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- EMDB-20969: Cryo-EM structure of a substrate-engaged Bam complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20969
TitleCryo-EM structure of a substrate-engaged Bam complex
Map data
Sample
  • Complex: BamABCDE bound to substrate BamA with loop 1 deleted
    • 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
    • Protein or peptide: Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA
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
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 BamD / Outer membrane protein assembly factor BamE / 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 / Outer membrane protein assembly factor BamC
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsTomasek D / Rawson S / Lee J / Wzorek JS / Harrison SC / Li Z / Kahne D
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI081059 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM108258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31GM116210 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2020
Title: Structure of a nascent membrane protein as it folds on the BAM complex.
Authors: David Tomasek / Shaun Rawson / James Lee / Joseph S Wzorek / Stephen C Harrison / Zongli Li / Daniel Kahne /
Abstract: Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands ...Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP), we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.
History
DepositionNov 18, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseJun 10, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v05
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20969.png

Downloads & links

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Map

FileDownload / File: emd_20969.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.35 / Movie #1: 0.35
Minimum - Maximum-1.5148301 - 2.4822104
Average (Standard dev.)0.006291929 (±0.049227778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 309.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z364364364
origin x/y/z0.0000.0000.000
length x/y/z309.400309.400309.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS364364364
D min/max/mean-1.5152.4820.006

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Supplemental data

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Sample components

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Entire : BamABCDE bound to substrate BamA with loop 1 deleted

EntireName: BamABCDE bound to substrate BamA with loop 1 deleted
Components
  • Complex: BamABCDE bound to substrate BamA with loop 1 deleted
    • 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
    • Protein or peptide: Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA

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Supramolecule #1: BamABCDE bound to substrate BamA with loop 1 deleted

SupramoleculeName: BamABCDE bound to substrate BamA with loop 1 deleted / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: 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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 90.567227 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAMKKLLIAS LLFSSATVYG AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVK ERPTIASITF SGNKSVKDDM LKQNLEASGV RVGESLDRTT IADIEKGLED FYYSVGKYSA SVKAVVTPLP R NRVDLKLV ...String:
MAMKKLLIAS LLFSSATVYG AEGFVVKDIH FEGLQRVAVG AALLSMPVRT GDTVNDEDIS NTIRALFATG NFEDVRVLRD GDTLLVQVK ERPTIASITF SGNKSVKDDM LKQNLEASGV RVGESLDRTT IADIEKGLED FYYSVGKYSA SVKAVVTPLP R NRVDLKLV FQEGVSAEIQ QINIVGNHAF TTDELISHFQ LRDEVPWWNV VGDRKYQKQK LAGDLETLRS YYLDRGYARF NI DSTQVSL TPDKKGIYVT VNITEGDQYK LSGVEVSGNL AGHSAEIEQL TKIEPGELYN GTKVTKMEDD IKKLLGRYGY AYP RVQSMP EINDADKTVK LRVNVDAGNR FYVRKIRFEG NDTSKDAVLR REMRQMEGAW LGSDLVDQGK ERLNRLGFFE TVDT DTQRV PGSPDQVDVV YKVKERNTGS FNFGIGYGTE SGVSFQAGVQ QDNWLGTGYA VGINGTKNDY QTYAELSVTN PYFTV DGVS LGGRLFYNDF QADDADLSDY TNKSYGTDVT LGFPINEYNS LRAGLGYVHN SLSNMQPQVA MWRYLYSMGE HPSTSD QDN SFKTDDFTFN YGWTYNKLDR GYFPTDGSRV NLTGKVTIPG SDNEYYKVTL DTATYVPIDD DHKWVVLGRT RWGYGDG LG GKEMPFYENF YAGGSSTVRG FQSNTIGPKA VYFPHQASNY DPDYDYESAT QDGAKDLSKS DDAVGGNAMA VASLEFIT P TPFISDKYAN SVRTSFFWDM GTVWDTNWDS SQYSGYPDYS DPSNIRMSAG IALQWMSPLG PLVFSYAQPF KKYDGDKAE QFQCNIGKTW

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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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 41.918945 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQLRKLLLPG LLSVTLLSGC SLFNSEEDVV KMSPLPTVEN QFTPTTAWST SVGSGIGNFY SNLHPALADN VVYAADRAGL VKALNADDG KEIWSVSLAE KDGWFSKEPA LLSGGVTVSG GHVYIGSEKA QVYALNTSDG TVAWQTKVAG EALSRPVVSD G LVLIHTSN ...String:
MQLRKLLLPG LLSVTLLSGC SLFNSEEDVV KMSPLPTVEN QFTPTTAWST SVGSGIGNFY SNLHPALADN VVYAADRAGL VKALNADDG KEIWSVSLAE KDGWFSKEPA LLSGGVTVSG GHVYIGSEKA QVYALNTSDG TVAWQTKVAG EALSRPVVSD G LVLIHTSN GQLQALNEAD GAVKWTVNLD MPSLSLRGES APTTAFGAAV VGGDNGRVSA VLMEQGQMIW QQRISQATGS TE IDRLSDV DTTPVVVNGV VFALAYNGNL TALDLRSGQI MWKRELGSVN DFIVDGNRIY LVDQNDRVMA LTIDGGVTLW TQS DLLHRL LTSPVLYNGN LVVGDSEGYL HWINVEDGRF VAQQKVDSSG FQTEPVAADG KLLIQAKDGT VYSITR

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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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 36.875277 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAYSVQKSRL AKVAGVSLVL LLAACSSDSR YKRQVSGDEA YLEAAPLAEL HAPAGMILPV TSGDYAIPVT NGSGAVGKAL DIRPPAQPL ALVSGARTQF TGDTASLLVE NGRGNTLWPQ VVSVLQAKNY TITQRDDAGQ TLTTDWVQWN RLDEDEQYRG R YQISVKPQ ...String:
MAYSVQKSRL AKVAGVSLVL LLAACSSDSR YKRQVSGDEA YLEAAPLAEL HAPAGMILPV TSGDYAIPVT NGSGAVGKAL DIRPPAQPL ALVSGARTQF TGDTASLLVE NGRGNTLWPQ VVSVLQAKNY TITQRDDAGQ TLTTDWVQWN RLDEDEQYRG R YQISVKPQ GYQQAVTVKL LNLEQAGKPV ADAASMQRYS TEMMNVISAG LDKSATDAAN AAQNRASTTM DVQSAADDTG LP MLVVRGP FNVVWQRLPA ALEKVGMKVT DSTRSQGNMA VTYKPLSDSD WQELGASDPG LASGDYKLQV GDLDNRSSLQ FID PKGHTL TQSQNDALVA VFQAAFSK

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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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 27.85835 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTRMKYLVAA ATLSLFLAGC SGSKEEVPDN PPNEIYATAQ QKLQDGNWRQ AITQLEALDN RYPFGPYSQQ VQLDLIYAYY KNADLPLAQ AAIDRFIRLN PTHPNIDYVM YMRGLTNMAL DDSALQGFFG VDRSDRDPQH ARAAFSDFSK LVRGYPNSQY T TDATKRLV ...String:
MTRMKYLVAA ATLSLFLAGC SGSKEEVPDN PPNEIYATAQ QKLQDGNWRQ AITQLEALDN RYPFGPYSQQ VQLDLIYAYY KNADLPLAQ AAIDRFIRLN PTHPNIDYVM YMRGLTNMAL DDSALQGFFG VDRSDRDPQH ARAAFSDFSK LVRGYPNSQY T TDATKRLV FLKDRLAKYE YSVAEYYTER GAWVAVVNRV EGMLRDYPDT QATRDALPLM ENAYRQMQMN AQAEKVAKII AA 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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 13.530256 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MRCKTLTAAA AVLLMLTAGC STLERVVYRP DINQGNYLTA NDVSKIRVGM TQQQVAYALG TPLMSDPFGT NTWFYVFRQQ PGHEGVTQQ TLTLTFNSSG VLTNIDNKPA LSGNGGHHHH HHHH

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Macromolecule #6: Outer membrane protein assembly factor BamA,Outer membrane protei...

MacromoleculeName: Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA
type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 64.010285 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAMKKLLIAS LLFSSATVYG AWSHPQFEKG GGSGGGSGGS AWSHPQFEKE GFVVKDIHFE GLQRVAVGAA LLSMPVRTGD TVNDEDISN TIRALFATGN FEDVRVLRDG DTLLVQVKER PTIASITFSG NKSVKDDMLK QNLEASGVRV GESLDRTTIA D IEKGLEDF ...String:
MAMKKLLIAS LLFSSATVYG AWSHPQFEKG GGSGGGSGGS AWSHPQFEKE GFVVKDIHFE GLQRVAVGAA LLSMPVRTGD TVNDEDISN TIRALFATGN FEDVRVLRDG DTLLVQVKER PTIASITFSG NKSVKDDMLK QNLEASGVRV GESLDRTTIA D IEKGLEDF YYSVGKYSAS VKAVVTPLPR NRVDLKLVFQ ENTGSFNFGF QAGVQQDNWL GTGYAVGING TKNDYQCYAE LS VTNPYFT VDGVSLGGRL FYNDFQADDA DLSDYTNKSY GTDVTLGFPI NEYNSLRAGL GYVHNSLSNM QPQVAMWRYL YSM GEHPST SDQDNSFKTD DFTFNYGWTY NKLDRGYFPT DGSRVNLTGK VTIPGSDNEY YKVTLDTATY VPIDDDHKWV VLGR TRWGY GDGLGGKEMP FYENFYAGGS STVRGFQSNT IGPKAVYFPH QASNYDPDYD YESATQDGAK DLSKSDDAVG GNAMA VASL EFITPTPFIS DKYANSVRTS FFWDMGTVWD TNWDSSQYSG YPDYSDPSNI RMSAGIALQW MSPLGPLVFS YAQPFK KYD GDKAEQFQFN IGKTW

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HCltris(hydroxymethyl)aminomethane hydrochloride
150.0 mMNaClsodium chloride
0.02 %GDNglyco-diosgenin
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 25 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4097 / Average exposure time: 3.0 sec. / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.8 µm / Calibrated defocus min: 1.1 µm / Calibrated magnification: 58717 / Illumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2054956
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 223353
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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