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- PDB-8fai: Cryo-EM structure of the Agrobacterium T-pilus -

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Basic information

Entry
Database: PDB / ID: 8fai
TitleCryo-EM structure of the Agrobacterium T-pilus
ComponentsProtein virB2
KeywordsPROTEIN FIBRIL / VirB/D4 T4SS / Bacterial conjugation / T-pilus / helical filament
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 / : / Protein virB2
Function and homology information
Biological speciesAgrobacterium tumefaciens (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 Å
AuthorsKreida, S. / Narita, A. / Johnson, M.D. / Tocheva, E.I. / Das, A. / Jensen, G.J. / Ghosal, D.
Funding support United States, Australia, Canada, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI127401 United States
National Health and Medical Research Council (NHMRC, Australia)APP1196924 Australia
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN 04345 Canada
CitationJournal: Structure / Year: 2023
Title: Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly.
Authors: Stefan Kreida / Akihiro Narita / Matthew D Johnson / Elitza I Tocheva / Anath Das / Debnath Ghosal / Grant J Jensen /
Abstract: Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an ...Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.
History
DepositionNov 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein virB2
B: Protein virB2
C: Protein virB2
D: Protein virB2
E: Protein virB2
F: Protein virB2
G: Protein virB2
H: Protein virB2
I: Protein virB2
J: Protein virB2
K: Protein virB2
L: Protein virB2
M: Protein virB2
N: Protein virB2
O: Protein virB2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,10130
Polymers184,89715
Non-polymers11,20515
Water0
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.: 15 / Source method: isolated from a natural source / Source: (natural) Agrobacterium tumefaciens (bacteria) / Strain: NT1REB(pJK270) / References: UniProt: P17792
#2: Chemical
ChemComp-XL0 / (7Z,19R,22S,25R)-22,25,26-trihydroxy-16,22-dioxo-17,21,23-trioxa-22lambda~5~-phosphahexacos-7-en-19-yl (9Z)-octadec-9-enoate


Mass: 746.991 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C40H75O10P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1T-pilusCOMPLEX#10NATURAL
2VirB2ORGANELLE OR CELLULAR COMPONENT#11NATURAL
3Phosphatidylglycerol 16:1/18:1ORGANELLE OR CELLULAR COMPONENT1NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
2145.964 kDa/nmNO
312.787 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Agrobacterium tumefaciens (bacteria)358NT1REB(pJK270)
32Agrobacterium tumefaciens (bacteria)358NT1REB(pJK270)
43Agrobacterium tumefaciens (bacteria)358NT1REB(pJK270)
Buffer solutionpH: 5.5
Buffer componentConc.: 50 mM / Name: 2-(N-morpholino)ethanesulfonic acid / Formula: MES
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 295 K

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1RELION4.0beta2particle selection
2SerialEM3-9-0beta7image acquisition
4RELION4.0beta2CTF correction
7Rosetta3model fitting
9PHENIX1.19.2model refinement
10RELION4.0beta2initial Euler assignment
11RELION4.0beta2final Euler assignment
12RELION4.0beta2classification
13RELION4.0beta23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
1132.6213.44C5
2132.6213.44C1
3132.6213.44C1
Particle selectionNum. of particles selected: 10934
Details: Filaments were manually picked using EMAN2 e2helixboxer and particles were extracted by RELION.
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3543 / Num. of class averages: 2 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037965
ELECTRON MICROSCOPYf_angle_d0.57210620
ELECTRON MICROSCOPYf_dihedral_angle_d15.1291545
ELECTRON MICROSCOPYf_chiral_restr0.0341230
ELECTRON MICROSCOPYf_plane_restr0.0031245

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