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- PDB-8bvq: E. coli BAM complex (BamABCDE) bound to darobactin B -

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Basic information

Entry
Database: PDB / ID: 8bvq
TitleE. coli BAM complex (BamABCDE) bound to darobactin B
Components
  • (Outer membrane protein assembly factor ...) x 5
  • Darobactin-B
KeywordsMEMBRANE PROTEIN / Outer Membrane Protein / Protein Folding / Antibiotic / beta barrel
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 / identical protein binding / membrane
Similarity search - Function
Beta-lactamase Inhibitory Protein; Chain:B, domain 1 / Beta-lactamase Inhibitory Protein; Chain:B, domain 1 - #10 / membrane protein fhac: a member of the omp85/tpsb transporter family / membrane protein fhac / Outer membrane protein assembly factor BamB / Outer membrane protein assembly factor BamC / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamB ...Beta-lactamase Inhibitory Protein; Chain:B, domain 1 / Beta-lactamase Inhibitory Protein; Chain:B, domain 1 - #10 / membrane protein fhac: a member of the omp85/tpsb transporter family / membrane protein fhac / Outer membrane protein assembly factor BamB / Outer membrane protein assembly factor BamC / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamB / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane protein assembly factor BamE domain / Outer membrane protein assembly factor BamD / Outer membrane lipoprotein BamD-like / Outer membrane lipoprotein / BamE-like / Pyrrolo-quinoline quinone repeat / Outer membrane protein assembly factor BamA / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / Surface antigen D15-like / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat / POTRA domain / POTRA domain profile. / Bacterial surface antigen (D15) / Omp85 superfamily domain / Porin / Quinoprotein alcohol dehydrogenase-like superfamily / Ubiquitin-like (UB roll) / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Roll / Beta Barrel / 2-Layer Sandwich / Mainly Beta / Alpha Beta
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)
Photorhabdus heterorhabditis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHorne, J.E. / Fenn, K.L. / Radford, S.E. / Ranson, N.A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P018491/1 United Kingdom
CitationJournal: Angew Chem Int Ed Engl / Year: 2023
Title: Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.
Authors: Samuel F Haysom / Jonathan Machin / James M Whitehouse / Jim E Horne / Katherine Fenn / Yue Ma / Hassane El Mkami / Nils Böhringer / Till F Schäberle / Neil A Ranson / Sheena E Radford / Christos Pliotas /
Abstract: The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational ...The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.
History
DepositionDec 5, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Sep 6, 2023Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
G: Darobactin-B


Theoretical massNumber of molelcules
Total (without water)201,2816
Polymers201,2816
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14370 Å2
ΔGint-73 kcal/mol
Surface area68080 Å2
MethodPISA

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Components

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Outer membrane protein assembly factor ... , 5 types, 5 molecules ABCDE

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


Mass: 88514.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A940
#2: Protein Outer membrane protein assembly factor BamB


Mass: 39882.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77774
#3: Protein Outer membrane protein assembly factor BamC


Mass: 34401.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / 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: 25816.818 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamD, yfiO, b2595, JW2577 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AC02
#5: Protein Outer membrane protein assembly factor BamE


Mass: 11610.833 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bamE, smpA, b2617, JW2598 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A937

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Protein/peptide , 1 types, 1 molecules G

#6: Protein/peptide Darobactin-B


Type: Peptide-like / Mass: 1055.189 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus heterorhabditis (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: BIRD: PRD_002342

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Details

Has ligand of interestY

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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: E. coli BAM complex (BamABCDE) bound to darobactin B / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.20098298 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMSodium ChlorideNaCl1
30.02 % w/vDDM1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 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 FIELD / Nominal magnification: 96000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 34.7 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3732

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.8.0particle selectionParticles picked using the general model
2EPU3image acquisition
4CTFFIND4.1.14CTF correction
9PHENIX1.20.1-4487model refinement
10RELION4initial Euler assignment
CTF correctionType: NONE
Particle selectionNum. of particles selected: 640909
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56124 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212135
ELECTRON MICROSCOPYf_angle_d0.56416495
ELECTRON MICROSCOPYf_dihedral_angle_d3.7461668
ELECTRON MICROSCOPYf_chiral_restr0.0431797
ELECTRON MICROSCOPYf_plane_restr0.0032172

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