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- EMDB-12263: structure of a POTRA-locked BAM complex in a lateral-open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-12263
Titlestructure of a POTRA-locked BAM complex in a lateral-open conformation
Map dataPostprocessed masked map of a POTRA-locked BAM complex
Sample
  • Complex: POTRA-locked beta-barrel assembly machinery (BAM) complex
    • Protein or peptide: Outer membrane protein assembly factor BamA
    • Protein or peptide: Outer membrane protein assembly factor BamB
    • 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
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 (strain K12) (bacteria) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsMachin JM / Haysom SF
Funding support United Kingdom, Belgium, 7 items
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, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJul 21, 2021-
Current statusJul 21, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00732
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.00732
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12263.png

Downloads & links

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Map

FileDownload / File: emd_12263.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed masked map of a POTRA-locked BAM complex
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.00732 / Movie #1: 0.00732
Minimum - Maximum-0.0044050794 - 0.014795905
Average (Standard dev.)0.00015388339 (±0.0009780419)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 299.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z299.200299.200299.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0040.0150.000

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Supplemental data

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Mask #1

Fileemd_12263_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 1 from Refine3D (RELION 3.1)

Fileemd_12263_half_map_1.map
AnnotationHalfmap 1 from Refine3D (RELION 3.1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 2 from Refine3D (RELION 3.1)

Fileemd_12263_half_map_2.map
AnnotationHalfmap 2 from Refine3D (RELION 3.1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : POTRA-locked beta-barrel assembly machinery (BAM) complex

EntireName: POTRA-locked beta-barrel assembly machinery (BAM) complex
Components
  • Complex: POTRA-locked beta-barrel assembly machinery (BAM) complex
    • Protein or peptide: Outer membrane protein assembly factor BamA
    • Protein or peptide: Outer membrane protein assembly factor BamB
    • 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

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Supramolecule #1: POTRA-locked beta-barrel assembly machinery (BAM) complex

SupramoleculeName: POTRA-locked beta-barrel assembly machinery (BAM) complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: beta-barrel assembly machinery (BAM) complex (BamABCDE) with POTRA-lock in BamA (G393C G584C) and natural cysteines removed (C690S C700S)
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 203 KDa

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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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAMKKLLIAS LLFSSATVYG AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVKE RPTIASITFS GNKSVKDDML KQNLEASGVR VGESLDRTTI ADIEKGLEDF YYSVGKYSAS VKAVVTPLPR NRVDLKLVFQ ...String:
MAMKKLLIAS LLFSSATVYG AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVKE RPTIASITFS GNKSVKDDML KQNLEASGVR VGESLDRTTI ADIEKGLEDF YYSVGKYSAS VKAVVTPLPR NRVDLKLVFQ EGVSAEIQQI NIVGNHAFTT DELISHFQLR DEVPWWNVVG DRKYQKQKLA GDLETLRSYY LDRGYARFNI DSTQVSLTPD KKGIYVTVNI TEGDQYKLSG VEVSGNLAGH SAEIEQLTKI EPGELYNGTK VTKMEDDIKK LLGRYGYAYP RVQSMPEIND ADKTVKLRVN VDAGNRFYVR KIRFEGNDTS KDAVLRREMR QMEGAWLGSD LVDQGKERLN RLGFFETVDT DTQRVPGSPD QVDVVYKVKE RNTGSFNFGI GYGTESGVSF QAGVQQDNWL GTGYAVGING TKNDYQTYAE LSVTNPYFTV DGVSLGGRLF YNDFQADDAD LSDYTNKSYG TDVTLGFPIN EYNSLRAGLG YVHNSLSNMQ PQVAMWRYLY SMGEHPSTSD QDNSFKTDDF TFNYGWTYNK LDRGYFPTDG SRVNLTGKVT IPGSDNEYYK VTLDTATYVP IDDDHKWVVL GRTRWGYGDG LGGKEMPFYE NFYAGGSSTV RGFQSNTIGP KAVYFPHQAS NYDPDYDYEC ATQDGAKDLC KSDDAVGGNA MAVASLEFIT PTPFISDKYA NSVRTSFFWD MGTVWDTNWD SSQYSGYPDY SDPSNIRMSA GIALQWMSPL GPLVFSYAQP FKKYDGDKAE QFQFNIGKTW

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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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQLRKLLLPG LLSVTLLSGC SLFNSEEDVV KMSPLPTVEN QFTPTTAWST SVGSGIGNFY SNLHPALADN VVYAADRAGL VKALNADDGK EIWSVSLAEK DGWFSKEPAL LSGGVTVSGG HVYIGSEKAQ VYALNTSDGT VAWQTKVAGE ALSRPVVSDG LVLIHTSNGQ ...String:
MQLRKLLLPG LLSVTLLSGC SLFNSEEDVV KMSPLPTVEN QFTPTTAWST SVGSGIGNFY SNLHPALADN VVYAADRAGL VKALNADDGK EIWSVSLAEK DGWFSKEPAL LSGGVTVSGG HVYIGSEKAQ VYALNTSDGT VAWQTKVAGE ALSRPVVSDG LVLIHTSNGQ LQALNEADGA VKWTVNLDMP SLSLRGESAP TTAFGAAVVG GDNGRVSAVL MEQGQMIWQQ RISQATGSTE IDRLSDVDTT PVVVNGVVFA LAYNGNLTAL DLRSGQIMWK RELGSVNDFI VDGNRIYLVD QNDRVMALTI DGGVTLWTQS DLLHRLLTSP VLYNGNLVVG DSEGYLHWIN VEDGRFVAQQ KVDSSGFQTE PVAADGKLLI QAKDGTVYSI TR

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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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAYSVQKSRL AKVAGVSLVL LLAACSSDSR YKRQVSGDEA YLEAAPLAEL HAPAGMILPV TSGDYAIPVT NGSGAVGKAL DIRPPAQPLA LVSGARTQFT GDTASLLVEN GRGNTLWPQV VSVLQAKNYT ITQRDDAGQT LTTDWVQWNR LDEDEQYRGR YQISVKPQGY ...String:
MAYSVQKSRL AKVAGVSLVL LLAACSSDSR YKRQVSGDEA YLEAAPLAEL HAPAGMILPV TSGDYAIPVT NGSGAVGKAL DIRPPAQPLA LVSGARTQFT GDTASLLVEN GRGNTLWPQV VSVLQAKNYT ITQRDDAGQT LTTDWVQWNR LDEDEQYRGR YQISVKPQGY QQAVTVKLLN LEQAGKPVAD AASMQRYSTE MMNVISAGLD KSATDAANAA QNRASTTMDV QSAADDTGLP MLVVRGPFNV VWQRLPAALE KVGMKVTDST RSQGNMAVTY KPLSDSDWQE LGASDPGLAS GDYKLQVGDL DNRSSLQFID PKGHTLTQSQ NDALVAVFQA AFSK

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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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTRMKYLVAA ATLSLFLAGC SGSKEEVPDN PPNEIYATAQ QKLQDGNWRQ AITQLEALDN RYPFGPYSQQ VQLDLIYAYY KNADLPLAQA AIDRFIRLNP THPNIDYVMY MRGLTNMALD DSALQGFFGV DRSDRDPQHA RAAFSDFSKL VRGYPNSQYT TDATKRLVFL ...String:
MTRMKYLVAA ATLSLFLAGC SGSKEEVPDN PPNEIYATAQ QKLQDGNWRQ AITQLEALDN RYPFGPYSQQ VQLDLIYAYY KNADLPLAQA AIDRFIRLNP THPNIDYVMY MRGLTNMALD DSALQGFFGV DRSDRDPQHA RAAFSDFSKL VRGYPNSQYT TDATKRLVFL KDRLAKYEYS VAEYYTERGA WVAVVNRVEG MLRDYPDTQA TRDALPLMEN AYRQMQMNAQ AEKVAKIIAA NSSNT

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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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
SequenceString:
MRCKTLTAAA AVLLMLTAGC STLERVVYRP DINQGNYLTA NDVSKIRVGM TQQQVAYALG TPLMSDPFGT NTWFYVFRQQ PGHEGVTQQT LTLTFNSSGV LTNIDNKPAL SGN

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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

Concentration2.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HClTrisTris
150.0 mMNaClSodium chloridesodium chloride
0.05 %DDMn-dodecyl-beta-D-maltoside
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Details: Glow discharge using GloqQube Plus, 30s, 60 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy #1

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: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Microscopy ID1
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

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: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Microscopy ID1
Image recordingImage recording ID: 2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 95848
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: OTHER
Details: Initial model generated by stochastic gradient descent from the cleaned particle stack using RELION 3.0
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: Refine3D
Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 19044
Image recording ID1
FSC plot (resolution estimation)

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