+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8015 | |||||||||
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Title | Cryo-EM structure of the Lysenin Pore | |||||||||
Map data | None | |||||||||
Sample |
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Function / homology | other 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 species | Eisenia foetida (common brandling worm) / Eisenia fetida (common brandling worm) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Savva CG / Bokori-Brown M / Martin TG / Titball RW / Naylor CE / Basak AK | |||||||||
Funding support | United Kingdom, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8015.map.gz | 3.5 MB | EMDB map data format | |
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Header (meta data) | emd-8015-v30.xml emd-8015.xml | 17.3 KB 17.3 KB | Display Display | EMDB header |
Images | emd_8015_1.png emd_8015_2.png | 202.3 KB 252.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8015 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8015 | HTTPS FTP |
-Related structure data
Related structure data | 5gaqMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8015.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.43 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Membrane-inserted form of the nonameric pore forming protein Lysenin
Entire | Name: Membrane-inserted form of the nonameric pore forming protein Lysenin |
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Components |
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-Supramolecule #1: Membrane-inserted form of the nonameric pore forming protein Lysenin
Supramolecule | Name: Membrane-inserted form of the nonameric pore forming protein Lysenin type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Eisenia foetida (common brandling worm) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pHis-Parallel 1 |
Molecular weight | Theoretical: 315 KDa |
-Macromolecule #1: Lysenin
Macromolecule | Name: Lysenin / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO |
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Source (natural) | Organism: Eisenia fetida (common brandling worm) |
Molecular weight | Theoretical: 34.977246 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.2 mg/mL | ||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: 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. | ||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 4.7 µm / Calibrated defocus min: 0.7 µm / Calibrated magnification: 34965 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Specialist optics | Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Temperature | Min: 85.0 K / Max: 85.0 K |
Details | Direct 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 recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / 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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 53779 Details: Autopicking performed with RELION after manually picking 1000 particles to create 2D Classes (references) for autopicking. |
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CTF correction | Software - Name: Gctf Software - details: This is a new GPU based software that was just published recently. http://www.ncbi.nlm.nih.gov/pubmed/26592709 |
Startup model | Type of model: OTHER / Details: Using EMAN 2 makeinitialmodel |
Initial angle assignment | Type: PROJECTION MATCHING Projection matching processing - Angular sampling: 7.5 degrees Software - Name: RELION (ver. 1.4) |
Final 3D classification | Number classes: 3 / Avg.num./class: 15000 / Software - Name: RELION (ver. 1.4) Details: 3D classification was used to reduced particle heterogeneity in final reconstruction |
Final angle assignment | Type: PROJECTION MATCHING Projection matching processing - Angular sampling: 0.47 degrees Software - Name: RELION (ver. 1.4) |
Final reconstruction | Applied 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 |
Details | Super-resolution images were binned by 2 for further processing. |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A / Chain - Residue range: 150-297 |
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Details | Initial docking of C-terminal region of lysenin monomer to EM map was performed using UCSF Chimera followed by model N-terminal extension in COOT. |
Refinement | Space: REAL / Protocol: AB INITIO MODEL |
Output model | PDB-5gaq: |