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- EMDB-9800: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-9800 | |||||||||
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Title | Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) in complex with Mg2+, NADPH, and CPD at pH7.5 | |||||||||
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Function / homology | ![]() ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.35 Å | |||||||||
![]() | Chen CY / Chang YC / Lin KF / Huang CH / Lin BL / Ko TP / Hsieh DL / Tsai MD | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 169.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.5 KB 14.5 KB | Display Display | ![]() |
Images | ![]() | 190.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6jczMC ![]() 9798C ![]() 9799C ![]() 9801C ![]() 6jcvC ![]() 6jcwC ![]() 6jd1C ![]() 6jd2C M: atomic model generated by this map C: citing same article ( |
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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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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
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Sample components
-Entire : Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois...
Entire | Name: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+, NAPDH, and CPD at pH7.5 |
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Components |
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-Supramolecule #1: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois...
Supramolecule | Name: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+, NAPDH, and CPD at pH7.5 type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Molecular weight | Experimental: 446 KDa |
-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: ![]() ![]() Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 |
Molecular weight | Theoretical: 37.229855 KDa |
Recombinant expression | Organism: ![]() ![]() |
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 |
-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: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
Macromolecule | Name: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE type: ligand / ID: 3 / Number of copies: 12 / Formula: NDP |
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Molecular weight | Theoretical: 745.421 Da |
Chemical component information | ![]() ChemComp-NDP: |
-Macromolecule #4: cyclopropane-1,1-dicarboxylic acid
Macromolecule | Name: cyclopropane-1,1-dicarboxylic acid / type: ligand / ID: 4 / Number of copies: 12 / Formula: 9TY |
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Molecular weight | Theoretical: 130.099 Da |
Chemical component information | ![]() ChemComp-9TY: |
-Macromolecule #5: water
Macromolecule | Name: water / type: ligand / ID: 5 / Number of copies: 99 / 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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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.2 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 |
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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: 1.4 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |