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- EMDB-12272: lateral-open conformation of the wild-type BAM complex (BamABCDE)... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-12272 | ||||||||||||||||||||||||
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Title | lateral-open conformation of the wild-type BAM complex (BamABCDE) bound to a bactericidal Fab fragment | ||||||||||||||||||||||||
![]() | Postprocessed masked map of the BAM (BamABCDE) 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: 5.2 Å | ||||||||||||||||||||||||
![]() | Iadanza MG | ||||||||||||||||||||||||
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 | ![]() | 8.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 35.2 KB 35.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 12.5 KB | Display | ![]() |
Images | ![]() | 106.1 KB | ||
Masks | ![]() | 163.6 MB | ![]() | |
Others | ![]() ![]() | 129.4 MB 129.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7nd0MC ![]() 7bm5C ![]() 7bnqC ![]() 7nbxC ![]() 7ncsC 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 BAM (BamABCDE) complex bound by a bactericidal Fab fragment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.065 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_12272_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_12272_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 : wild-type beta-barrel assembly machinery (BAM) complex (BamABCDE)...
+Supramolecule #1: wild-type beta-barrel assembly machinery (BAM) complex (BamABCDE)...
+Supramolecule #2: wild-type 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 R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE Details: grids glow discharged for 60 sec at 20 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 | #0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Average electron dose: 74.9 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Average electron dose: 59.8 e/Å2 / #2 - Image recording ID: 3 / #2 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #2 - Detector mode: COUNTING / #2 - Average electron dose: 60.9 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: 3.25 µm / Nominal defocus min: 1.75 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 | 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. | ||||||||||||||||||
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 183 | ||||||||||||||||||
Output model | ![]() PDB-7nd0: |