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- PDB-8p5w: Single particle cryo-EM structure of homohexameric 2-oxoglutarate... -

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Basic information

Entry
Database: PDB / ID: 8p5w
TitleSingle particle cryo-EM structure of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum following reaction with the 2-oxoglutarate analogue succinyl phosphonate
Components2-oxoglutarate dehydrogenase E1/E2 component
KeywordsOXIDOREDUCTASE / 2-oxoglutarate dehydrogenase / ODH
Function / homology
Function and homology information


oxoglutarate dehydrogenase (succinyl-transferring) / oxoglutarate dehydrogenase (succinyl-transferring) activity / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / oxoglutarate dehydrogenase complex / thiamine pyrophosphate binding / tricarboxylic acid cycle / magnesium ion binding / cytosol
Similarity search - Function
2-oxoglutarate dehydrogenase E1 component, N-terminal domain / 2-oxoglutarate dehydrogenase N-terminus / Multifunctional 2-oxoglutarate metabolism enzyme, C-terminal / Multifunctional 2-oxoglutarate metabolism enzyme, C-terminal domain superfamily / 2-oxoglutarate dehydrogenase C-terminal / 2-oxoglutarate dehydrogenase E1 component / Dehydrogenase, E1 component / Dehydrogenase E1 component / 2-oxoacid dehydrogenase acyltransferase, catalytic domain / 2-oxoacid dehydrogenases acyltransferase (catalytic domain) ...2-oxoglutarate dehydrogenase E1 component, N-terminal domain / 2-oxoglutarate dehydrogenase N-terminus / Multifunctional 2-oxoglutarate metabolism enzyme, C-terminal / Multifunctional 2-oxoglutarate metabolism enzyme, C-terminal domain superfamily / 2-oxoglutarate dehydrogenase C-terminal / 2-oxoglutarate dehydrogenase E1 component / Dehydrogenase, E1 component / Dehydrogenase E1 component / 2-oxoacid dehydrogenase acyltransferase, catalytic domain / 2-oxoacid dehydrogenases acyltransferase (catalytic domain) / Transketolase-like, pyrimidine-binding domain / Transketolase, pyrimidine binding domain / Transketolase, pyrimidine binding domain / Chloramphenicol acetyltransferase-like domain superfamily / Thiamin diphosphate-binding fold
Similarity search - Domain/homology
ACETYL COENZYME *A / Chem-QSP / 2-oxoglutarate dehydrogenase E1/E2 component
Similarity search - Component
Biological speciesCorynebacterium glutamicum ATCC 13032 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.26 Å
AuthorsYang, L. / Mechaly, A.M. / Bellinzoni, M.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-13-JSV8-0003 France
Agence Nationale de la Recherche (ANR)ANR-18-CE92-0003 France
Citation
Journal: Nat Commun / Year: 2023
Title: High resolution cryo-EM and crystallographic snapshots of the actinobacterial two-in-one 2-oxoglutarate dehydrogenase.
Authors: Lu Yang / Tristan Wagner / Ariel Mechaly / Alexandra Boyko / Eduardo M Bruch / Daniela Megrian / Francesca Gubellini / Pedro M Alzari / Marco Bellinzoni /
Abstract: Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), ...Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), actinobacteria rely on a two-in-one protein (OdhA) in which both the oxidative decarboxylation and succinyl transferase steps are carried out by the same polypeptide. Here we describe high-resolution cryo-EM and crystallographic snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that assembles into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the signalling protein OdhI.
#1: Journal: Biorxiv / Year: 2023
Title: High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties
Authors: Yang, L. / Wagner, T. / Mechaly, A. / Boyko, A. / Bruch, E.M. / Megrian, D. / Gubellini, F. / Alzari, P.M. / Bellinzoni, M.
History
DepositionMay 24, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 2-oxoglutarate dehydrogenase E1/E2 component
B: 2-oxoglutarate dehydrogenase E1/E2 component
C: 2-oxoglutarate dehydrogenase E1/E2 component
D: 2-oxoglutarate dehydrogenase E1/E2 component
E: 2-oxoglutarate dehydrogenase E1/E2 component
F: 2-oxoglutarate dehydrogenase E1/E2 component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)818,48624
Polymers809,8326
Non-polymers8,65418
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
2-oxoglutarate dehydrogenase E1/E2 component / ODH E1/E2 component


Mass: 134972.078 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Corynebacterium glutamicum ATCC 13032 (bacteria)
Gene: odhA, Cgl1129, cg1280 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q8NRC3, oxoglutarate dehydrogenase (succinyl-transferring), dihydrolipoyllysine-residue succinyltransferase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-ACO / ACETYL COENZYME *A


Mass: 809.571 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C23H38N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-QSP / (4~{S})-4-[(2~{R})-3-[(4-azanyl-2-methyl-pyrimidin-5-yl)methyl]-4-methyl-5-[2-[oxidanyl(phosphonooxy)phosphoryl]oxyethyl]-2~{H}-1,3-thiazol-2-yl]-4-oxidanyl-4-phosphono-butanoic acid


Mass: 608.391 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C16H27N4O13P3S / 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: Homohexameric 2-oxoglutarate dehydrogenase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.808 MDa / Experimental value: NO
Source (natural)Organism: Corynebacterium glutamicum ATCC 13032 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES-Na1
2500 mMNaCl1
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.4 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16647 / Details: Each image was composed by 60 frames

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9REFMACmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 646352 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 8P5T

8p5t


Accession code: 8P5T / Source name: PDB / Type: experimental model
RefinementResolution: 2.26→2.26 Å / Cor.coef. Fo:Fc: 0.88 / SU B: 6.094 / SU ML: 0.129 / ESU R: 0.143
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31511 --
obs0.31511 1572575 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 43.874 Å2
Refinement stepCycle: 1 / Total: 52656
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01253766
ELECTRON MICROSCOPYr_bond_other_d00.01650352
ELECTRON MICROSCOPYr_angle_refined_deg1.0111.65272930
ELECTRON MICROSCOPYr_angle_other_deg0.3151.581115884
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.82456708
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.3365384
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.505108940
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0510.27944
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0264140
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0212330
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.4894.04926856
ELECTRON MICROSCOPYr_mcbond_other4.4894.04926855
ELECTRON MICROSCOPYr_mcangle_it7.2087.3333552
ELECTRON MICROSCOPYr_mcangle_other7.2087.3333553
ELECTRON MICROSCOPYr_scbond_it5.5084.67426910
ELECTRON MICROSCOPYr_scbond_other5.5084.67426909
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.1098.26439379
ELECTRON MICROSCOPYr_long_range_B_refined12.25136.9555627
ELECTRON MICROSCOPYr_long_range_B_other12.25136.9555628
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.481 116494 -
obs--100 %

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