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- EMDB-8797: SpoIIIAG 83-229 -

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Basic information

Entry
Database: EMDB / ID: EMD-8797
TitleSpoIIIAG 83-229
Map dataSpoIIIAG
Sample
  • Complex: SpoIIIAG
    • Protein or peptide: SpoIIIAG 83-229
Function / homologySporulation stage III, protein AG / membrane => GO:0016020 / Stage III sporulation engulfment assemblyprotein
Function and homology information
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZeytuni N / Hong C / Worrall LJ / Huang RK / Yu Z / Strynadka NCJ
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 30, 2017-
Header (metadata) releaseJul 12, 2017-
Map releaseAug 16, 2017-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.097
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.097
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8797.png

Downloads & links

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Map

FileDownload / File: emd_8797.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSpoIIIAG
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.64 Å/pix.
x 220 pix.
= 360.8 Å		1.64 Å/pix.
x 220 pix.
= 360.8 Å		1.64 Å/pix.
x 220 pix.
= 360.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.097 / Movie #1: 0.097
Minimum - Maximum-0.36317122 - 0.6316508
Average (Standard dev.)0.00085397094 (±0.014751099)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 360.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z360.800360.800360.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.3630.6320.001

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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 83-229

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Supramolecule #1: SpoIIIAG

SupramoleculeName: SpoIIIAG / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: residues 83-229
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET28
Molecular weightExperimental: 580 KDa

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Macromolecule #1: SpoIIIAG 83-229

MacromoleculeName: SpoIIIAG 83-229 / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ASKSDKPKDS IDDYEKEYEN QLKEILETII GVDDVSVVVN VDATSLKVYE KNKSNKNTTT EETDKEGGKR SVTDQSSEEE IVMIKNGDKE TPVVVQTKKP DIRGVLVVAQ GVDNVQIKQT IIEAVTRVLD VPSHRVAVAP KKIKEDS

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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

Concentration9 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
0.3 MNaClSodium Chloride
0.01 M(HOCH2)3CNH2Tris
GridModel: Quantifoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / 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
TemperatureMin: 80.0 K / Max: 80.0 K
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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2435 / Average exposure time: 0.3 sec. / Average electron dose: 1.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.1 µm / Calibrated defocus min: 1.4 µm / Calibrated magnification: 30488 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 277000
CTF correctionSoftware - Name: CTFFIND4 (ver. 4)
Startup modelType of model: OTHER / Details: EMAN2
Final reconstructionApplied symmetry - Point group: C30 (30 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 129000
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: Relion 1.4
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: Relion 1.4
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)

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