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Yorodumi- PDB-6w6m: Single particle cryoEM structure of V. cholerae Type IV competenc... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6w6m | ||||||
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Title | Single particle cryoEM structure of V. cholerae Type IV competence pilus secretin PilQ | ||||||
Components | Type IV pilus secretin PilQ family protein | ||||||
Keywords | TRANSPORT PROTEIN / secretion system / outer membrane 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 | ||||||
Biological species | Vibrio cholerae (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||
Authors | Sazinsky, M.H. / Weaver, S.J. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 6w6m.cif.gz | 1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb6w6m.ent.gz | 849.8 KB | Display | PDB format |
PDBx/mmJSON format | 6w6m.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6w6m_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 6w6m_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 6w6m_validation.xml.gz | 161.5 KB | Display | |
Data in CIF | 6w6m_validation.cif.gz | 241.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/w6/6w6m ftp://data.pdbj.org/pub/pdb/validation_reports/w6/6w6m | HTTPS FTP |
-Related structure data
Related structure data | 21559MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: 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, UniProt: Q9KNV0*PLUS |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Vibrio cholerae Type IV competence pilus secretin PilQ Type: COMPLEX / Entity ID: all / Source: NATURAL | |||||||||||||||
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Molecular weight | Value: 0.826 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Vibrio cholerae (bacteria) / Strain: TND1751 | |||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES 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 | |||||||||||||||
Specimen support | Details: Pelco EasiGlow, 20 mA, 60 seconds / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K / Details: blot force -6, blot time 4 s |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 30000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
-Processing
Software | Name: PHENIX / Version: 1.16_3549: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | 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. | |||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. 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. | |||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C14 (14 fold cyclic) | |||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100543 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | |||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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