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Yorodumi- PDB-7ncs: Lateral-open conformation of the lid-locked BAM complex (BamA E43... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7ncs | ||||||||||||||||||||||||
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Title | Lateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) bound by a bactericidal Fab fragment | ||||||||||||||||||||||||
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Keywords | MEMBRANE 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 / identical protein binding / membrane Similarity search - Function | ||||||||||||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) Homo sapiens (human) | ||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 Å | ||||||||||||||||||||||||
Authors | Haysom, S.F. / Machin, J.M. | ||||||||||||||||||||||||
Funding support | United Kingdom, Belgium, 7items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ncs.cif.gz | 616.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ncs.ent.gz | 522.7 KB | Display | PDB format |
PDBx/mmJSON format | 7ncs.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ncs_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 7ncs_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7ncs_validation.xml.gz | 69.9 KB | Display | |
Data in CIF | 7ncs_validation.cif.gz | 103.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nc/7ncs ftp://data.pdbj.org/pub/pdb/validation_reports/nc/7ncs | HTTPS FTP |
-Related structure data
Related structure data | 12271MC 7bm5C 7bnqC 7nbxC 7nd0C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Outer membrane protein assembly factor ... , 5 types, 5 molecules ABCDE
#1: Protein | Mass: 90601.352 Da / Num. of mol.: 1 / Mutation: E435C S665C C690S C700S Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A940 |
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#2: Protein | Mass: 41918.945 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77774 |
#3: Protein | Mass: 36875.277 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 / Gene: bamC, dapX, nlpB, b2477, JW2462 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A903 |
#4: Protein | Mass: 27858.350 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 / Gene: bamD, yfiO, b2595, JW2577 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AC02 |
#5: Protein | Mass: 13530.256 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 / Gene: bamE, smpA, b2617, JW2598 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A937 |
-Antibody , 2 types, 2 molecules HL
#6: Antibody | 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) |
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#7: Antibody | 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) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Buffer solution | pH: 8 | ||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Details: Grids were glow discharged for 30s at 60 mA in a GlowQube Plus Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 49.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 162844 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61777 Details: Half maps from non-uniform refinement in cryoSPARC were postprocessed in RELION 3.0 to generate the final map Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 183 / Protocol: FLEXIBLE FIT / Space: REAL Details: 7NBX (lateral-open BAM-LL cryoEM structure) and 7BM5 (Fab1 crystal structure) were rigid fitted into the electron density in UCSF Chimera. One round of molecular dynamics based flexible ...Details: 7NBX (lateral-open BAM-LL cryoEM structure) and 7BM5 (Fab1 crystal structure) were rigid fitted into the electron density in UCSF Chimera. One round of molecular dynamics based flexible fitting was then set up in VMD and run in NAMD to fit the structures into the BAM-LL-Fab1 density. The resulting structure was finally real-space refined in phenix to generate the final model. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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