+Open data
-Basic information
Entry | Database: PDB / ID: 5wc3 | ||||||
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Title | SpoIIIAG | ||||||
Components | SpoIIIAG, Stage III sporulation engulfment assemblyprotein | ||||||
Keywords | PROTEIN TRANSPORT / Secretion system / Sporulation channel / Ring Building Motif (RBM) | ||||||
Function / homology | Sporulation stage III, protein AG / membrane => GO:0016020 / Stage III sporulation engulfment assemblyprotein Function and homology information | ||||||
Biological species | Bacillus subtilis BEST7613 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
Authors | Zeytuni, N. / Hong, C. / Worrall, L.J. / Huang, R.K. / Yu, Z. / Strynadka, N.C.J. | ||||||
Funding support | Canada, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2017 Title: Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in . Authors: Natalie Zeytuni / Chuan Hong / Kelly A Flanagan / Liam J Worrall / Kate A Theiltges / Marija Vuckovic / Rick K Huang / Shawn C Massoni / Amy H Camp / Zhiheng Yu / Natalie C Strynadka / Abstract: Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the ...Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5wc3.cif.gz | 1.5 MB | Display | PDBx/mmCIF format |
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PDB format | pdb5wc3.ent.gz | 1.2 MB | Display | PDB format |
PDBx/mmJSON format | 5wc3.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5wc3_validation.pdf.gz | 913.4 KB | Display | wwPDB validaton report |
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Full document | 5wc3_full_validation.pdf.gz | 977.8 KB | Display | |
Data in XML | 5wc3_validation.xml.gz | 107.5 KB | Display | |
Data in CIF | 5wc3_validation.cif.gz | 136.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wc/5wc3 ftp://data.pdbj.org/pub/pdb/validation_reports/wc/5wc3 | HTTPS FTP |
-Related structure data
Related structure data | 8795MC 8797C 8798C 8800C 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: 19729.086 Da / Num. of mol.: 30 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Bacillus subtilis BEST7613 (bacteria) / Gene: spoIIIAG, BEST7613_3991 / Plasmid: pET28 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: L8AN07 |
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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: SpoIIIAG / Type: COMPLEX / Details: residues 55-229 / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.58 MDa / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: Bacillus subtilis (bacteria) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pET28 | |||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Cs corrector |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 37037 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 2100 nm / Cs: 0.01 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K |
Image recording | Average exposure time: 0.2 sec. / Electron dose: 1.1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2876 |
EM imaging optics | Energyfilter name: Gatan GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV |
Image scans | Sampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 50 / Used frames/image: 1-50 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 770000 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C30 (30 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55000 / Algorithm: FOURIER SPACE / Symmetry type: POINT |