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- PDB-8cue: CryoEM structure of the T-pilus from Agrobacterium tumefaciens -

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Basic information

Entry
Database: PDB / ID: 8cue
TitleCryoEM structure of the T-pilus from Agrobacterium tumefaciens
ComponentsProtein virB2
KeywordsSTRUCTURAL PROTEIN / conjugation / VirB2 / type IV secretion system / pili
Function / homologyConjugal transfer TrbC/type IV secretion VirB2 / TrbC/VIRB2 pilin / type IV secretion system complex / protein secretion by the type IV secretion system / cell outer membrane / Chem-CPL / Protein virB2
Function and homology information
Biological speciesAgrobacterium fabrum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBui, K.H. / Black, C.S.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: 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
DepositionMay 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year
Revision 1.2Apr 17, 2024Group: Data collection / Source and taxonomy / Category: chem_comp_atom / chem_comp_bond / entity_src_nat
Item: _entity_src_nat.pdbx_ncbi_taxonomy_id / _entity_src_nat.pdbx_organism_scientific

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1A: Protein virB2
1B: Protein virB2
1C: Protein virB2
1D: Protein virB2
1E: Protein virB2
1F: Protein virB2
1G: Protein virB2
1H: Protein virB2
1I: Protein virB2
1J: Protein virB2
1K: Protein virB2
1L: Protein virB2
1M: Protein virB2
1N: Protein virB2
2A: Protein virB2
2B: Protein virB2
2C: Protein virB2
2D: Protein virB2
2E: Protein virB2
2F: Protein virB2
2G: Protein virB2
2H: Protein virB2
2I: Protein virB2
2J: Protein virB2
2K: Protein virB2
2L: Protein virB2
2M: Protein virB2
2N: Protein virB2
3A: Protein virB2
3B: Protein virB2
3C: Protein virB2
3D: Protein virB2
3E: Protein virB2
3F: Protein virB2
3G: Protein virB2
3H: Protein virB2
3I: Protein virB2
3J: Protein virB2
3K: Protein virB2
3L: Protein virB2
3M: Protein virB2
3N: Protein virB2
4A: Protein virB2
4B: Protein virB2
4C: Protein virB2
4D: Protein virB2
4E: Protein virB2
4F: Protein virB2
4G: Protein virB2
4H: Protein virB2
4I: Protein virB2
4J: Protein virB2
4K: Protein virB2
4L: Protein virB2
4M: Protein virB2
4N: Protein virB2
5A: Protein virB2
5B: Protein virB2
5C: Protein virB2
5D: Protein virB2
5E: Protein virB2
5F: Protein virB2
5G: Protein virB2
5H: Protein virB2
5I: Protein virB2
5J: Protein virB2
5K: Protein virB2
5L: Protein virB2
5M: Protein virB2
5N: Protein virB2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)915,915140
Polymers862,85170
Non-polymers53,06470
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Protein virB2


Mass: 12326.439 Da / Num. of mol.: 70 / Source method: isolated from a natural source
Source: (natural) Agrobacterium fabrum (strain C58 / ATCC 33970) (bacteria)
References: UniProt: P17792
#2: Chemical...
ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE


Mass: 758.060 Da / Num. of mol.: 70 / Source method: obtained synthetically / Formula: C42H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: T-pilus of the type IV secretion system of Agrobacterium tumefaciens.
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 24.7 kDa/nm / Experimental value: YES
Source (natural)Organism: Agrobacterium tumefaciens (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.3/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Linux / Type: package
EM software
IDNameVersionCategory
1cryoSPARC3.1particle selection
2SerialEM3.8image acquisition
4cryoSPARC3.1CTF correction
9cryoSPARC3.1initial Euler assignment
10cryoSPARC3.1final Euler assignment
12cryoSPARC3.13D reconstruction
13PHENIX1.19.2model refinement
14Coot0.9.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 615500 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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