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Yorodumi- PDB-6kou: 277 K cryoEM structure of Sso-KARI in complex with magnesium ions -
+Open data
-Basic information
Entry | Database: PDB / ID: 6kou | ||||||
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Title | 277 K cryoEM structure of Sso-KARI in complex with magnesium ions | ||||||
Components | Ketol-acid reductoisomerase | ||||||
Keywords | ISOMERASE / Complex | ||||||
Function / homology | Function and homology information ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process / isomerase activity / metal ion binding / cytosol Similarity search - Function | ||||||
Biological species | Saccharolobus solfataricus (archaea) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å | ||||||
Authors | Chen, C.Y. / Chang, Y.C. / Lin, B.L. / Huang, C.H. / Tsai, M.D. | ||||||
Citation | Journal: J Am Chem Soc / Year: 2019 Title: Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions. Authors: Chin-Yu Chen / Yuan-Chih Chang / Bo-Lin Lin / Chun-Hsiang Huang / Ming-Daw Tsai / Abstract: Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein ...Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 °C, and show that structures of both the Mg form (KARI:2Mg) and its ternary complex (KARI:2Mg:NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions. | ||||||
History |
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-Structure visualization
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Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 6kou.cif.gz | 1.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb6kou.ent.gz | 1 MB | Display | PDB format |
PDBx/mmJSON format | 6kou.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6kou_validation.pdf.gz | 946.1 KB | Display | wwPDB validaton report |
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Full document | 6kou_full_validation.pdf.gz | 968 KB | Display | |
Data in XML | 6kou_validation.xml.gz | 99.9 KB | Display | |
Data in CIF | 6kou_validation.cif.gz | 151.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ko/6kou ftp://data.pdbj.org/pub/pdb/validation_reports/ko/6kou | HTTPS FTP |
-Related structure data
Related structure data | 0740MC 0742C 0743C 0746C 0747C 0748C 0749C 0750C 0751C 0752C 0753C 0754C 6kpaC 6kpeC 6kphC 6kpiC 6kpjC 6kpkC 6kq4C 6kq8C 6kqjC 6kqkC 6kqoC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 37229.855 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Gene: SSOP1_1436 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A157T175, UniProt: Q97YJ9*PLUS #2: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: KARI-Mg2+ complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Saccharolobus solfataricus (archaea) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 / Details: 20 mM Tris-Cl, pH 7.5, 50 mM NaCl, 5 mM MgCl2 |
Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Microscopy | Model: FEI TITAN |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 52 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING ONLY |
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3D reconstruction | Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87959 / Symmetry type: POINT |