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- PDB-8p5u: Single particle cryo-EM structure of homohexameric 2-oxoglutarate... -

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Basic information

Entry
Database: PDB / ID: 8p5u
TitleSingle particle cryo-EM structure of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum with Coenzyme A bound to the E2o domain
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 / COENZYME A / THIAMINE DIPHOSPHATE / 2-oxoglutarate dehydrogenase E1/E2 component
Similarity search - Component
Biological speciesCorynebacterium glutamicum ATCC 13032 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.17 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)821,99330
Polymers809,8326
Non-polymers12,16024
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A
32A
42A
53A
63A
74A
84A
95A
105A
116A
126A
137A
147A
158A
168A
179A
189A
1910A
2010A
2111A
2211A
2312A
2412A
2513A
2613A
2714A
2814A
2915A
3015A

NCS domain segments:

Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: GLU / End label comp-ID: GLU / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 102 - 1220 / Label seq-ID: 104 - 1222

Dom-IDComponent-IDEns-ID
111
211
322
422
533
633
744
844
955
1055
1166
1266
1377
1477
1588
1688
1799
1899
191010
201010
211111
221111
231212
241212
251313
261313
271414
281414
291515
301515

NCS ensembles :
IDDetails
1Local NCS retraints between domains: 1 2
2Local NCS retraints between domains: 3 4
3Local NCS retraints between domains: 5 6
4Local NCS retraints between domains: 7 8
5Local NCS retraints between domains: 9 10
6Local NCS retraints between domains: 11 12
7Local NCS retraints between domains: 13 14
8Local NCS retraints between domains: 15 16
9Local NCS retraints between domains: 17 18
10Local NCS retraints between domains: 19 20
11Local NCS retraints between domains: 21 22
12Local NCS retraints between domains: 23 24
13Local NCS retraints between domains: 25 26
14Local NCS retraints between domains: 27 28
15Local NCS retraints between domains: 29 30

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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-COA / COENZYME A


Mass: 767.534 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C21H36N7O16P3S / Feature type: SUBJECT OF INVESTIGATION
#4: 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
#5: Chemical
ChemComp-TPP / THIAMINE DIPHOSPHATE


Mass: 425.314 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C12H19N4O7P2S / 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
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.82 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: 12202 / Details: Each image was composed of 40 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: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1849406 / 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: 2.17→2.17 Å / Cor.coef. Fo:Fc: 0.878 / SU B: 5.148 / SU ML: 0.115 / ESU R: 0.125
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31199 --
obs0.31199 1796886 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 42.016 Å2
Refinement stepCycle: 1 / Total: 52878
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01254000
ELECTRON MICROSCOPYr_bond_other_d00.01650502
ELECTRON MICROSCOPYr_angle_refined_deg1.1431.6573266
ELECTRON MICROSCOPYr_angle_other_deg0.3581.585116226
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.72256708
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.7435384
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.066108940
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0620.27962
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0264524
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0212378
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_it5.5383.75426856
ELECTRON MICROSCOPYr_mcbond_other5.5383.75426855
ELECTRON MICROSCOPYr_mcangle_it7.7076.83333552
ELECTRON MICROSCOPYr_mcangle_other7.7076.83333553
ELECTRON MICROSCOPYr_scbond_it6.7934.51527144
ELECTRON MICROSCOPYr_scbond_other6.7924.51527137
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other10.717.93239715
ELECTRON MICROSCOPYr_long_range_B_refined13.63735.3855484
ELECTRON MICROSCOPYr_long_range_B_other13.63735.3855485
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AELECTRON MICROSCOPYLocal ncs0.013240.05013
12AELECTRON MICROSCOPYLocal ncs0.013240.05013
23AELECTRON MICROSCOPYLocal ncs0.012560.05013
24AELECTRON MICROSCOPYLocal ncs0.012560.05013
35AELECTRON MICROSCOPYLocal ncs0.017230.05013
36AELECTRON MICROSCOPYLocal ncs0.017230.05013
47AELECTRON MICROSCOPYLocal ncs0.016020.05013
48AELECTRON MICROSCOPYLocal ncs0.016020.05013
59AELECTRON MICROSCOPYLocal ncs0.017690.05013
510AELECTRON MICROSCOPYLocal ncs0.017690.05013
611AELECTRON MICROSCOPYLocal ncs0.01520.05013
612AELECTRON MICROSCOPYLocal ncs0.01520.05013
713AELECTRON MICROSCOPYLocal ncs0.018960.05013
714AELECTRON MICROSCOPYLocal ncs0.018960.05013
815AELECTRON MICROSCOPYLocal ncs0.013460.05013
816AELECTRON MICROSCOPYLocal ncs0.013460.05013
917AELECTRON MICROSCOPYLocal ncs0.019040.05013
918AELECTRON MICROSCOPYLocal ncs0.019040.05013
1019AELECTRON MICROSCOPYLocal ncs0.019320.05013
1020AELECTRON MICROSCOPYLocal ncs0.019320.05013
1121AELECTRON MICROSCOPYLocal ncs0.012460.05013
1122AELECTRON MICROSCOPYLocal ncs0.012460.05013
1223AELECTRON MICROSCOPYLocal ncs0.014710.05013
1224AELECTRON MICROSCOPYLocal ncs0.014710.05013
1325AELECTRON MICROSCOPYLocal ncs0.020980.05013
1326AELECTRON MICROSCOPYLocal ncs0.020980.05013
1427AELECTRON MICROSCOPYLocal ncs0.018670.05013
1428AELECTRON MICROSCOPYLocal ncs0.018670.05013
1529AELECTRON MICROSCOPYLocal ncs0.017090.05013
1530AELECTRON MICROSCOPYLocal ncs0.017090.05013
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.475 133069 -
obs--100 %

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