Dalia / Ankur B Dalia / Grant J Jensen /
Abstract: Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of ...Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
Validation ReportPDB-ID: 6w6m

SummaryFull reportAbout validation report
History
DepositionMar 17, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateOct 21, 2020-
Current statusOct 21, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w6m
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21559.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 400 pix.
= 441.6 Å
1.1 Å/pix.
x 400 pix.
= 441.6 Å
1.1 Å/pix.
x 400 pix.
= 441.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.104 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.10304258 - 0.21682216
Average (Standard dev.)0.0000827912 (±0.0044978512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 441.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1041.1041.104
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z441.600441.600441.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ161186271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.1030.2170.000

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Supplemental data

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Segmentation: #1

Fileemd_21559_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Type IV competence pilus secretin PilQ Relion Refine3D...

Fileemd_21559_additional_1.map
AnnotationType IV competence pilus secretin PilQ Relion Refine3D output map (run_class001.mrc)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Type IV competence pilus secretin PilQ Relion Refine3D half map 1

Fileemd_21559_half_map_1.map
AnnotationType IV competence pilus secretin PilQ Relion Refine3D half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Type IV competence pilus secretin PilQ Relion Refine3D half map 2

Fileemd_21559_half_map_2.map
AnnotationType IV competence pilus secretin PilQ Relion Refine3D half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Vibrio cholerae Type IV competence pilus secretin PilQ

EntireName: Vibrio cholerae Type IV competence pilus secretin PilQ
Number of components: 2

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Component #1: protein, Vibrio cholerae Type IV competence pilus secretin PilQ

ProteinName: Vibrio cholerae Type IV competence pilus secretin PilQ
Recombinant expression: No
MassTheoretical: 826 kDa
SourceSpecies: Vibrio cholerae (bacteria) / Strain: TND1751

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Component #2: protein, Type IV pilus secretin PilQ family protein

ProteinName: Type IV pilus secretin PilQ family protein / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 62.459188 kDa
SourceSpecies: Vibrio cholerae (bacteria) / Strain: TND1751

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.8 mg/mL / pH: 8
Support filmPelco EasiGlow, 20 mA, 60 seconds
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293.15 K / Humidity: 100 % / Details: blot force -6, blot time 4 s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 10000.0 - 30000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 3808

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C14 (14 fold cyclic) / Number of projections: 100543
Details: MotionCor2 was used for motion correction and dose weighting of 3,808 movies. CTF correction was used to evaluate micrograph quality. 2,510 micrographs were selected for particle picking.
3D reconstructionSoftware: RELION
CTF correction: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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