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Yorodumi- EMDB-33219: Cryo-EM reconstruction of partial transmembrane channel E289A mut... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-33219 | |||||||||
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Title | Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin | |||||||||
Map data | Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin | |||||||||
Sample |
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Biological species | Vibrio cholerae (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.4 Å | |||||||||
Authors | Mondal AK / Sengupta N / Singh M / Lata K / Lahiri I / Dutta S / Chattopadhyay K | |||||||||
Funding support | India, 2 items
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Citation | Journal: J Biol Chem / Year: 2022 Title: Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation. Authors: Anish Kumar Mondal / Nayanika Sengupta / Mahendra Singh / Rupam Biswas / Kusum Lata / Indrajit Lahiri / Somnath Dutta / Kausik Chattopadhyay / Abstract: Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and ...Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_33219.map.gz | 44.8 MB | EMDB map data format | |
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Header (meta data) | emd-33219-v30.xml emd-33219.xml | 12.8 KB 12.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_33219_fsc.xml | 8.8 KB | Display | FSC data file |
Images | emd_33219.png | 47.3 KB | ||
Others | emd_33219_half_map_1.map.gz emd_33219_half_map_2.map.gz | 44.2 MB 44.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-33219 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-33219 | HTTPS FTP |
-Validation report
Summary document | emd_33219_validation.pdf.gz | 820.9 KB | Display | EMDB validaton report |
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Full document | emd_33219_full_validation.pdf.gz | 820.4 KB | Display | |
Data in XML | emd_33219_validation.xml.gz | 15.5 KB | Display | |
Data in CIF | emd_33219_validation.cif.gz | 19.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-33219 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-33219 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_33219.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.92 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half-map 1 of partial transmembrane channel E289A mutant...
File | emd_33219_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2 of partial transmembrane channel E289A mutant...
File | emd_33219_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cryo-EM reconstruction of partial transmembrane channel E289A mut...
Entire | Name: Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin |
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Components |
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-Supramolecule #1: Cryo-EM reconstruction of partial transmembrane channel E289A mut...
Supramolecule | Name: Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin type: complex / Chimera: Yes / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Vibrio cholerae (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |