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- EMDB-2979: Cryo EM structure of suilysin prepore -

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Basic information

Entry
Database: EMDB / ID: EMD-2979
TitleCryo EM structure of suilysin prepore
Map dataDisulphide-locked suilysin Gly52Cys/Ser187Cys
Sample
  • Sample: Suilysin prepore on liposomes trapped with engineered disulphide lock (Gly52Cys/Ser187Cys)
  • Protein or peptide: Suilysin
Keywordscholesterol dependent cytolysin / hemolysin / toxin
Function / homology:
Function and homology information
Biological speciesStreptococcus suis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsDudkina NV / Leung C / Lukoyanova N / Hodel AW / Farabella I / Pandurangan AP / Jahan N / Damaso MP / Osmanovic D / Reboul CF ...Dudkina NV / Leung C / Lukoyanova N / Hodel AW / Farabella I / Pandurangan AP / Jahan N / Damaso MP / Osmanovic D / Reboul CF / Dunstone MA / Andrew PW / Lonnen R / Topf M / Saibil HR / Hoogenboom BW
CitationJournal: Elife / Year: 2014
Title: Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin.
Authors: Carl Leung / Natalya V Dudkina / Natalya Lukoyanova / Adrian W Hodel / Irene Farabella / Arun P Pandurangan / Nasrin Jahan / Mafalda Pires Damaso / Dino Osmanović / Cyril F Reboul / ...Authors: Carl Leung / Natalya V Dudkina / Natalya Lukoyanova / Adrian W Hodel / Irene Farabella / Arun P Pandurangan / Nasrin Jahan / Mafalda Pires Damaso / Dino Osmanović / Cyril F Reboul / Michelle A Dunstone / Peter W Andrew / Rana Lonnen / Maya Topf / Helen R Saibil / Bart W Hoogenboom /
Abstract: Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in ...Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
History
DepositionApr 18, 2015-
Header (metadata) releaseApr 29, 2015-
Map releaseApr 29, 2015-
UpdateApr 29, 2015-
Current statusApr 29, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0021
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0021
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

SlyPrepore.jpg

Downloads & links

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Map

FileDownload / File: emd_2979.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDisulphide-locked suilysin Gly52Cys/Ser187Cys
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 400 pix.
= 800. Å		2 Å/pix.
x 400 pix.
= 800. Å		2 Å/pix.
x 400 pix.
= 800. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0021 / Movie #1: 0.0021
Minimum - Maximum-0.01393337 - 0.0108655
Average (Standard dev.)0.0000178 (±0.00063224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-199-199-199
Dimensions400400400
Spacing400400400
CellA=B=C: 800.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z800.000800.000800.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-199-199-199
NC/NR/NS400400400
D min/max/mean-0.0140.0110.000

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Supplemental data

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Sample components

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Entire : Suilysin prepore on liposomes trapped with engineered disulphide ...

EntireName: Suilysin prepore on liposomes trapped with engineered disulphide lock (Gly52Cys/Ser187Cys)
Components
  • Sample: Suilysin prepore on liposomes trapped with engineered disulphide lock (Gly52Cys/Ser187Cys)
  • Protein or peptide: Suilysin

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Supramolecule #1000: Suilysin prepore on liposomes trapped with engineered disulphide ...

SupramoleculeName: Suilysin prepore on liposomes trapped with engineered disulphide lock (Gly52Cys/Ser187Cys)
type: sample / ID: 1000
Details: The protein was incubated with lipid vesicles (PC:Cholesterol, molar ratio 45:55) for 10 minutes at 37oC.
Oligomeric state: 37 / Number unique components: 1
Molecular weightExperimental: 2.072 MDa / Theoretical: 2.072 MDa / Method: Calculated from the molecular weight of the monomer

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Macromolecule #1: Suilysin

MacromoleculeName: Suilysin / type: protein_or_peptide / ID: 1 / Name.synonym: SLY / Number of copies: 37 / Oligomeric state: 37 / Recombinant expression: Yes
Source (natural)Organism: Streptococcus suis (bacteria)
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta-2 / Recombinant plasmid: pEHISTEV
SequenceUniProtKB: UNIPROTKB: C6GNG6

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES, 100 mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 91 K / Instrument: FEI VITROBOT MARK III
Method: Liposomes with the protein were applied to glow-discharged lacey carbon coated copper grids and blotted for 5 s.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000x magnification
DateDec 2, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 1309 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Details37-fold symmetrised reconstruction. Image regions containing the prepores with small surrounding areas of membrane were selected manually using Boxer (EMAN 1.9) software.
CTF correctionDetails: Phase flipping for each particle
Final reconstructionApplied symmetry - Point group: C37 (37 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: SPIDER, IMAGIC, EMAN / Details: Final maps were calculated by BP RP in SPIDER / Number images used: 450

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Atomic model buiding 1

Initial modelPDB ID:

3hvn


Chain - Chain ID: A
SoftwareName: Chimera
DetailsRigid body fitting of domain models created as described in Leung et al (2014) eLife 3:e04247
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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