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Open data
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Basic information
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Title | CryoEM structure of the N-pilus from Escherichia coli | |||||||||
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![]() | conjugation / TraM / self transmissable plasmid / Pili / STRUCTURAL PROTEIN | |||||||||
Function / homology | membrane / Conjugal transfer protein TraM![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
![]() | Bui KH / Black CS | |||||||||
Funding support | ![]()
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![]() | ![]() 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 | ![]() | 59.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.4 KB 16.4 KB | Display Display | ![]() |
Images | ![]() | 110.7 KB | ||
Masks | ![]() | 64 MB | ![]() | |
Filedesc metadata | ![]() | 5.8 KB | ||
Others | ![]() ![]() | 59.4 MB 59.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1.2 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 12.5 KB | Display | |
Data in CIF | ![]() | 14.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8cw4MC ![]() 8cueC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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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
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Density Histograms |
-Half map: #1
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Density Histograms |
-Half map: #2
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Density Histograms |
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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. |
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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: ![]() ![]() |
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: ![]() ![]() |
Molecular weight | Theoretical: 10.121063 KDa |
Recombinant expression | Organism: ![]() ![]() |
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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![]() | single particle reconstruction |
Aggregation state | helical array |
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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.. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: NONE |
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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 |
Initial angle assignment | Type: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) |
-Atomic model buiding 1
Refinement | Protocol: AB INITIO MODEL |
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Output model | ![]() PDB-8cw4: |