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- EMDB-27023: CryoEM structure of the N-pilus from Escherichia coli -

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Basic information

Entry
Database: EMDB / ID: EMD-27023
TitleCryoEM structure of the N-pilus from Escherichia coli
Map data
Sample
  • Complex: N-pilus of the type IV secretion system of Escherichia coli.Secretion
    • Protein or peptide: Conjugal transfer protein TraM
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
Function / homologymembrane / Conjugal transfer protein TraM
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsBui KH / Black CS
Funding support Canada, 1 items
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, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27023.png

Downloads & links

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Map

FileDownload / File: emd_27023.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.364
Minimum - Maximum-0.96351814 - 2.0087733
Average (Standard dev.)0.005658618 (±0.09371535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 279.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27023_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27023_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27023_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : N-pilus of the type IV secretion system of Escherichia coli.

EntireName: N-pilus of the type IV secretion system of Escherichia coli.Secretion
Components
  • Complex: N-pilus of the type IV secretion system of Escherichia coli.Secretion
    • Protein or peptide: Conjugal transfer protein TraM
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: N-pilus of the type IV secretion system of Escherichia coli.

SupramoleculeName: N-pilus of the type IV secretion system of Escherichia coli.
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 24.8 kDa/nm

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Macromolecule #1: Conjugal transfer protein TraM

MacromoleculeName: Conjugal transfer protein TraM / type: protein_or_peptide / ID: 1 / Number of copies: 70 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.121063 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTTLFKKYGP AVVMGVLSIA LPQIALAAGT DTGESTATSI QTWLSTWIPI GCAIAIMVSC FMWMLHVIPA SFIPRIVISL IGIGSASYL VSLTGVGS

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Macromolecule #2: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (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
Molecular weightTheoretical: 749.007 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.5
GridModel: 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: 5.0 nm / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GOLD / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 recordingFilm 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

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 134660

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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