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- EMDB-12271: Lateral-open conformation of the lid-locked BAM complex (BamA E43... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-12271 | ||||||||||||||||||||||||
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Title | Lateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) bound by a bactericidal Fab fragment | ||||||||||||||||||||||||
![]() | Postprocessed masked map of the of a lid-locked BAM complex bound by a bactericidal Fab fragment | ||||||||||||||||||||||||
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Function / homology | ![]() 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 | ![]() ![]() ![]() | ||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.1 Å | ||||||||||||||||||||||||
![]() | Machin JM / Haysom SF | ||||||||||||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 9.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 33.2 KB 33.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.8 KB | Display | ![]() |
Images | ![]() | 107 KB | ||
Masks | ![]() | 103 MB | ![]() | |
Others | ![]() ![]() | 95.5 MB 95.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 545.5 KB | Display | ![]() |
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Full document | ![]() | 544.6 KB | Display | |
Data in XML | ![]() | 18.5 KB | Display | |
Data in CIF | ![]() | 23.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7ncsMC ![]() 7bm5C ![]() 7bnqC ![]() 7nbxC ![]() 7nd0C C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Postprocessed masked map of the of a lid-locked BAM complex bound by a bactericidal Fab fragment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Half map: Halfmap 2 from non-uniform refinement (cryoSPARC 2.2.0) of...
File | emd_12271_half_map_1.map | ||||||||||||
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Annotation | Halfmap 2 from non-uniform refinement (cryoSPARC 2.2.0) of the polished particle stack | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Halfmap 1 from non-uniform refinement (cryoSPARC 2.2.0) of...
File | emd_12271_half_map_2.map | ||||||||||||
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Annotation | Halfmap 1 from non-uniform refinement (cryoSPARC 2.2.0) of the polished particle stack | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : lid-locked beta-barrel assembly machinery (BAM) complex (BamABCDE...
+Supramolecule #1: lid-locked beta-barrel assembly machinery (BAM) complex (BamABCDE...
+Supramolecule #2: lid-locked beta-barrel assembly machinery (BAM) complex (BamABCDE)
+Supramolecule #3: Light and heavy chains of bactericidal Fab fragment Fab1
+Macromolecule #1: Outer membrane protein assembly factor BamA
+Macromolecule #2: Outer membrane protein assembly factor BamB
+Macromolecule #3: Outer membrane protein assembly factor BamC
+Macromolecule #4: Outer membrane protein assembly factor BamD
+Macromolecule #5: Outer membrane protein assembly factor BamE
+Macromolecule #6: Fab1 heavy chain
+Macromolecule #7: Fab1 light chain
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 3.3 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE Details: Grids were glow discharged for 30s at 60 mA in a GlowQube Plus | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 49.1 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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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. | ||||||||||||||||
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 183 | ||||||||||||||||
Output model | ![]() PDB-7ncs: |