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- PDB-6kqo: 328 K cryoEM structure of Sso-KARI in complex with Mg2+, NADH and CPD -

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Basic information

Entry
Database: PDB / ID: 6kqo
Title328 K cryoEM structure of Sso-KARI in complex with Mg2+, NADH and CPD
ComponentsKetol-acid reductoisomerase
KeywordsISOMERASE / Complex
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 Rossmann-like Domain / NAD(P)-binding domain superfamily ...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 Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
cyclopropane-1,1-dicarboxylic acid / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / Ketol-acid reductoisomerase / Putative ketol-acid reductoisomerase 2
Similarity search - Component
Biological speciesSaccharolobus solfataricus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsChen, C.Y. / Chang, Y.C. / Lin, B.L. / Huang, C.H. / Tsai, M.D.
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, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)456,88860
Polymers446,75812
Non-polymers10,13048
Water00
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


Mass: 37229.855 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Gene: SSOP1_1436 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A157T175, UniProt: Q97YJ9*PLUS
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NAI / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / NADH


Mass: 665.441 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C21H29N7O14P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-9TY / cyclopropane-1,1-dicarboxylic acid


Mass: 130.099 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C5H6O4 / 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: KARI-Mg2+/NADH/CPD complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Saccharolobus solfataricus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 20 mM Tris-Cl, pH 7.5, 50 mM NaCl and 5 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135584 / Symmetry type: POINT

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