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- EMDB-12262: Lateral-open conformation of the lid-locked BAM complex (BamA E43... -

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Basic information

Entry
Database: EMDB / ID: EMD-12262
TitleLateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) by cryoEM
Map dataPostprocessed masked map of the lateral-open conformation of a lid-locked BAM complex
Sample
  • Complex: beta-barrel assembly machinery (BAM) complex (BamABCDE)
    • 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 K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsHaysom 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: 0.323
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.323
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7nbx
  • Surface level: 0.323
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12262.png

Downloads & links

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Map

FileDownload / File: emd_12262.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed masked map of the lateral-open conformation of a lid-locked BAM complex
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.323 / Movie #1: 0.323
Minimum - Maximum-0.6408634 - 1.4038843
Average (Standard dev.)0.003962451 (±0.036806487)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z321.000321.000321.000
α/β/γ90.00090.00090.000
start NX/NY/NZ7297100
NX/NY/NZ181127145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.6411.4040.004

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Supplemental data

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Mask #1

Fileemd_12262_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 2 from non-uniform refinement (cryoSPARC 2.2.0) of...

Fileemd_12262_half_map_1.map
AnnotationHalfmap 2 from non-uniform refinement (cryoSPARC 2.2.0) of the polished particle stack
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 1 from non-uniform refinement (cryoSPARC 2.2.0) of...

Fileemd_12262_half_map_2.map
AnnotationHalfmap 1 from non-uniform refinement (cryoSPARC 2.2.0) of the polished particle stack
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : beta-barrel assembly machinery (BAM) complex (BamABCDE)

EntireName: beta-barrel assembly machinery (BAM) complex (BamABCDE)
Components
  • Complex: beta-barrel assembly machinery (BAM) complex (BamABCDE)
    • 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: beta-barrel assembly machinery (BAM) complex (BamABCDE)

SupramoleculeName: beta-barrel assembly machinery (BAM) complex (BamABCDE)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 90.601352 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 FNFGIGYGTC 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 FQCNTIGPKA VYFPHQASNY DPDYDYESAT QDGAKDLSKS DDAVGGNAMA VASLEFIT P TPFISDKYAN SVRTSFFWDM GTVWDTNWDS SQYSGYPDYS DPSNIRMSAG IALQWMSPLG PLVFSYAQPF KKYDGDKAE 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 / 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 (strain K12) (bacteria) / Strain: K12
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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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:
ConcentrationFormulaName
50.0 mMTris-HClTrisTris
150.0 mMNaClSodium chloridesodium chloride
0.05 %DDMn-dodecyl-beta-D-maltoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
Details: Grids were glow discharged in a GlowQube Plus for 30s at 60 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 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 opticsC2 aperture diameter: 100.0 µm / Illumination 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: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 49.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 793444
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. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.2.0)
Software - details: final refinement used the non-uniform refinement in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
RELION (ver. 3.0)half maps from non-uniform refinement were postprocessed in RELION 3.0
cryoSPARC (ver. 2.2.0)final refinement used the non-uniform refinement in cryoSPARC

Number images used: 141612
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7bnq

chain_id: A, residue_range: 24-675

7bnq

chain_id: A, residue_range: 702-810

5ljo

chain_id: A, residue_range: 720-734

5ljo

chain_id: A, residue_range: 807-808

7bnq

chain_id: B

7bnq

chain_id: C

7bnq

chain_id: D

7bnq

chain_id: E
DetailsInitial 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
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 211
Output model

PDB-7nbx:
Lateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) by cryoEM

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