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- PDB-6w6m: Single particle cryoEM structure of V. cholerae Type IV competenc... -

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Basic information

Entry
Database: PDB / ID: 6w6m
TitleSingle particle cryoEM structure of V. cholerae Type IV competence pilus secretin PilQ
ComponentsType IV pilus secretin PilQ family protein
KeywordsTRANSPORT PROTEIN / secretion system / outer membrane 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSazinsky, M.H. / Weaver, S.J.
Funding support United States, 1items
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 Report
SummaryFull reportAbout validation report
History
DepositionMar 17, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Type IV pilus secretin PilQ family protein
B: Type IV pilus secretin PilQ family protein
C: Type IV pilus secretin PilQ family protein
D: Type IV pilus secretin PilQ family protein
E: Type IV pilus secretin PilQ family protein
F: Type IV pilus secretin PilQ family protein
G: Type IV pilus secretin PilQ family protein
H: Type IV pilus secretin PilQ family protein
I: Type IV pilus secretin PilQ family protein
J: Type IV pilus secretin PilQ family protein
K: Type IV pilus secretin PilQ family protein
L: Type IV pilus secretin PilQ family protein
M: Type IV pilus secretin PilQ family protein
N: Type IV pilus secretin PilQ family protein


Theoretical massNumber of molelcules
Total (without water)874,42914
Polymers874,42914
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, single particle cryogenic electron microscopy
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area128550 Å2
ΔGint-448 kcal/mol
Surface area275400 Å2

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Components

#1: Protein
Type IV pilus secretin PilQ family protein


Mass: 62459.188 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Source: (natural) Vibrio cholerae (bacteria) / Strain: TND1751 / References: UniProt: A0A2V4P274

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vibrio cholerae Type IV competence pilus secretin PilQ
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.826 MDa / Experimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: TND1751
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris HCl1
2300 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.8 mg/ml
Details: The 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, ...Details: The 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
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco EasiGlow, 20 mA, 60 seconds / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K / Details: blot force -6, blot time 4 s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 30000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.7 sec. / Electron dose: 1.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3808
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correctionwhole micrographs
5RELIONCTF correctionPer particle CtfRefine
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
20PHENIX1.16-dev3549model refinement
Image processingDetails: 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.
CTF correctionDetails: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C14 (14 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100543 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00844226
ELECTRON MICROSCOPYf_angle_d0.84760046
ELECTRON MICROSCOPYf_dihedral_angle_d13.42527146
ELECTRON MICROSCOPYf_chiral_restr0.0537392
ELECTRON MICROSCOPYf_plane_restr0.0067798

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