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- EMDB-4072: Cryo-EM 3D reconstruction of rings formed by the extracellular do... -

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Entry
Database: EMDB / ID: EMD-4072
TitleCryo-EM 3D reconstruction of rings formed by the extracellular domain of SpoIIIAG from Bacillus subtilis
Map dataCryo-EM 3D reconstruction of SpoIIIAG rings from Bacillus subtilis.
Sample
  • Complex: Oligomeric rings formed by the D1+D2 extracellular domains of SpoIIIAG from Bacillus subtilis
    • Protein or peptide: SpoIIIAG
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 35.0 Å
AuthorsRodrigues C / Henry X / Neumann E / Schoehn G / Rudner D / Morlot C
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis.
Authors: Christopher D A Rodrigues / Xavier Henry / Emmanuelle Neumann / Vilius Kurauskas / Laure Bellard / Yann Fichou / Paul Schanda / Guy Schoehn / David Z Rudner / Cecile Morlot /
Abstract: During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is ...During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems.
History
DepositionJul 26, 2016-
Header (metadata) releaseOct 5, 2016-
Map releaseOct 5, 2016-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.253
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.253
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4072.png

Downloads & links

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Map

FileDownload / File: emd_4072.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM 3D reconstruction of SpoIIIAG rings from Bacillus subtilis.
Voxel sizeX=Y=Z: 2.38 Å
Density
Contour LevelBy AUTHOR: 0.253 / Movie #1: 0.253
Minimum - Maximum0. - 0.96044
Average (Standard dev.)0.023103384 (±0.102511354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 304.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.382.382.38
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z304.640304.640304.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean0.0000.9600.023

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Supplemental data

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Sample components

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Entire : Oligomeric rings formed by the D1+D2 extracellular domains of Spo...

EntireName: Oligomeric rings formed by the D1+D2 extracellular domains of SpoIIIAG from Bacillus subtilis
Components
  • Complex: Oligomeric rings formed by the D1+D2 extracellular domains of SpoIIIAG from Bacillus subtilis
    • Protein or peptide: SpoIIIAG

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Supramolecule #1: Oligomeric rings formed by the D1+D2 extracellular domains of Spo...

SupramoleculeName: Oligomeric rings formed by the D1+D2 extracellular domains of SpoIIIAG from Bacillus subtilis
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: ROSETTA pLysS RARE / Recombinant plasmid: pCR094
Molecular weightExperimental: 19 KDa

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Macromolecule #1: SpoIIIAG

MacromoleculeName: SpoIIIAG / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString:
SSPEKTENAK TITAVSSQH SADSKEKTAE VFKASKSDKP KDSIDDYEKE YENQLKEILE TIIGVDDVSV V VNVDATSL KVYEKNKSNK NTTTEETDKE GGKRSVTDQS SEEEIVMIKN GDKETPVVVQ TK KPDIRGV LVVAQGVDNV QIKQTIIEAV TRVLDVPSHR VAVAPKKIKE DS

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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

Concentration0.1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: KODAK SO-163 FILM / Average electron dose: 15.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER
Details: The initial model was generated by back-projecting a side view and imposing C24 symmetry (as determined by ultracentrifugation experiments)
Final reconstructionApplied symmetry - Point group: C24 (24 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 6125
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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