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- EMDB-12708: I-Layer C16 at 3.08A - Local refinement with C16 symmetry of I-la... -

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Basic information

Entry
Database: EMDB / ID: EMD-12708
TitleI-Layer C16 at 3.08A - Local refinement with C16 symmetry of I-layer of the outer membrane core complex from the fully-assembled R388 type IV secretion system.
Map dataI-layer focus refinement C16 symmetry sharpened map
Sample
  • Complex: Type IV secretion system complex
    • Protein or peptide: TrwE protein
    • Protein or peptide: TrwF protein
Keywordstype IV secretion system / type 4 secretion system / T4SS / I-layer / core complex / outer membrane complex / R388 plasmid / conjugation / bacterial secretion / secretion / secretion system / protein complex / VirB10 / VirB9 / TrwE / TrwF / MEMBRANE PROTEIN
Function / homology
Function and homology information


: / Conjugal transfer, TrbG/VirB9/CagX / VirB9/CagX/TrbG, C-terminal / VirB9/CagX/TrbG, C-terminal domain superfamily / Conjugal transfer protein / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein / Type IV secretion system, VirB10 / TraB / TrbI
Similarity search - Domain/homology
TrwF protein / TrwE protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMace K / Vadakkepat AK
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Wellcome Trust098302 United Kingdom
Wellcome Trust217089 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Nature / Year: 2022
Title: Cryo-EM structure of a type IV secretion system.
Authors: Kévin Macé / Abhinav K Vadakkepat / Adam Redzej / Natalya Lukoyanova / Clasien Oomen / Nathalie Braun / Marta Ukleja / Fang Lu / Tiago R D Costa / Elena V Orlova / David Baker / Qian Cong ...Authors: Kévin Macé / Abhinav K Vadakkepat / Adam Redzej / Natalya Lukoyanova / Clasien Oomen / Nathalie Braun / Marta Ukleja / Fang Lu / Tiago R D Costa / Elena V Orlova / David Baker / Qian Cong / Gabriel Waksman /
Abstract: Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell. It is the primary means by which antibiotic resistance ...Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell. It is the primary means by which antibiotic resistance genes spread among bacterial populations. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
History
DepositionApr 3, 2021-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_12708.map.gz / Format: CCP4 / Size: 17.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationI-layer focus refinement C16 symmetry sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 80 pix.
= 85.36 Å
1.07 Å/pix.
x 241 pix.
= 257.147 Å
1.07 Å/pix.
x 242 pix.
= 258.214 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.067 Å
Density
Contour LevelBy AUTHOR: 10.0
Minimum - Maximum-27.673960000000001 - 30.828892
Average (Standard dev.)0.017591923 (±1.6233896)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin13214270
Dimensions24124280
Spacing24224180
CellA: 258.21402 Å / B: 257.147 Å / C: 85.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_12708_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: I-layer focus refinement C16 symmetry unsharpened map

Fileemd_12708_additional_1.map
AnnotationI-layer focus refinement C16 symmetry unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: I-layer focus refinement C16 symmetry - Half-B

Fileemd_12708_half_map_1.map
AnnotationI-layer focus refinement C16 symmetry - Half-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: I-layer focus refinement C16 symmetry - Half-A

Fileemd_12708_half_map_2.map
AnnotationI-layer focus refinement C16 symmetry - Half-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Type IV secretion system complex

EntireName: Type IV secretion system complex
Components
  • Complex: Type IV secretion system complex
    • Protein or peptide: TrwE protein
    • Protein or peptide: TrwF protein

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Supramolecule #1: Type IV secretion system complex

SupramoleculeName: Type IV secretion system complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: R388 plasmid
Molecular weightTheoretical: 2.808 MDa

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Macromolecule #1: TrwE protein

MacromoleculeName: TrwE protein / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 42.443785 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFGRKKGDVI DAGAELERAE QERIEGEYGA SELASERRPH TPGARTLLMV LLCVIAVVLV TLSYKAYKVR GVVEDDDAQP QQVVRQVIP GYTPRPIRPE PENVPEPPQP TTSVPAIQPA PVTQPVRPQP TGPREKTPYE LARERMLRSG LTAGSGGGED L PRPQGGDV ...String:
MFGRKKGDVI DAGAELERAE QERIEGEYGA SELASERRPH TPGARTLLMV LLCVIAVVLV TLSYKAYKVR GVVEDDDAQP QQVVRQVIP GYTPRPIRPE PENVPEPPQP TTSVPAIQPA PVTQPVRPQP TGPREKTPYE LARERMLRSG LTAGSGGGED L PRPQGGDV PAGGLMGGGG GGGELAEKLQ PMRLSGSSAG RLGNRDMLIT QGTQLDCVLE TRLVTTQPGM TTCHLTRDVY ST SGRVVLL DRGSKVVGFY QGGLRQGQAR IFVQWSRIET PSGVVINLDS PGTGPLGEAG LGGWIDRHFW ERFGGAIMIS LIG DLGDWA SRQGSRQGDN SIQFSNTANG VESAAAEALR NSINIPPTLY KNQGERVNIL VARDLDFSDV YSLESIPTK

UniProtKB: TrwE protein

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Macromolecule #2: TrwF protein

MacromoleculeName: TrwF protein / type: protein_or_peptide / ID: 2 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 29.749586 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKLAIVALL ASLHAVPALA LDVPSSSRYD HRIRYVTYNP ADVVQVDTVL GVATHIMLEE GEQYLTHAFG DSEAYAFARK GRHIFIKPQ AELANTNLIV VTDRRSYKFR LQMRNDRNGA MYELAFRYPD TQARQTREAN ARAAVEAAFE QRVGAYYNLK Y MMSGDKDI ...String:
MKKLAIVALL ASLHAVPALA LDVPSSSRYD HRIRYVTYNP ADVVQVDTVL GVATHIMLEE GEQYLTHAFG DSEAYAFARK GRHIFIKPQ AELANTNLIV VTDRRSYKFR LQMRNDRNGA MYELAFRYPD TQARQTREAN ARAAVEAAFE QRVGAYYNLK Y MMSGDKDI APVNAWDDGR FTYFKFSANA DLPSIYFVDA EGNESLVPRT TVGSSNNIIA VHKVNPKWMI RLGNRALAIF NE AYDPNGV PNDTGTASPA VRRVNKGGN

UniProtKB: TrwF protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: CryoSPARC ab-initio
Final reconstructionApplied symmetry - Point group: C16 (16 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1280606
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER / Details: Stochastic gradient descent (SGD)

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