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- EMDB-21313: Cryo-EM structure of a substrate-engaged Bam complex with alterna... -

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Basic information

Entry
Database: EMDB / ID: EMD-21313
TitleCryo-EM structure of a substrate-engaged Bam complex with alternative cysteine-cysteine crosslink
Map data
Sample
  • Complex: BamABCDE bound to substrate BamA with loop 1 deleted
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsTomasek D / Rawson S / Lee J / Wzorek JS / Harrison SC / Li Z / Kahne D
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI081059 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM108258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31GM116210 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2020
Title: Structure of a nascent membrane protein as it folds on the BAM complex.
Authors: David Tomasek / Shaun Rawson / James Lee / Joseph S Wzorek / Stephen C Harrison / Zongli Li / Daniel Kahne /
Abstract: Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands ...Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP), we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.
History
DepositionFeb 3, 2020-
Header (metadata) releaseMar 25, 2020-
Map releaseJun 10, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21313.png

Downloads & links

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Map

FileDownload / File: emd_21313.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.017799594 - 0.053419784
Average (Standard dev.)0.00003521115 (±0.0023964406)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 309.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z364364364
origin x/y/z0.0000.0000.000
length x/y/z309.400309.400309.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS364364364
D min/max/mean-0.0180.0530.000

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Supplemental data

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Sample components

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Entire : BamABCDE bound to substrate BamA with loop 1 deleted

EntireName: BamABCDE bound to substrate BamA with loop 1 deleted
Components
  • Complex: BamABCDE bound to substrate BamA with loop 1 deleted

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Supramolecule #1: BamABCDE bound to substrate BamA with loop 1 deleted

SupramoleculeName: BamABCDE bound to substrate BamA with loop 1 deleted / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: contains a cysteine crosslink between S439C in BamA and E800C in the substrate
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTristris(hydroxymethyl)aminomethane hydrochloride
150.0 mMNaClSodium chloridesodium chloride
0.02 %GDNglyco-diosgenin
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Detailscontains a cysteine crosslink between S439C in BamA and E800C in the substrate

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.2 µm / Calibrated defocus min: 1.1 µm / Calibrated magnification: 58717 / Illumination mode: OTHER / Imaging mode: OTHER
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 25 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 690143
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 233064
FSC plot (resolution estimation)

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