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- PDB-5ljo: E. coli BAM complex (BamABCDE) by cryoEM -

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Basic information

Entry
Database: PDB / ID: 5ljo
TitleE. coli BAM complex (BamABCDE) by cryoEM
Components
  • Outer membrane protein assembly factor BamA
  • Outer membrane protein assembly factor BamB
  • Outer membrane protein assembly factor BamC
  • Outer membrane protein assembly factor BamD
  • Outer membrane protein assembly factor BamE
KeywordsMEMBRANE PROTEIN / BAM / OMP / Beta barrel / Outer membrane / Gram negative
Function / homology
Function and homology information


Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane / cell outer membrane / protein-macromolecule adaptor activity / cell adhesion / response to antibiotic / cell surface / membrane / identical protein binding
Similarity search - Function
PQQ enzyme repeat / Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like ...PQQ enzyme repeat / Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like / SmpA / OmlA family / Outer membrane lipoprotein / BamE-like / Outer membrane protein assembly factor BamA / Pyrrolo-quinoline quinone repeat / PQQ-like domain / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / POTRA domain / POTRA domain profile. / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat / Surface antigen D15-like / Bacterial surface antigen (D15) / Omp85 superfamily domain / Quinoprotein alcohol dehydrogenase-like superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamB
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsIadanza, M.G. / Ranson, N.A. / Radford, S.E. / Higgins, A.J. / Schffrin, B. / Calabrese, A.N. / Ashcroft, A.E. / Brockwell, D.J.
Funding support United Kingdom, 8items
OrganizationGrant numberCountry
European Research CouncilFP7/2007-2013)/ERC Grant Agreement 32240 United Kingdom
Wellcome Trust105220/Z/14/Z United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/K000659/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/J014443/1 United Kingdom
Wellcome Trust090932/Z/09/Z United Kingdom
Wellcome Trust094232/Z/10/Z United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/E012558/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M012573/1 United Kingdom
CitationJournal: Nat Commun / Year: 2016
Title: Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM.
Authors: Matthew G Iadanza / Anna J Higgins / Bob Schiffrin / Antonio N Calabrese / David J Brockwell / Alison E Ashcroft / Sheena E Radford / Neil A Ranson /
Abstract: The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins ...The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a 'lateral gating' motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.
History
DepositionJul 19, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 12, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2017Group: Database references
Revision 1.2Aug 30, 2017Group: Author supporting evidence / Experimental preparation
Category: em_sample_support / pdbx_audit_support
Item: _em_sample_support.grid_type / _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Outer membrane protein assembly factor BamB
C: Outer membrane protein assembly factor BamC
D: Outer membrane protein assembly factor BamD
E: Outer membrane protein assembly factor BamE
A: Outer membrane protein assembly factor BamA


Theoretical massNumber of molelcules
Total (without water)180,0735
Polymers180,0735
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13910 Å2
ΔGint-66 kcal/mol
Surface area69720 Å2
MethodPISA

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Components

#1: Protein Outer membrane protein assembly factor BamB


Mass: 39692.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli (E. coli) / References: UniProt: P77774
#2: Protein Outer membrane protein assembly factor BamC


Mass: 17858.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: bamC, dapX, nlpB, b2477, JW2462 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A903
#3: Protein Outer membrane protein assembly factor BamD


Mass: 25008.967 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamD, yfiO, Z3889, ECs3458 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AC04, UniProt: P0AC02*PLUS
#4: Protein Outer membrane protein assembly factor BamE


Mass: 9728.837 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamE, smpA, c3139 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A938, UniProt: P0A937*PLUS
#5: Protein Outer membrane protein assembly factor BamA


Mass: 87783.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamA, yaeT, ECS88_0187 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MBF8, UniProt: P0A940*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BAM complexBam A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pJH114
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTRISHOCH2)3CNH21
20.05 % (w/v)Dodecyl maltopyranosideC24H46O111
3150 mMSodium ChlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco Easyglo / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: Double blot. 3 ul sampel applied and blotted by hand, then additional 3 ul sample applied, blotted, and plunge frozen

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3500 nm / Cs: 2.6 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansSampling size: 5 µm / Width: 3480 / Height: 3712 / Movie frames/image: 20 / Used frames/image: 4-14

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4particle selectionAutopicking
2EPUimage acquisition
4CTFFIND4CTF correctionRun from RELION
5RELION1.4CTF correction
8MDFFmodel fitting
11RELION1.4final Euler assignment
13RELION1.43D reconstruction
14RosettaEMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 472857
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95878 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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