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- EMDB-11240: Cryo-EM structure of the E.coli HTL-BAm complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11240
TitleCryo-EM structure of the E.coli HTL-BAm complex
Map dataCryo-EM of the E. coli HTL-BAM complex in detergent. 18,23A resolution.
Sample
  • Complex: E. coli inner membrane holotranslocon in complex with the outer membrane complex BAM
    • Complex: E coli HTL
    • Complex: E. coli BAM
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.23 Å
AuthorsAlvira S / Collinson S
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S008349/1 United Kingdom
Wellcome Trust206181/Z/17/Z United Kingdom
Wellcome Trust202904/Z/16/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R000484/1 United Kingdom
CitationJournal: Elife / Year: 2020
Title: Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.
Authors: Sara Alvira / Daniel W Watkins / Luca Troman / William J Allen / James S Lorriman / Gianluca Degliesposti / Eli J Cohen / Morgan Beeby / Bertram Daum / Vicki Am Gold / J Mark Skehel / Ian Collinson /
Abstract: The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic β-barrel uter-embrane roteins ...The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic β-barrel uter-embrane roteins (OMPs) - are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-arrel ssembly achinery (BAM) in the outer-membrane for insertion and folding. We show the olo-ransocon (HTL) - an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC - contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.
History
DepositionJun 29, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0066
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0066
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11240.png

Downloads & links

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Map

FileDownload / File: emd_11240.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM of the E. coli HTL-BAM complex in detergent. 18,23A resolution.
Voxel sizeX=Y=Z: 1.75 Å
Density
Contour LevelBy AUTHOR: 0.0066 / Movie #1: 0.0066
Minimum - Maximum-0.0033417405 - 0.025266567
Average (Standard dev.)0.0007380842 (±0.0025354407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 437.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.751.751.75
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z437.500437.500437.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0030.0250.001

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Supplemental data

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Sample components

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Entire : E. coli inner membrane holotranslocon in complex with the outer m...

EntireName: E. coli inner membrane holotranslocon in complex with the outer membrane complex BAM
Components
  • Complex: E. coli inner membrane holotranslocon in complex with the outer membrane complex BAM
    • Complex: E coli HTL
    • Complex: E. coli BAM

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Supramolecule #1: E. coli inner membrane holotranslocon in complex with the outer m...

SupramoleculeName: E. coli inner membrane holotranslocon in complex with the outer membrane complex BAM
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 205 KDa

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Supramolecule #2: E coli HTL

SupramoleculeName: E coli HTL / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: E. coli BAM

SupramoleculeName: E. coli BAM / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 8
Details: 20mM HEPES, pH 8.0 250 mM NaCl, 0.03% (w/v) DDM/0.003% (w/v) CL
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.02 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 79000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average exposure time: 20.0 sec. / Average electron dose: 1.127 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: OTHER / Details: Negative staining model from the same sample.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 18.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 8448
FSC plot (resolution estimation)

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