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- EMDB-8015: Cryo-EM structure of the Lysenin Pore -

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Basic information

Entry
Database: EMDB / ID: EMD-8015
TitleCryo-EM structure of the Lysenin Pore
Map dataNone
Sample
  • Complex: Membrane-inserted form of the nonameric pore forming protein Lysenin
    • Protein or peptide: Lysenin
KeywordsPore forming protein / aerolysin / toxin
Function / homologyother organism cell membrane / monoatomic ion transport / toxin activity / killing of cells of another organism / defense response to bacterium / extracellular region / membrane / Lysenin
Function and homology information
Biological speciesEisenia foetida (common brandling worm) / Eisenia fetida (common brandling worm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSavva CG / Bokori-Brown M
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC-A021-53019 United Kingdom
Wellcome TrustWT089618MA United Kingdom
CitationJournal: Nat Commun / Year: 2016
Title: Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein.
Authors: Monika Bokori-Brown / Thomas G Martin / Claire E Naylor / Ajit K Basak / Richard W Titball / Christos G Savva /
Abstract: Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. ...Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel.
History
DepositionJan 5, 2016-
Header (metadata) releaseMar 9, 2016-
Map releaseApr 6, 2016-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5gaq
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8015_1.png

emd_8015_2.png

Downloads & links

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Map

FileDownload / File: emd_8015.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 224 pix.
= 320.32 Å		1.43 Å/pix.
x 224 pix.
= 320.32 Å		1.43 Å/pix.
x 224 pix.
= 320.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.17919779 - 0.3905925
Average (Standard dev.)0.0005988714 (±0.009276755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 320.31998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z320.320320.320320.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.1790.3910.001

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Supplemental data

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Sample components

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Entire : Membrane-inserted form of the nonameric pore forming protein Lysenin

EntireName: Membrane-inserted form of the nonameric pore forming protein Lysenin
Components
  • Complex: Membrane-inserted form of the nonameric pore forming protein Lysenin
    • Protein or peptide: Lysenin

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Supramolecule #1: Membrane-inserted form of the nonameric pore forming protein Lysenin

SupramoleculeName: Membrane-inserted form of the nonameric pore forming protein Lysenin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Eisenia foetida (common brandling worm)
Molecular weightTheoretical: 315 KDa

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Macromolecule #1: Lysenin

MacromoleculeName: Lysenin / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Eisenia fetida (common brandling worm)
Molecular weightTheoretical: 34.977246 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSAKAAEGYE QIEVDVVAVW KEGYVYENRG STSVDQKITI TKGMKNVNSE TRTVTATHSI GSTISTGDAF EIGSVEVSYS HSHEESQVS MTETEVYESK VIEHTITIPP TSKFTRWQLN ADVGGADIEY MYLIDEVTPI GGTQSIPQVI TSRAKIIVGR Q IILGKTEI ...String:
MSAKAAEGYE QIEVDVVAVW KEGYVYENRG STSVDQKITI TKGMKNVNSE TRTVTATHSI GSTISTGDAF EIGSVEVSYS HSHEESQVS MTETEVYESK VIEHTITIPP TSKFTRWQLN ADVGGADIEY MYLIDEVTPI GGTQSIPQVI TSRAKIIVGR Q IILGKTEI RIKHAERKEY MTVVSRKSWP AATLGHSKLF KFVLYEDWGG FRIKTLNTMY SGYEYAYSSD QGGIYFDQGT DN PKQRWAI NKSLPLRHGD VVTFMNKYFT RSGLCYDDGP ATNVYCLDKR EDKWILEVVG LVPRGSGHHH HHH

UniProtKB: Lysenin

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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

Concentration0.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
150.0 mMSodium Chloride
50.0 mMTris
500.0 mMImidazole
0.02 %Dodecyl Maltoside
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS
Details: Grids were glow-discharged prior to deposition of Graphene-oxide.No further treatment was performed after graphene-oxide deposition.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 85.0 K / Max: 85.0 K
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
DetailsDirect alignments: Beam tilt pivot points, Beam shift, Comma Free. C2 aperture centering, C2 lens astigmatism correction. Objective aperture centering and objective lens astigmatism correction. Energy filter Tuning and occasional ZLP centering.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 0-20 / Number grids imaged: 1 / Number real images: 280 / Average exposure time: 18.0 sec. / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 4.7 µm / Calibrated defocus min: 0.7000000000000001 µm / Calibrated magnification: 34965 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

DetailsSuper-resolution images were binned by 2 for further processing.
Particle selectionNumber selected: 53779
Details: Autopicking performed with RELION after manually picking 1000 particles to create 2D Classes (references) for autopicking.
Startup modelType of model: OTHER / Details: Using EMAN 2 makeinitialmodel
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 29329
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 0.47 degrees
Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 3 / Avg.num./class: 15000 / Software - Name: RELION (ver. 1.4)
Details: 3D classification was used to reduced particle heterogeneity in final reconstruction

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Atomic model buiding 1

Initial modelPDB ID:

3zxd


Chain - Chain ID: A / Chain - Residue range: 150-297 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial docking of C-terminal region of lysenin monomer to EM map was performed using UCSF Chimera followed by model N-terminal extension in COOT.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-5gaq:
Cryo-EM structure of the Lysenin Pore

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