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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 dataType IV competence pilus secretin PilQ Relion PostProcess map (postprocess.mrc)
Sample
  • Complex: Vibrio cholerae Type IV competence pilus secretin PilQ
    • Protein or peptide: Type IV pilus secretin PilQ family protein
Keywordssecretion system / outer membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


pilus assembly / protein secretion by the type II secretion system / protein secretion / cell outer membrane
Similarity search - Function
Type IV pilus secretin PilQ / Secretin/TonB, short N-terminal domain / Secretin and TonB N terminus short domain / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
Type IV pilus secretin PilQ family protein / Fimbrial assembly protein
Similarity search - Component
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.
History
DepositionMar 17, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing 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
  • Download
  • 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

emd_21559.png

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)
AnnotationType IV competence pilus secretin PilQ Relion PostProcess map (postprocess.mrc)
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.)0.00008279121 (±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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Mask #1

Fileemd_21559_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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
Components
  • Complex: Vibrio cholerae Type IV competence pilus secretin PilQ
    • Protein or peptide: Type IV pilus secretin PilQ family protein

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Supramolecule #1: Vibrio cholerae Type IV competence pilus secretin PilQ

SupramoleculeName: Vibrio cholerae Type IV competence pilus secretin PilQ
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: TND1751
Molecular weightTheoretical: 826 KDa

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Macromolecule #1: Type IV pilus secretin PilQ family protein

MacromoleculeName: Type IV pilus secretin PilQ family protein / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: TND1751
Molecular weightTheoretical: 62.459188 KDa
SequenceString: MRNGLKTYVA QTWLTLWVGL ALCASSTVFS AEFATTNQLE NIDFRVNKEK AAVLIVELAS PSAVVDVQKV QEGLNIELLK TDVADDKLY LLDVKDFSTP VESVEVFRKA PSTQLVVTVD GEFQHDYTLK GKYLEVVISK LKADEKPKPK SVLEKEGKLI S INFQDIPV ...String:
MRNGLKTYVA QTWLTLWVGL ALCASSTVFS AEFATTNQLE NIDFRVNKEK AAVLIVELAS PSAVVDVQKV QEGLNIELLK TDVADDKLY LLDVKDFSTP VESVEVFRKA PSTQLVVTVD GEFQHDYTLK GKYLEVVISK LKADEKPKPK SVLEKEGKLI S INFQDIPV RNVLQLIADY NGFNLVVSDS VVGNLTLRLD GVPWQQVLDI ILQVKGLDKR VDGNVILIAP KEELDLREKQ AL EKARLAE ELGDLKSEII KINFAKASDI AAMIGGEGNV NMLSERGSIS IDERTNSLLI RELPDNIAVI REIIESLDIP VKQ VQIEAR IVTVKEGNLE ELGVRWGVMS TNGSHSVGGS IESNLWQKGL LADDEFPVDE FLNVNLASTS ANASSIAFQV AKLG SGTLL DLELSALQNE SKAEIISSPR LITTNKQPAY IEQGTEIPYL ESSSSGASTV AFKKAVLSLK VTPQITPDNR LVLDL SVTQ DRRGETVKTG TGEAVSIDTQ RIGTQVLVNN GETVVLGGIF QHSINNSVDK VPLLGDLPVL GALFRRTYEQ MGKSEL LIF VTPKVVIQ

UniProtKB: Type IV pilus secretin PilQ family protein

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris HCl
300.0 mMsodium chlorideNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: Pelco EasiGlow, 20 mA, 60 seconds
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot force -6, blot time 4 s.
DetailsThe protein was purified using Ni NTA agarose beads (Anatrace, SUPER-NINTA25) and concentrated to ~1 mg/mL. PilQ was then exchanged into Amphipol A8-35 (0.585 mg for a 3:1 ratio, Anatrace, A835). BioBeads were used to remove excess DDM and the sample was concentrated to ~0.8 mg/mL

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 30.0 µm / Nominal defocus min: 10.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3808 / Average exposure time: 3.7 sec. / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 252319
Details: CryoSPARC blob picking on 2,510 micrographs yielded 3,100,353 potential particles. After inspection in the cryoSPARC GUI, the 252,319 particles were extracted.
Startup modelType of model: OTHER / Details: cryoSPARC ab initio initial model generation
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C14 (14 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 100543
DetailsMotionCor2 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.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6w6m:
Single particle cryoEM structure of V. cholerae Type IV competence pilus secretin PilQ

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