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- PDB-7nbx: Lateral-open conformation of the lid-locked BAM complex (BamA E43... -

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Basic information

Entry
Database: PDB / ID: 7nbx
TitleLateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) 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 / Outer membrane protein assembly / beta-barrel / Gram negative bacteria / protein foldase
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
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 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 BamC / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsHaysom, S.F.
Funding support United Kingdom, Belgium, 7items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P018491/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011151/1 United Kingdom
Wellcome Trust222373/Z/21/Z United Kingdom
Wellcome Trust105220/Z/14/Z United Kingdom
Research Foundation - Flanders (FWO)G0G0818N Belgium
Royal SocietyRSRP/R1/211057 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.
Authors: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / ...Authors: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / Kelly M Storek / Steven T Rutherford / David J Brockwell / Neil A Ranson / Sheena E Radford /
Abstract: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit ...The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
History
DepositionJan 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-12262
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Outer membrane protein assembly factor BamA
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


Theoretical massNumber of molelcules
Total (without water)210,7845
Polymers210,7845
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10920 Å2
ΔGint-34 kcal/mol
Surface area74910 Å2
MethodPISA

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Components

#1: Protein Outer membrane protein assembly factor BamA / Omp85


Mass: 90601.352 Da / Num. of mol.: 1 / Mutation: E435C S665C C690S C700S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A940
#2: Protein Outer membrane protein assembly factor BamB


Mass: 41918.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77774
#3: Protein Outer membrane protein assembly factor BamC


Mass: 36875.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamC, dapX, nlpB, b2477, JW2462 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A903
#4: Protein Outer membrane protein assembly factor BamD


Mass: 27858.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamD, yfiO, b2595, JW2577 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AC02
#5: Protein Outer membrane protein assembly factor BamE


Mass: 13530.256 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamE, smpA, b2617, JW2598 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A937

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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: beta-barrel assembly machinery (BAM) complex (BamABCDE)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.203 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
30.05 %n-dodecyl-beta-D-maltosideDDM1
SpecimenConc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged in a GlowQube Plus for 30s at 60 mA
Grid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.3.5particle selectionpicked with the general model
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimeramodel fittingUsed for initial fitting of map to density
8VMDmodel fittingUsed to setup cMDFF simulations
10RELION3initial Euler assignment
11cryoSPARC2.2.0final Euler assignmentfinal refinement used the non-uniform refinement in cryoSPARC
12RELION3classification
13RELION33D reconstructionhalf maps from non-uniform refinement were postprocessed in RELION 3.0
14cryoSPARC2.2.03D reconstructionfinal refinement used the non-uniform refinement in cryoSPARC
15NAMDmodel refinementcMDFF was used for flexible fitting as implemented in NAMD
16PHENIX1.14model refinement
17Coot0.9model refinementEM optimised version from ccpem bundle
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 793444
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141612 / Symmetry type: POINT
Atomic model buildingB value: 211 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Initial local fitting accomplished in Chimera. VMD was then used to setup cMDFF simulations that were run using NAMD. Both 7BNQ and 5LJO were used as initial models, with the best fitting ...Details: Initial local fitting accomplished in Chimera. VMD was then used to setup cMDFF simulations that were run using NAMD. Both 7BNQ and 5LJO were used as initial models, with the best fitting parts of each then combined and flexibly fit again to generate the final conformation. This was then Real Space Refined in Phenix to generate the final structure
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
17BNQ

7bnq

A124-675
27BNQ

7bnq

A1702-810
35LJO

5ljo

A1720-734
45LJO

5ljo

A1807-808
57BNQ

7bnq

B1
67BNQ

7bnq

C1
77BNQ

7bnq

D1
87BNQ

7bnq

E1

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