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- PDB-8s6h: Cryo-EM Structure of the R388 plasmid conjugative pilus reveals a... -

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Basic information

Entry
Database: PDB / ID: 8s6h
TitleCryo-EM Structure of the R388 plasmid conjugative pilus reveals a helical polymer characterised by an unusual pilin/phospholipid binary complex
ComponentsTrwL protein
KeywordsPROTEIN FIBRIL / Conjugal transfer protein / VirB2 / Conjugative pilus / Type-4 secretion system fibre / Conduit for horizontal gene transfer / anti-microbial resistance
Function / homologyConjugal transfer TrbC/type IV secretion VirB2 / TrbC/VIRB2 pilin / membrane / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / TrwL protein
Function and homology information
Biological speciesEscherichia coli HB101 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsVadakkepat, A.K. / Waksman, G. / Redzej, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust217089 United Kingdom
CitationJournal: Structure / Year: 2024
Title: Cryo-EM structure of the R388 plasmid conjugative pilus reveals a helical polymer characterized by an unusual pilin/phospholipid binary complex.
Authors: Abhinav K Vadakkepat / Songlin Xue / Adam Redzej / Terry K Smith / Brian T Ho / Gabriel Waksman /
Abstract: Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial ...Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.
History
DepositionFeb 27, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TrwL protein
B: TrwL protein
C: TrwL protein
D: TrwL protein
E: TrwL protein
F: TrwL protein
G: TrwL protein
H: TrwL protein
I: TrwL protein
J: TrwL protein
K: TrwL protein
L: TrwL protein
M: TrwL protein
N: TrwL protein
3: TrwL protein
4: TrwL protein
5: TrwL protein
6: TrwL protein
7: TrwL protein
8: TrwL protein
9: TrwL protein
A1: TrwL protein
A2: TrwL protein
A3: TrwL protein
A4: TrwL protein
A5: TrwL protein
A6: TrwL protein
A7: TrwL protein
0: TrwL protein
1: TrwL protein
2: TrwL protein
p: TrwL protein
q: TrwL protein
r: TrwL protein
s: TrwL protein
t: TrwL protein
u: TrwL protein
v: TrwL protein
w: TrwL protein
x: TrwL protein
y: TrwL protein
z: TrwL protein
c: TrwL protein
d: TrwL protein
e: TrwL protein
f: TrwL protein
g: TrwL protein
h: TrwL protein
i: TrwL protein
j: TrwL protein
k: TrwL protein
l: TrwL protein
m: TrwL protein
n: TrwL protein
o: TrwL protein
O: TrwL protein
P: TrwL protein
Q: TrwL protein
R: TrwL protein
S: TrwL protein
T: TrwL protein
U: TrwL protein
V: TrwL protein
W: TrwL protein
X: TrwL protein
Y: TrwL protein
Z: TrwL protein
a: TrwL protein
b: TrwL protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)560,259138
Polymers510,37569
Non-polymers49,88569
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area375000 Å2
ΔGint-3628 kcal/mol
Surface area134740 Å2
MethodPISA

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Components

#1: Protein ...
TrwL protein


Mass: 7396.732 Da / Num. of mol.: 69 / Source method: isolated from a natural source
Details: The first 43 amino acids have been cleaved off from the pro-pilin TrwL/VirB2 during post-translational processing for maturation.
Source: (natural) Escherichia coli HB101 (bacteria) / Plasmid details: Wild type R388 / References: UniProt: O50328
#2: Chemical...
ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE


Mass: 722.970 Da / Num. of mol.: 69 / Source method: obtained synthetically / Formula: C38H75O10P / 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: helical reconstruction

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Sample preparation

ComponentName: Conjugative pilus from the E. coli R388 plasmid / Type: COMPLEX
Details: Helical filament called the conjugative pilus. This is part of the type-4 secretion system. Monomeric unit of the complex comprises of the protein VirB2/TrwL (for R388 pilus) and a lipid PG 32.1
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 7.39 kDa/nm / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli) / Strain: HB101 / Cellular location: Cell Surface
Buffer solutionpH: 7.4 / Details: Phosphate buffer saline
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMSodium PhosphateNa2HPO41
21.8 mMPotassium PhosphateKH2PO41
32.7 mMPotassium ChlorideKCl1
42.7 mMSodium ChlorideNaCl1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This specimen was purified to high levels.
Specimen supportGrid material: COPPER/PALLADIUM / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: C-flat grids (Protochips, US 1.2/1.3 400 mesh) were negatively glow discharged using PELCO Easiglow (Ted Pella, USA) and coated with graphene oxide. 3 microliter of the purified pili sample ...Details: C-flat grids (Protochips, US 1.2/1.3 400 mesh) were negatively glow discharged using PELCO Easiglow (Ted Pella, USA) and coated with graphene oxide. 3 microliter of the purified pili sample was applied on each grid and a Vitrobot Mark IV (Thermo Fisher Scientific, USA) operating at 4C and 100 percent humidity was used to incubate the sample on the grid for 30 secs and blotting for 16 secs (blot force -10) prior to vitrification in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: The R388 pilus data were collected at the ISMB Birkbeck EM facility using a Titan Krios microscope (Thermo Fisher Scientific, USA) operated at 300 keV and equipped with a BioQuantum energy ...Details: The R388 pilus data were collected at the ISMB Birkbeck EM facility using a Titan Krios microscope (Thermo Fisher Scientific, USA) operated at 300 keV and equipped with a BioQuantum energy filter (Gatan, USA) with a slit width of 20 eV. The images were collected with a post-GIF K3 direct electron detector (Gatan, USA) operating in super resolution mode, at a magnification of 81,000 corresponding to a pixel size of 1.067 A. The dose rate was set to 14.62 e per pixel per second and a total dose of 34.67 e per A2 was fractionated over 50 frames.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 2400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingElectron dose: 34.67 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 4884
Details: The R388 pilus data were collected at the ISMB Birkbeck EM facility using a Titan Krios microscope (Thermo Fisher Scientific, USA) operated at 300 keV and equipped with a BioQuantum energy ...Details: The R388 pilus data were collected at the ISMB Birkbeck EM facility using a Titan Krios microscope (Thermo Fisher Scientific, USA) operated at 300 keV and equipped with a BioQuantum energy filter (Gatan, USA) with a slit width of 20 eV. The images were collected with a post-GIF K3 direct electron detector (Gatan, USA) operating in super resolution mode, at a magnification of 81,000 corresponding to a pixel size of 1.067 A. The dose rate was set to 14.62 e per pixel per second and a total dose of 34.67 e per A2 was fractionated over 50 frames. Data were collected using the EPU software with a defocus range 0.9 micrometer to 2.4 micrometer and a total of 4884 movies were collected.

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Processing

EM software
IDNameVersionCategory
1cryoSPARCCRYOSPARC v4.3.1particle selection
2EPUimage acquisition
4cryoSPARCCRYOSPARC v4.3.1CTF correction
7CootCOOT v0.9.3model fitting
9cryoSPARCCRYOSPARC v4.3.1initial Euler assignment
10cryoSPARCCRYOSPARC v4.3.1final Euler assignment
11cryoSPARCCRYOSPARC v4.3.1classification
12cryoSPARCCRYOSPARC v4.3.13D reconstruction
13PHENIX1.21rc1_5058model refinement
Image processingDetails: Find more details in the Methods section of the publication
CTF correctionDetails: Find more details in the Methods section of the publication
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 28.983 ° / Axial rise/subunit: 13.241 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1158759
Details: Find more details in the Methods section of the publication
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209930
Details: Find more details in the Methods section of the publication
Num. of class averages: 3 / Symmetry type: HELICAL
Atomic model buildingB value: 109.7 / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation in Phenix
Details: Find more details in the Methods section of the publication
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003339537
ELECTRON MICROSCOPYf_angle_d0.378652923
ELECTRON MICROSCOPYf_chiral_restr0.03496555
ELECTRON MICROSCOPYf_plane_restr0.00246072
ELECTRON MICROSCOPYf_dihedral_angle_d10.260614973

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