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- PDB-8cy8: apo form Cryo-EM structure of Campylobacter jejune ketol-acid red... -

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Basic information

Entry
Database: PDB / ID: 8cy8
Titleapo form Cryo-EM structure of Campylobacter jejune ketol-acid reductoisommerase crosslinked by Glutaraldehyde
ComponentsKetol-acid reductoisomerase (NADP(+))
KeywordsISOMERASE / ketol-acid reductoisomerase / enzyme stability / dodecamer / branched-chain amino acid
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process / isomerase activity / NADP binding / magnesium ion binding
Similarity search - Function
Ketol-acid reductoisomerase, prokaryotic / 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
PENTANEDIAL / Ketol-acid reductoisomerase (NADP(+))
Similarity search - Component
Biological speciesCampylobacter jejuni (Campylobacter)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.94 Å
AuthorsZheng, S. / Guddat, L.W.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210101802 Australia
CitationJournal: Appl Biosci / Year: 2022
Title: Enhancing the Thermal and Kinetic Stability of Ketol-Acid Reductoisomerase, a Central Catalyst of a Cell-Free Enzyme Cascade for the Manufacture of Platform Chemicals
Authors: Lv, Y. / Zheng, S. / Goldenzweig, A. / Liu, F. / Gao, Y. / Yang, X. / Kandale, A. / McGeary, R.P. / Williams, S. / Kobe, B. / Schembri, M.A. / Landsberg, M.J. / Wu, B. / Bruck, T.B. / ...Authors: Lv, Y. / Zheng, S. / Goldenzweig, A. / Liu, F. / Gao, Y. / Yang, X. / Kandale, A. / McGeary, R.P. / Williams, S. / Kobe, B. / Schembri, M.A. / Landsberg, M.J. / Wu, B. / Bruck, T.B. / Sieber, V. / Boden, M. / Rao, Z. / Fleishman, S.J. / Schenk, G. / Guddat, L.W.
History
DepositionMay 23, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ketol-acid reductoisomerase (NADP(+))
B: Ketol-acid reductoisomerase (NADP(+))
C: Ketol-acid reductoisomerase (NADP(+))
D: Ketol-acid reductoisomerase (NADP(+))
E: Ketol-acid reductoisomerase (NADP(+))
F: Ketol-acid reductoisomerase (NADP(+))
G: Ketol-acid reductoisomerase (NADP(+))
H: Ketol-acid reductoisomerase (NADP(+))
I: Ketol-acid reductoisomerase (NADP(+))
J: Ketol-acid reductoisomerase (NADP(+))
K: Ketol-acid reductoisomerase (NADP(+))
L: Ketol-acid reductoisomerase (NADP(+))
hetero molecules


Theoretical massNumber of molelcules
Total (without water)429,19324
Polymers427,99112
Non-polymers1,20112
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Ketol-acid reductoisomerase (NADP(+)) / KARI / Acetohydroxy-acid isomeroreductase / AHIR / Alpha-keto-beta-hydroxylacyl reductoisomerase


Mass: 35665.953 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni (Campylobacter) / Gene: ilvC, CW563_00670 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5T0UG45
#2: Chemical
ChemComp-PTD / PENTANEDIAL / Glutaraldehyde


Mass: 100.116 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C5H8O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Campylobacter jejuni ketol-acid reductoisomerase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 450 kDa/nm / Experimental value: YES
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74899 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0129244
ELECTRON MICROSCOPYf_angle_d0.77139360
ELECTRON MICROSCOPYf_dihedral_angle_d13.3710836
ELECTRON MICROSCOPYf_chiral_restr0.0564452
ELECTRON MICROSCOPYf_plane_restr0.0065076

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