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- PDB-5wc3: SpoIIIAG -

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Basic information

Entry
Database: PDB / ID: 5wc3
TitleSpoIIIAG
ComponentsSpoIIIAG, Stage III sporulation engulfment assemblyprotein
KeywordsPROTEIN TRANSPORT / Secretion system / Sporulation channel / Ring Building Motif (RBM)
Function / homologySporulation stage III, protein AG / membrane => GO:0016020 / Stage III sporulation engulfment assemblyprotein
Function and homology information
Biological speciesBacillus subtilis BEST7613 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZeytuni, N. / Hong, C. / Worrall, L.J. / Huang, R.K. / Yu, Z. / Strynadka, N.C.J.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: 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
DepositionJun 29, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 16, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Aug 30, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
B: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
C: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
D: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
E: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
F: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
G: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
H: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
I: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
J: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
K: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
L: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
M: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
N: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
O: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
P: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
Q: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
R: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
S: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
T: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
U: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
V: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
W: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
X: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
Y: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
Z: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
0: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
1: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
2: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
3: SpoIIIAG, Stage III sporulation engulfment assemblyprotein


Theoretical massNumber of molelcules
Total (without water)591,87330
Polymers591,87330
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, CryoEM single particle
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
SpoIIIAG, Stage III sporulation engulfment assemblyprotein


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

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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: SpoIIIAG / Type: COMPLEX / Details: residues 55-229 / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.58 MDa / Experimental value: YES
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pET28
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
10.3 MSodium ChlorideNaClSodium chloride1
20.01 MTris(HOCH2)3CNH21
SpecimenConc.: 13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrector
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage 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 opticsEnergyfilter name: Gatan GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1EMAN22particle selection
2RELION1.4particle selection
3SerialEMimage acquisition
5CTFFIND44CTF correction
11RELION1.4initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 770000
SymmetryPoint symmetry: C30 (30 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55000 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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