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- EMDB-8795: SpoIIIAG stage III sporulation engulfment assembly protein -

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Basic information

Entry
Database: EMDB / ID: EMD-8795
TitleSpoIIIAG stage III sporulation engulfment assembly protein
Map data
Sample
  • Complex: SpoIIIAG
    • Protein or peptide: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
KeywordsSecretion system / Sporulation channel / Ring Building Motif (RBM) / PROTEIN TRANSPORT
Function / homologySporulation stage III, protein AG / membrane => GO:0016020 / Stage III sporulation engulfment assemblyprotein
Function and homology information
Biological speciesBacillus subtilis (bacteria) / Bacillus subtilis BEST7613 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZeytuni N / Hong C
Funding support Canada, 1 items
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, 2017-
Header (metadata) releaseJul 12, 2017-
Map releaseAug 16, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wc3
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8795.png

Downloads & links

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Map

FileDownload / File: emd_8795.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.1715358 - 0.22656336
Average (Standard dev.)0.0012546782 (±0.009449681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1720.2270.001

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Supplemental data

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Sample components

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Entire : SpoIIIAG

EntireName: SpoIIIAG
Components
  • Complex: SpoIIIAG
    • Protein or peptide: SpoIIIAG, Stage III sporulation engulfment assemblyprotein

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Supramolecule #1: SpoIIIAG

SupramoleculeName: SpoIIIAG / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: residues 55-229
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 580 KDa

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Macromolecule #1: SpoIIIAG, Stage III sporulation engulfment assemblyprotein

MacromoleculeName: SpoIIIAG, Stage III sporulation engulfment assemblyprotein
type: protein_or_peptide / ID: 1 / Number of copies: 30 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis BEST7613 (bacteria)
Molecular weightTheoretical: 19.729086 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMKTENAK TITAVSSQHS ADSKEKTAEV FKASKSDKPK DSIDDYEKEY ENQLKEILET IIGVDDVSVV VNVDATSLKV YEKNKSNKN TTTEETDKEG GKRSVTDQSS EEEIVMIKNG DKETPVVVQT KKPDIRGVLV VAQGVDNVQI KQTIIEAVTR V LDVPSHRV AVAPKKIKED S

UniProtKB: Stage III sporulation engulfment assemblyprotein

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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

Concentration13 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
0.3 MNaClSodium chlorideSodium Chloride
0.01 M(HOCH2)3CNH2Tris
GridModel: Quantifoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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 / Calibrated defocus max: 2.1 µm / Calibrated defocus min: 0.9 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: Gatan GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
DetailsCs corrector
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2876 / Average exposure time: 0.2 sec. / Average electron dose: 1.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 770000
Startup modelType of model: OTHER / Details: EMAN2
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: Relion 1.4
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: Relion 1.4
Final reconstructionApplied symmetry - Point group: C30 (30 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 55000

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