+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27023 | |||||||||
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Title | CryoEM structure of the N-pilus from Escherichia coli | |||||||||
Map data | ||||||||||
Sample |
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Keywords | conjugation / TraM / self transmissable plasmid / Pili / STRUCTURAL PROTEIN | |||||||||
Function / homology | membrane / Conjugal transfer protein TraM Function and homology information | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Bui KH / Black CS | |||||||||
Funding support | Canada, 1 items
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Citation | Journal: Structure / Year: 2023 Title: Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen. Authors: Jaafar Amro / Corbin Black / Zakaria Jemouai / Nathan Rooney / Caroline Daneault / Natalie Zeytuni / Matthieu Ruiz / Khanh Huy Bui / Christian Baron / Abstract: Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a ...Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27023.map.gz | 59.5 MB | EMDB map data format | |
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Header (meta data) | emd-27023-v30.xml emd-27023.xml | 16.4 KB 16.4 KB | Display Display | EMDB header |
Images | emd_27023.png | 110.7 KB | ||
Masks | emd_27023_msk_1.map | 64 MB | Mask map | |
Filedesc metadata | emd-27023.cif.gz | 5.8 KB | ||
Others | emd_27023_half_map_1.map.gz emd_27023_half_map_2.map.gz | 59.4 MB 59.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27023 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27023 | HTTPS FTP |
-Related structure data
Related structure data | 8cw4MC 8cueC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_27023.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.09 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_27023_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27023_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_27023_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : N-pilus of the type IV secretion system of Escherichia coli.
Entire | Name: N-pilus of the type IV secretion system of Escherichia coli.Secretion |
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Components |
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-Supramolecule #1: N-pilus of the type IV secretion system of Escherichia coli.
Supramolecule | Name: N-pilus of the type IV secretion system of Escherichia coli. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 24.8 kDa/nm |
-Macromolecule #1: Conjugal transfer protein TraM
Macromolecule | Name: Conjugal transfer protein TraM / type: protein_or_peptide / ID: 1 / Number of copies: 70 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 10.121063 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTTLFKKYGP AVVMGVLSIA LPQIALAAGT DTGESTATSI QTWLSTWIPI GCAIAIMVSC FMWMLHVIPA SFIPRIVISL IGIGSASYL VSLTGVGS UniProtKB: Conjugal transfer protein TraM |
-Macromolecule #2: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...
Macromolecule | Name: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate type: ligand / ID: 2 / Number of copies: 70 / Formula: PGW |
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Molecular weight | Theoretical: 749.007 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | helical array |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #0 - Film thickness: 5 / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GOLD / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: 3 uL of the sample was repeatedly applied and manually blotted three times using the multiple blotting technique prior to the Vitrobot step to increase the concentration of pili.. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Initial angle assignment | Type: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 134660 |
-Atomic model buiding 1
Refinement | Protocol: AB INITIO MODEL |
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Output model | PDB-8cw4: |