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- PDB-8cw4: CryoEM structure of the N-pilus from Escherichia coli -

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Basic information

Entry
Database: PDB / ID: 8cw4
TitleCryoEM structure of the N-pilus from Escherichia coli
ComponentsConjugal transfer protein TraM
KeywordsSTRUCTURAL PROTEIN / conjugation / TraM / self transmissable plasmid / Pili
Function / homologyConjugal transfer TrbC/type IV secretion VirB2 / TrbC/VIRB2 pilin / membrane / Chem-PGW / TraM
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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 18, 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 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)760,905140
Polymers708,47470
Non-polymers52,43070
Water00
1


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

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Components

#1: Protein ...
Conjugal transfer protein TraM / TraM / TraM protein


Mass: 10121.063 Da / Num. of mol.: 70
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: traM, BON75_23740, CDC27_28740, DNQ45_22575, EIZ93_17580, ELT32_23125, GFY34_23075, GFY34_26255, JFD_05214, pMUR050_034, RCS44_P0033
Production host: Escherichia coli (E. coli) / References: UniProt: Q46696
#2: Chemical...
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 70 / Source method: obtained synthetically / Formula: C40H77O10P / 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: N-pilus of the type IV secretion system of Escherichia coli.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 24.8 kDa/nm / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
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
9PHENIX1.19.2model refinement
10Coot0.9.5model refinement
11cryoSPARC3.1initial Euler assignment
12cryoSPARC3.1final Euler assignment
14cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134660 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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