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Yorodumi- EMDB-2757: Cryo-electron microscopy of TibC12-TibA6 octadecamer in active state -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2757 | |||||||||
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Title | Cryo-electron microscopy of TibC12-TibA6 octadecamer in active state | |||||||||
Map data | Reconstruction of TibC12-TibA6 complex in active conformation | |||||||||
Sample |
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Biological species | Escherichia coli ETEC H10407 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.2 Å | |||||||||
Authors | Yao Q / Lu QH / Wan XB / Song F / Xu Y / Zamyatina A / Huang N / Zhu P / Shao F | |||||||||
Citation | Journal: Elife / Year: 2014 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 |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2757.map.gz | 20 MB | EMDB map data format | |
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Header (meta data) | emd-2757-v30.xml emd-2757.xml | 10.1 KB 10.1 KB | Display Display | EMDB header |
Images | emd_2757.png emd_2757_1.png | 240.7 KB 171.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2757 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2757 | HTTPS FTP |
-Validation report
Summary document | emd_2757_validation.pdf.gz | 234.9 KB | Display | EMDB validaton report |
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Full document | emd_2757_full_validation.pdf.gz | 234 KB | Display | |
Data in XML | emd_2757_validation.xml.gz | 5.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2757 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2757 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2757.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of TibC12-TibA6 complex in active conformation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.778 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of TibC12-TibA6 octadecamer
Entire | Name: Complex of TibC12-TibA6 octadecamer |
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Components |
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-Supramolecule #1000: Complex of TibC12-TibA6 octadecamer
Supramolecule | Name: Complex of TibC12-TibA6 octadecamer / type: sample / ID: 1000 / Oligomeric state: One TibA monomer binds to one TibC dimer / Number unique components: 2 |
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Molecular weight | Theoretical: 727 KDa |
-Macromolecule #1: TibC
Macromolecule | Name: TibC / type: protein_or_peptide / ID: 1 Details: Ferric ions were attached to specific cysteine residues. Lys230 was substituted by alanine to generate the catalytically inactive mutant. Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli ETEC H10407 (bacteria) |
Molecular weight | Theoretical: 46 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Gold / Recombinant plasmid: pACYCDuet |
-Macromolecule #2: TibA
Macromolecule | Name: TibA / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli ETEC H10407 (bacteria) |
Molecular weight | Theoretical: 29 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pGEX-6p-2 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 / Details: 10mM Tris-HCl, 100mM NaCl, 2mM DTT |
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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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 155,000 times magnification |
Details | Energy filter turned-off |
Date | May 1, 2013 |
Image recording | Category: CCD / Film or detector model: OTHER / Average electron dose: 18 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: OTHER / Software - Name: EMAN2, Relion / Number images used: 35300 |
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-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: NAMD |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |