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- PDB-7nd0: lateral-open conformation of the wild-type BAM complex (BamABCDE)... -

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Basic information

Entry
Database: PDB / ID: 7nd0
Titlelateral-open conformation of the wild-type BAM complex (BamABCDE) bound to a bactericidal Fab fragment
Components
  • (Outer membrane protein assembly factor ...) x 5
  • Fab1 heavy chainPIKFYVE
  • Fab1 light chainPIKFYVE
KeywordsMEMBRANE PROTEIN / Outer membrane protein assembly / beta-barrel / Gram negative bacteria / protein foldase
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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsIadanza, M.G. / Haysom, S.H.
Funding support United Kingdom, Belgium, 7items
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 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-12272
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
H: Fab1 heavy chain
L: Fab1 light chain


Theoretical massNumber of molelcules
Total (without water)258,9707
Polymers258,9707
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 90643.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A940
#2: Protein Outer membrane protein assembly factor BamB


Mass: 41918.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77774
#3: Protein Outer membrane protein assembly factor BamC


Mass: 36875.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / 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: 27858.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: bamD, yfiO, b2595, JW2577 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AC02
#5: Protein Outer membrane protein assembly factor BamE


Mass: 13530.256 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: bamE, smpA, b2617, JW2598 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A937

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Antibody , 2 types, 2 molecules HL

#6: Antibody Fab1 heavy chain / PIKFYVE


Mass: 24501.666 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#7: Antibody Fab1 light chain / PIKFYVE


Mass: 23642.215 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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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

Component
IDNameTypeEntity IDParent-IDSource
1wild-type beta-barrel assembly machinery (BAM) complex (BamABCDE) bound by a bactericidal Fab fragmentCOMPLEXall0MULTIPLE SOURCES
2wild-type beta-barrel assembly machinery (BAM) complex (BamABCDE)COMPLEX#1-#51RECOMBINANT
3Light and heavy chains of bactericidal Fab fragment Fab1COMPLEX#6-#71RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.251 MDaNO
210.203 MDaNO
310.048 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (strain K12) (bacteria)83333K12
32Escherichia coli (strain K12) (bacteria)83333K12
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
30.05 %n-dodecyl-beta-D-maltosideDDM1
SpecimenConc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: grids glow discharged for 60 sec at 20 mA in a GlowQube Plus
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3250 nm / Nominal defocus min: 1750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1174.9COUNTINGGATAN K2 SUMMIT (4k x 4k)
2159.8COUNTINGGATAN K2 SUMMIT (4k x 4k)
3160.9COUNTINGGATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10cryoSPARC2.2.0final Euler assignmentfinal refinement used the non-uniform refinement in cryoSPARC
11RELION3classification
12RELION33D reconstructionhalf maps from non-uniform refinement were postprocessed in RELION 3.0
13cryoSPARC2.2.03D reconstructionfinal refinement used the non-uniform refinement in cryoSPARC
14NAMDmodel refinement
15MDFFmodel refinement
16VMDmodel refinement
17PHENIX1.14model refinement
18Coot0.9model refinementEM optimised version from ccpem bundle
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 703997
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131853 / Symmetry type: POINT
Atomic model buildingB value: 183 / Protocol: FLEXIBLE FIT / Space: REAL
Details: The starting BAM model was created from 5LJO (lateral-open BAM-WT cryoEM structure) with BamA residues 687-700 replaced by those from 5EKQ (crystal structure of BamACDE in a lateral-open ...Details: The starting BAM model was created from 5LJO (lateral-open BAM-WT cryoEM structure) with BamA residues 687-700 replaced by those from 5EKQ (crystal structure of BamACDE in a lateral-open conformation). This and 7BM5 (Fab1 crystal structure) were rigid fitted into the BAM-WT Fab1 complex electron density in UCSF Chimera. Molecular dynamics based flexible fitting was then set up in VMD and run in NAMD to fit the structures into the density. The resulting structure was finally real-space refined in phenix to generate the final model.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15LJO

5ljo

A124-806
25EKQ

5ekq

A1687-700
35LJO

5ljo

B1
45LJO

5ljo

C1
55LJO

5ljo

D1
65LJO

5ljo

E1
77BM5

7bm5

H1
87BM5

7bm5

L1

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