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- EMDB-0752: 309 K cryoEM structure of Sso-KARI in complex with Mg2+, NADH and CPD -

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Basic information

Entry
Database: EMDB / ID: EMD-0752
Title309 K cryoEM structure of Sso-KARI in complex with Mg2+, NADH and CPD
Map dataSso-KARI dodecameric enzyme in complex with Mg2 and cryoEM sample was prepared at 309 K.
Sample
  • Complex: KARI-Mg2+/NADH/CPD complex
    • Protein or peptide: Ketol-acid reductoisomerase
  • Ligand: MAGNESIUM ION
  • Ligand: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE
  • Ligand: cyclopropane-1,1-dicarboxylic acid
KeywordsComplex / ISOMERASE
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / L-valine biosynthetic process / isoleucine biosynthetic process / isomerase activity / metal ion binding
Similarity search - Function
Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Ketol-acid reductoisomerase, C-terminal 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
Ketol-acid reductoisomerase / Putative ketol-acid reductoisomerase 2
Similarity search - Component
Biological speciesSaccharolobus solfataricus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsChen CY / Chang YC / Lin BL / Huang CH / Tsai MD
CitationJournal: 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
DepositionAug 18, 2019-
Header (metadata) releaseMar 25, 2020-
Map releaseMar 25, 2020-
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: 1.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kqj
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0752.png

Downloads & links

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Map

FileDownload / File: emd_0752.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSso-KARI dodecameric enzyme in complex with Mg2 and cryoEM sample was prepared at 309 K.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 352 pix.
= 295.68 Å		0.84 Å/pix.
x 352 pix.
= 295.68 Å		0.84 Å/pix.
x 352 pix.
= 295.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.55 / Movie #1: 1.55
Minimum - Maximum-3.0105672 - 9.179881999999999
Average (Standard dev.)-0.0049249516 (±0.36966163)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 295.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z295.680295.680295.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-3.0119.180-0.005

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Supplemental data

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Sample components

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Entire : KARI-Mg2+/NADH/CPD complex

EntireName: KARI-Mg2+/NADH/CPD complex
Components
  • Complex: KARI-Mg2+/NADH/CPD complex
    • Protein or peptide: Ketol-acid reductoisomerase
  • Ligand: MAGNESIUM ION
  • Ligand: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE
  • Ligand: cyclopropane-1,1-dicarboxylic acid

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Supramolecule #1: KARI-Mg2+/NADH/CPD complex

SupramoleculeName: KARI-Mg2+/NADH/CPD complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharolobus solfataricus (archaea)

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Macromolecule #1: Ketol-acid reductoisomerase

MacromoleculeName: Ketol-acid reductoisomerase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Saccharolobus solfataricus (archaea)
Molecular weightTheoretical: 37.229855 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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: Ketol-acid reductoisomerase

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 24 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20 mM Tris-Cl, pH 7.5, 50 mM NaCl and 5 mM MgCl2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 102422
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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