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- EMDB-19758: Cryo-EM Structure of the R388 plasmid conjugative pilus reveals a... -

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Basic information

Entry
Database: EMDB / ID: EMD-19758
TitleCryo-EM Structure of the R388 plasmid conjugative pilus reveals a helical polymer characterised by an unusual pilin/phospholipid binary complex
Map dataSharpened map of the R388 Pilus filament generated using cryosparc
Sample
  • Complex: Conjugative pilus from the E. coli R388 plasmid
    • Protein or peptide: TrwL protein
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
KeywordsConjugal transfer protein / VirB2 / Conjugative pilus / Type-4 secretion system fibre / Conduit for horizontal gene transfer / anti-microbial resistance / PROTEIN FIBRIL
Function / homologyConjugal transfer TrbC/type IV secretion VirB2 / TrbC/VIRB2 pilin / membrane / TrwL protein
Function and homology information
Biological speciesEscherichia coli (E. coli) / Escherichia coli HB101 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsVadakkepat AK / Waksman G / Redzej A
Funding support United Kingdom, 1 items
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, 2024-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateSep 18, 2024-
Current statusSep 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19758.png

Downloads & links

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Map

FileDownload / File: emd_19758.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the R388 Pilus filament generated using cryosparc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 400 pix.
= 426.8 Å		1.07 Å/pix.
x 400 pix.
= 426.8 Å		1.07 Å/pix.
x 400 pix.
= 426.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.321
Minimum - Maximum-1.0331122 - 2.2909944
Average (Standard dev.)0.0002951389 (±0.08620985)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 426.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Halfmap-B of the R388 Pilus filament generated using...

Fileemd_19758_half_map_1.map
AnnotationHalfmap-B of the R388 Pilus filament generated using cryosparc and used to calculate the FSC curve using LocalResolution in Cryosparc. The resulting FSC curve shown in the associated publication.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap-A of the R388 Pilus filament generated using...

Fileemd_19758_half_map_2.map
AnnotationHalfmap-A of the R388 Pilus filament generated using cryosparc and used to calculate the FSC curve using LocalResolution in Cryosparc. The resulting FSC curve shown in the associated publication.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Conjugative pilus from the E. coli R388 plasmid

EntireName: Conjugative pilus from the E. coli R388 plasmid
Components
  • Complex: Conjugative pilus from the E. coli R388 plasmid
    • Protein or peptide: TrwL protein
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

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Supramolecule #1: Conjugative pilus from the E. coli R388 plasmid

SupramoleculeName: Conjugative pilus from the E. coli R388 plasmid / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
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
Source (natural)Organism: Escherichia coli (E. coli) / Strain: HB101 / Location in cell: Cell Surface
Molecular weightTheoretical: 7.39 kDa/nm

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Macromolecule #1: TrwL protein

MacromoleculeName: TrwL protein / type: protein_or_peptide / ID: 1
Details: The first 43 amino acids have been cleaved off from the pro-pilin TrwL/VirB2 during post-translational processing for maturation.
Number of copies: 69 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli HB101 (bacteria)
Molecular weightTheoretical: 7.396732 KDa
SequenceString:
AQGLEKARSV LETLQQELTT IVPIAAAVIL LCLGIAYAGR FIEKDTFVRW SIGVIIAGSA VQITAMLFT

UniProtKB: TrwL protein

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Macromolecule #2: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 2 / Number of copies: 69 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMNa2HPO4Sodium Phosphate
1.8 mMKH2PO4Potassium Phosphate
2.7 mMKClPotassium Chloride
2.7 mMNaClSodium Chloride

Details: Phosphate buffer saline
GridModel: C-flat-1.2/1.3 / Material: COPPER/PALLADIUM / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 100 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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..
DetailsThis specimen was purified to high levels.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
DetailsThe 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.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4884 / Average electron dose: 34.67 e/Å2
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.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.4 µm / Calibrated defocus min: 0.9 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFind more details in the Methods section of the publication
Final reconstructionNumber classes used: 3
Applied symmetry - Helical parameters - Δz: 13.241 Å
Applied symmetry - Helical parameters - Δ&Phi: 28.983 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. CRYOSPARC v4.3.1)
Details: Find more details in the Methods section of the publication
Number images used: 209930
Segment selectionNumber selected: 1158759 / Software - Name: cryoSPARC (ver. CRYOSPARC v4.3.1)
Details: Find more details in the Methods section of the publication
Startup modelType of model: NONE
Details: Find more details in the Methods section of the publication
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. CRYOSPARC v4.3.1)
Details: Find more details in the Methods section of the publication
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsFind more details in the Methods section of the publication
RefinementProtocol: RIGID BODY FIT / Overall B value: 109.7 / Target criteria: Cross-correlation in Phenix
Output model

PDB-8s6h:
Cryo-EM Structure of the R388 plasmid conjugative pilus reveals a helical polymer characterised by an unusual pilin/phospholipid binary complex

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