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- EMDB-9801: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois... -

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Basic information

Entry
Database: EMDB / ID: EMD-9801
TitleCryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) in complex with Mg2+, NADH, and CPD at pH7.5
Map data
Sample
  • Complex: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
    • Protein or peptide: Putative ketol-acid reductoisomerase 2
  • Ligand: MAGNESIUM ION
  • Ligand: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE
  • Ligand: cyclopropane-1,1-dicarboxylic acid
  • Ligand: water
KeywordsBi-specific / Thermostable / Reductoisomerase / Magnesium-dependent / Dodecamer / Knotted protein / ISOMERASE
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / L-valine biosynthetic process / isoleucine biosynthetic process
Similarity search - Function
Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Acetohydroxy acid isomeroreductase, catalytic domain / Acetohydroxy acid isomeroreductase, NADPH-binding domain / KARI N-terminal domain profile. / KARI C-terminal domain profile. / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Putative ketol-acid reductoisomerase 2
Similarity search - Component
Biological speciesSulfolobus solfataricus P2 (archaea) / Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsChen CY / Chang YC / Lin KF / Huang CH / Lin BL
Funding support Taiwan, 2 items
OrganizationGrant numberCountry
Ministry of Science and Technology (Taiwan)AS-KPQ-105-TPP Taiwan
Ministry of Science and Technology (Taiwan)OST 106-2113-M-008-011 Taiwan
CitationJournal: 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
DepositionJan 30, 2019-
Header (metadata) releaseApr 17, 2019-
Map releaseApr 17, 2019-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.88
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.88
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6jd1
  • Surface level: 1.88
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9801.png

Downloads & links

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Map

FileDownload / File: emd_9801.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 324 pix.
= 281.88 Å		0.87 Å/pix.
x 324 pix.
= 281.88 Å		0.87 Å/pix.
x 324 pix.
= 281.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.88 / Movie #1: 1.88
Minimum - Maximum-2.3328114 - 6.3463106
Average (Standard dev.)-0.0012305274 (±0.3605161)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 281.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z281.880281.880281.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean-2.3336.346-0.001

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Supplemental data

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Sample components

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Entire : Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois...

EntireName: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
Components
  • Complex: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
    • Protein or peptide: Putative ketol-acid reductoisomerase 2
  • Ligand: MAGNESIUM ION
  • Ligand: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE
  • Ligand: cyclopropane-1,1-dicarboxylic acid
  • Ligand: water

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Supramolecule #1: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois...

SupramoleculeName: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Sulfolobus solfataricus P2 (archaea)
Molecular weightTheoretical: 446 KDa

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Macromolecule #1: Putative ketol-acid reductoisomerase 2

MacromoleculeName: Putative ketol-acid reductoisomerase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: ketol-acid reductoisomerase (NADP+)
Source (natural)Organism: Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea)
Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2
Molecular weightTheoretical: 37.229855 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 36 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE

MacromoleculeName: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 12 / Formula: NAI
Molecular weightTheoretical: 665.441 Da
Chemical component information

ChemComp-NAI:
1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE

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Macromolecule #4: cyclopropane-1,1-dicarboxylic acid

MacromoleculeName: cyclopropane-1,1-dicarboxylic acid / type: ligand / ID: 4 / Number of copies: 12 / Formula: 9TY
Molecular weightTheoretical: 130.099 Da
Chemical component information

ChemComp-9TY:
cyclopropane-1,1-dicarboxylic acid

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 192 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris-HCl
50.0 mMNaClSodium chloride
5.0 mMMgCl2Magnesium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 1.31 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0-beta) / Number images used: 18522
Initial angle assignmentType: COMMON LINE / Software - Name: cisTEM (ver. 1.0.0-beta)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0.0-beta)
Final 3D classificationSoftware - Name: cisTEM (ver. 1.0.0-beta)

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Atomic model buiding 1

Initial modelPDB ID:

6jcv


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 117.95
Output model

PDB-6jd1:
Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) in complex with Mg2+, NADH, and CPD at pH7.5

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