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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2755 | |||||||||
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Title | Cryo-electron microscopy of TibC dodecamer | |||||||||
![]() | Reconstruction of TibC dodecamer | |||||||||
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![]() | Bacterial autotransporters / Glycosyltransferase / Bacterial pathogenesis / Cryo-EM / Enzyme complex | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.5 Å | |||||||||
![]() | Yao Q / Lu QH / Wan XB / Song F / Xu Y / Zamyatina A / Huang N / Zhu P / Shao F | |||||||||
![]() | ![]() Title: A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family. Authors: Qing Yao / Qiuhe Lu / Xiaobo Wan / Feng Song / Yue Xu / Mo Hu / Alla Zamyatina / Xiaoyun Liu / Niu Huang / Ping Zhu / Feng Shao / ![]() ![]() Abstract: A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. ...A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex. | |||||||||
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 | ![]() | 45.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 9.1 KB 9.1 KB | Display Display | ![]() |
Images | ![]() ![]() ![]() | 151 KB 151 KB 123.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 218.3 KB | Display | ![]() |
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Full document | ![]() | 217.4 KB | Display | |
Data in XML | ![]() | 6.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Reconstruction of TibC dodecamer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.196 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Complex of TibC
Entire | Name: Complex of TibC |
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Components |
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-Supramolecule #1000: Complex of TibC
Supramolecule | Name: Complex of TibC / type: sample / ID: 1000 / Oligomeric state: TibC forms a dodecamer of six dimers / Number unique components: 1 |
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Molecular weight | Theoretical: 555 KDa |
-Macromolecule #1: TibC
Macromolecule | Name: TibC / type: protein_or_peptide / ID: 1 Details: Ferric ions were attached to specific cysteine residues Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 46 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.6 / Details: 10mM Tris-HCl, 100mM NaCl, 2mM DTT |
Grid | Details: Quantifoil R2.1, 300 mesh |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV Method: 10 ug/ml bacitracin (Sigma) was added to the purified protein to obtain monodispersed particles and make the orientation distribution more anisotropic. Blot for 4 sec using blotting force 2 before plunging. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 155,000 times magnification |
Date | Oct 11, 2012 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3000 / Average electron dose: 18 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 75000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
CTF correction | Details: CTF correction of each particle |
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Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 8546 |