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- EMDB-16282: E. coli BAM complex (BamABCDE) wild-type -

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Basic information

Entry
Database: EMDB / ID: EMD-16282
TitleE. coli BAM complex (BamABCDE) wild-type
Map data
Sample
  • Complex: E. coli BAM complex (BamABCDE) wild-type
    • Complex: Outer membrane protein assembly factor BamA, BamC, BamD and BamE
      • Protein or peptide: Outer membrane protein assembly factor BamA
      • Protein or peptide: Outer membrane protein assembly factor BamC
      • Protein or peptide: Outer membrane protein assembly factor BamD
      • Protein or peptide: Outer membrane protein assembly factor BamE
    • Complex: Outer membrane protein assembly factor BamB
      • Protein or peptide: Outer membrane protein assembly factor BamB
KeywordsOuter Membrane Protein / Protein Folding / beta barrel / MEMBRANE PROTEIN
Function / homology
Function and homology information


Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane / cell outer membrane / 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 BamE / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamB
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMachin JM / Radford SE / Ranson NA
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust222373/Z/21/Z 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 6, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16282.png

Downloads & links

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Map

FileDownload / File: emd_16282.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.74 Å
Density
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.8525161 - 1.2350934
Average (Standard dev.)-0.013235591 (±0.031297915)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 266.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16282_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16282_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli BAM complex (BamABCDE) wild-type

EntireName: E. coli BAM complex (BamABCDE) wild-type
Components
  • Complex: E. coli BAM complex (BamABCDE) wild-type
    • Complex: Outer membrane protein assembly factor BamA, BamC, BamD and BamE
      • Protein or peptide: Outer membrane protein assembly factor BamA
      • Protein or peptide: Outer membrane protein assembly factor BamC
      • Protein or peptide: Outer membrane protein assembly factor BamD
      • Protein or peptide: Outer membrane protein assembly factor BamE
    • Complex: Outer membrane protein assembly factor BamB
      • Protein or peptide: Outer membrane protein assembly factor BamB

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Supramolecule #1: E. coli BAM complex (BamABCDE) wild-type

SupramoleculeName: E. coli BAM complex (BamABCDE) wild-type / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 200.98298 KDa

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Supramolecule #2: Outer membrane protein assembly factor BamA, BamC, BamD and BamE

SupramoleculeName: Outer membrane protein assembly factor BamA, BamC, BamD and BamE
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3-#5
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: Outer membrane protein assembly factor BamB

SupramoleculeName: Outer membrane protein assembly factor BamB / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Outer membrane protein assembly factor BamA

MacromoleculeName: Outer membrane protein assembly factor BamA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 88.514742 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVKE RPTIASITFS GNKSVKDDM LKQNLEASGV RVGESLDRTT IADIEKGLED FYYSVGKYSA SVKAVVTPLP RNRVDLKLVF QEGVSAEIQQ I NIVGNHAF ...String:
AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVKE RPTIASITFS GNKSVKDDM LKQNLEASGV RVGESLDRTT IADIEKGLED FYYSVGKYSA SVKAVVTPLP RNRVDLKLVF QEGVSAEIQQ I NIVGNHAF TTDELISHFQ LRDEVPWWNV VGDRKYQKQK LAGDLETLRS YYLDRGYARF NIDSTQVSLT PDKKGIYVTV NI TEGDQYK LSGVEVSGNL AGHSAEIEQL TKIEPGELYN GTKVTKMEDD IKKLLGRYGY AYPRVQSMPE INDADKTVKL RVN VDAGNR FYVRKIRFEG NDTSKDAVLR REMRQMEGAW LGSDLVDQGK ERLNRLGFFE TVDTDTQRVP GSPDQVDVVY KVKE RNTGS FNFGIGYGTE SGVSFQAGVQ QDNWLGTGYA VGINGTKNDY QTYAELSVTN PYFTVDGVSL GGRLFYNDFQ ADDAD LSDY TNKSYGTDVT LGFPINEYNS LRAGLGYVHN SLSNMQPQVA MWRYLYSMGE HPSTSDQDNS FKTDDFTFNY GWTYNK LDR GYFPTDGSRV NLTGKVTIPG SDNEYYKVTL DTATYVPIDD DHKWVVLGRT RWGYGDGLGG KEMPFYENFY AGGSSTV RG FQSNTIGPKA VYFPHQASNY DPDYDYECAT QDGAKDLCKS DDAVGGNAMA VASLEFITPT PFISDKYANS VRTSFFWD M GTVWDTNWDS SQYSGYPDYS DPSNIRMSAG IALQWMSPLG PLVFSYAQPF KKYDGDKAEQ FQFNIGKTW

UniProtKB: Outer membrane protein assembly factor BamA

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Macromolecule #2: Outer membrane protein assembly factor BamB

MacromoleculeName: Outer membrane protein assembly factor BamB / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 39.882375 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: CSLFNSEEDV VKMSPLPTVE NQFTPTTAWS TSVGSGIGNF YSNLHPALAD NVVYAADRAG LVKALNADDG KEIWSVSLAE KDGWFSKEP ALLSGGVTVS GGHVYIGSEK AQVYALNTSD GTVAWQTKVA GEALSRPVVS DGLVLIHTSN GQLQALNEAD G AVKWTVNL ...String:
CSLFNSEEDV VKMSPLPTVE NQFTPTTAWS TSVGSGIGNF YSNLHPALAD NVVYAADRAG LVKALNADDG KEIWSVSLAE KDGWFSKEP ALLSGGVTVS GGHVYIGSEK AQVYALNTSD GTVAWQTKVA GEALSRPVVS DGLVLIHTSN GQLQALNEAD G AVKWTVNL DMPSLSLRGE SAPTTAFGAA VVGGDNGRVS AVLMEQGQMI WQQRISQATG STEIDRLSDV DTTPVVVNGV VF ALAYNGN LTALDLRSGQ IMWKRELGSV NDFIVDGNRI YLVDQNDRVM ALTIDGGVTL WTQSDLLHRL LTSPVLYNGN LVV GDSEGY LHWINVEDGR FVAQQKVDSS GFQTEPVAAD GKLLIQAKDG TVYSITR

UniProtKB: Outer membrane protein assembly factor BamB

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Macromolecule #3: Outer membrane protein assembly factor BamC

MacromoleculeName: Outer membrane protein assembly factor BamC / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 34.40125 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: CSSDSRYKRQ VSGDEAYLEA APLAELHAPA GMILPVTSGD YAIPVTNGSG AVGKALDIRP PAQPLALVSG ARTQFTGDTA SLLVENGRG NTLWPQVVSV LQAKNYTITQ RDDAGQTLTT DWVQWNRLDE DEQYRGRYQI SVKPQGYQQA VTVKLLNLEQ A GKPVADAA ...String:
CSSDSRYKRQ VSGDEAYLEA APLAELHAPA GMILPVTSGD YAIPVTNGSG AVGKALDIRP PAQPLALVSG ARTQFTGDTA SLLVENGRG NTLWPQVVSV LQAKNYTITQ RDDAGQTLTT DWVQWNRLDE DEQYRGRYQI SVKPQGYQQA VTVKLLNLEQ A GKPVADAA SMQRYSTEMM NVISAGLDKS ATDAANAAQN RASTTMDVQS AADDTGLPML VVRGPFNVVW QRLPAALEKV GM KVTDSTR SQGNMAVTYK PLSDSDWQEL GASDPGLASG DYKLQVGDLD NRSSLQFIDP KGHTLTQSQN DALVAVFQAA FSK

UniProtKB: Outer membrane protein assembly factor BamC

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Macromolecule #4: Outer membrane protein assembly factor BamD

MacromoleculeName: Outer membrane protein assembly factor BamD / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.816818 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: CSGSKEEVPD NPPNEIYATA QQKLQDGNWR QAITQLEALD NRYPFGPYSQ QVQLDLIYAY YKNADLPLAQ AAIDRFIRLN PTHPNIDYV MYMRGLTNMA LDDSALQGFF GVDRSDRDPQ HARAAFSDFS KLVRGYPNSQ YTTDATKRLV FLKDRLAKYE Y SVAEYYTE ...String:
CSGSKEEVPD NPPNEIYATA QQKLQDGNWR QAITQLEALD NRYPFGPYSQ QVQLDLIYAY YKNADLPLAQ AAIDRFIRLN PTHPNIDYV MYMRGLTNMA LDDSALQGFF GVDRSDRDPQ HARAAFSDFS KLVRGYPNSQ YTTDATKRLV FLKDRLAKYE Y SVAEYYTE RGAWVAVVNR VEGMLRDYPD TQATRDALPL MENAYRQMQM NAQAEKVAKI IAANSSNT

UniProtKB: Outer membrane protein assembly factor BamD

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Macromolecule #5: Outer membrane protein assembly factor BamE

MacromoleculeName: Outer membrane protein assembly factor BamE / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.610833 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
CSTLERVVYR PDINQGNYLT ANDVSKIRVG MTQQQVAYAL GTPLMSDPFG TNTWFYVFRQ QPGHEGVTQQ TLTLTFNSSG VLTNIDNKP ALSGNGGHHH HHHHH

UniProtKB: Outer membrane protein assembly factor BamE

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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

Concentration3.3 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMSodium ChlorideNaClSodium chloride
0.025 % w/vDDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 6063 / Average exposure time: 3.58 sec. / Average electron dose: 44.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 656080
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 169599
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 82
Output model

PDB-8bwc:
E. coli BAM complex (BamABCDE) wild-type

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