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Yorodumi- EMDB-9798: Cryo-EM structure of Sulfolobus solfataricus ketol-acid reductois... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-9798 | |||||||||
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Title | Cryo-EM structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) with Mg2+ at pH7.5 | |||||||||
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Keywords | Bi-specific / Thermostable / Reductoisomerase / Magnesium-dependent / Dodecamer / Knot-structure / ISOMERASE | |||||||||
Function / homology | Function and homology information ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process Similarity search - Function | |||||||||
Biological species | Sulfolobus solfataricus P2 (archaea) / Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.92 Å | |||||||||
Authors | Chen CY / Chang YC / Lin KF / Huang CH / Lin BL / Ko TP / Hsieh DL / Tsai MD | |||||||||
Funding support | Taiwan, 2 items
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Citation | Journal: J Am Chem Soc / Year: 2019 Title: Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase. Authors: Chin-Yu Chen / Yuan-Chih Chang / Bo-Lin Lin / Kuan-Fu Lin / Chun-Hsiang Huang / Dong-Lin Hsieh / Tzu-Ping Ko / Ming-Daw Tsai / Abstract: While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 ...While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg. The results showed that the distances of the two catalytic Mg ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology. | |||||||||
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_9798.map.gz | 120 MB | EMDB map data format | |
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Header (meta data) | emd-9798-v30.xml emd-9798.xml | 13.4 KB 13.4 KB | Display Display | EMDB header |
Images | emd_9798.png | 213.5 KB | ||
Filedesc metadata | emd-9798.cif.gz | 5.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-9798 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-9798 | HTTPS FTP |
-Validation report
Summary document | emd_9798_validation.pdf.gz | 536.6 KB | Display | EMDB validaton report |
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Full document | emd_9798_full_validation.pdf.gz | 536.1 KB | Display | |
Data in XML | emd_9798_validation.xml.gz | 6.9 KB | Display | |
Data in CIF | emd_9798_validation.cif.gz | 7.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9798 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9798 | HTTPS FTP |
-Related structure data
Related structure data | 6jcvMC 9799C 9800C 9801C 6jcwC 6jczC 6jd1C 6jd2C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_9798.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.87 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 : Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Do...
Entire | Name: Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+ in pH7.5 |
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Components |
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-Supramolecule #1: Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Do...
Supramolecule | Name: Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+ in pH7.5 type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Sulfolobus solfataricus P2 (archaea) |
Molecular weight | Theoretical: 446 kDa/nm |
-Macromolecule #1: Putative ketol-acid reductoisomerase 2
Macromolecule | Name: Putative ketol-acid reductoisomerase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: ketol-acid reductoisomerase (NADP+) |
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Source (natural) | Organism: Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea) Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 |
Molecular weight | Theoretical: 37.229855 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MDKTVLDANL DPLKGKTIGV IGYGNQGRVQ ATIMRENGLN VIVGNVKDKY YELAKKEGFE VYEIDEAVRR SDVALLLIPD EVMKEVYEK KIAPVLQGKK EFVLDFASGY NVAFGLIRPP KSVDTIMVAP RMVGEGIMDL HKQGKGYPVL LGVKQDASGK A WDYAKAIA ...String: MDKTVLDANL DPLKGKTIGV IGYGNQGRVQ ATIMRENGLN VIVGNVKDKY YELAKKEGFE VYEIDEAVRR SDVALLLIPD EVMKEVYEK KIAPVLQGKK EFVLDFASGY NVAFGLIRPP KSVDTIMVAP RMVGEGIMDL HKQGKGYPVL LGVKQDASGK A WDYAKAIA KGIGAIPGGI AVISSFEEEA LLDLMSEHTW VPILFGAIKA CYDIAVKEYG VSPEAALLEF YASGELAEIA RL IAEEGIF NQMVHHSTTS QYGTLTRMFK YYDVVRRIVE NEAKYIWDGS FAKEWSLEQQ AGYPVFYRLW ELATQSEMAK AEK ELYKLL GRKVKND UniProtKB: Putative ketol-acid reductoisomerase 2 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 24 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: water
Macromolecule | Name: water / type: ligand / ID: 3 / Number of copies: 96 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 1.91 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 123.03 |
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Output model | PDB-6jcv: |