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- PDB-8p5x: Single particle cryo-EM structure of the complex between Coryneba... -

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Basic information

Entry
Database: PDB / ID: 8p5x
TitleSingle particle cryo-EM structure of the complex between Corynebacterium glutamicum homohexameric 2-oxoglutarate dehydrogenase OdhA and the FHA-protein inhibitor OdhI
Components
  • 2-oxoglutarate dehydrogenase E1/E2 component
  • Oxoglutarate dehydrogenase inhibitor
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 / mRNA binding / magnesium ion binding / cytoplasm / 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-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 / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Chloramphenicol acetyltransferase-like domain superfamily / Thiamin diphosphate-binding fold / SMAD/FHA domain superfamily
Similarity search - Domain/homology
THIAMINE DIPHOSPHATE / Oxoglutarate dehydrogenase inhibitor / 2-oxoglutarate dehydrogenase E1/E2 component
Similarity search - Component
Biological speciesCorynebacterium glutamicum ATCC 13032 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.29 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
G: Oxoglutarate dehydrogenase inhibitor
H: Oxoglutarate dehydrogenase inhibitor
I: Oxoglutarate dehydrogenase inhibitor
J: Oxoglutarate dehydrogenase inhibitor
K: Oxoglutarate dehydrogenase inhibitor
L: Oxoglutarate dehydrogenase inhibitor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)905,37524
Polymers902,67712
Non-polymers2,69812
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
21B
12A
22C
13A
23D
14A
24E
15A
25F
16B
26C
17B
27D
18B
28E
19B
29F
110C
210D
111C
211E
112C
212F
113D
213E
114D
214F
115E
215F
116G
216H
117G
217I
118G
218J
119G
219K
120G
220L
121H
221I
122H
222J
123H
223K
124H
224L
125I
225J
126I
226K
127I
227L
128J
228K
129J
229L
130K
230L

NCS domain segments:

Component-ID: _ / End auth comp-ID: ALA / End label comp-ID: ALA / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11PROPROAA102 - 1221104 - 1223
21PROPROBB102 - 1221104 - 1223
12PROPROAA102 - 1221104 - 1223
22PROPROCC102 - 1221104 - 1223
13PROPROAA102 - 1221104 - 1223
23PROPRODD102 - 1221104 - 1223
14PROPROAA102 - 1221104 - 1223
24PROPROEE102 - 1221104 - 1223
15PROPROAA102 - 1221104 - 1223
25PROPROFF102 - 1221104 - 1223
16PROPROBB102 - 1221104 - 1223
26PROPROCC102 - 1221104 - 1223
17PROPROBB102 - 1221104 - 1223
27PROPRODD102 - 1221104 - 1223
18PROPROBB102 - 1221104 - 1223
28PROPROEE102 - 1221104 - 1223
19PROPROBB102 - 1221104 - 1223
29PROPROFF102 - 1221104 - 1223
110PROPROCC102 - 1221104 - 1223
210PROPRODD102 - 1221104 - 1223
111PROPROCC102 - 1221104 - 1223
211PROPROEE102 - 1221104 - 1223
112PROPROCC102 - 1221104 - 1223
212PROPROFF102 - 1221104 - 1223
113PROPRODD102 - 1221104 - 1223
213PROPROEE102 - 1221104 - 1223
114PROPRODD102 - 1221104 - 1223
214PROPROFF102 - 1221104 - 1223
115PROPROEE102 - 1221104 - 1223
215PROPROFF102 - 1221104 - 1223
116GLUGLUGG40 - 14241 - 143
216GLUGLUHH40 - 14241 - 143
117GLUGLUGG40 - 14241 - 143
217GLUGLUII40 - 14241 - 143
118GLUGLUGG40 - 14241 - 143
218GLUGLUJJ40 - 14241 - 143
119GLUGLUGG40 - 14241 - 143
219GLUGLUKK40 - 14241 - 143
120GLUGLUGG40 - 14241 - 143
220GLUGLULL40 - 14241 - 143
121GLUGLUHH40 - 14241 - 143
221GLUGLUII40 - 14241 - 143
122GLUGLUHH40 - 14241 - 143
222GLUGLUJJ40 - 14241 - 143
123GLUGLUHH40 - 14241 - 143
223GLUGLUKK40 - 14241 - 143
124GLUGLUHH40 - 14241 - 143
224GLUGLULL40 - 14241 - 143
125GLUGLUII40 - 14241 - 143
225GLUGLUJJ40 - 14241 - 143
126GLUGLUII40 - 14241 - 143
226GLUGLUKK40 - 14241 - 143
127GLUGLUII40 - 14241 - 143
227GLUGLULL40 - 14241 - 143
128GLUGLUJJ40 - 14241 - 143
228GLUGLUKK40 - 14241 - 143
129GLUGLUJJ40 - 14241 - 143
229GLUGLULL40 - 14241 - 143
130GLUGLUKK40 - 14241 - 143
230GLUGLULL40 - 14241 - 143

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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: Protein
Oxoglutarate dehydrogenase inhibitor


Mass: 15474.066 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Corynebacterium glutamicum ATCC 13032 (bacteria)
Gene: odhI, Cgl1441, cg1630 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8NQJ3
#3: Chemical
ChemComp-TPP / THIAMINE DIPHOSPHATE


Mass: 425.314 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C12H19N4O7P2S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / 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: Complex of homohexameric 2-oxoglutarate dehydrogenase OdhA and the FHA-domain inhibitor OdhI
Type: COMPLEX / Details: 1:1 complex / Entity ID: #1-#2 / 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.: 11.5 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: 2.45 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: 19443 / Details: Each image was composed by 40 frames

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 958690 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeSource nameType
18P5T

8p5t

18P5TPDBexperimental model
24QCJ

4qcj

14QCJPDBexperimental model
RefinementResolution: 2.29→2.29 Å / Cor.coef. Fo:Fc: 0.872 / SU B: 6.25 / SU ML: 0.133 / ESU R: 0.142
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31969 --
obs0.31969 1697269 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 43.46 Å2
Refinement stepCycle: 1 / Total: 55346
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01356500
ELECTRON MICROSCOPYr_bond_other_d00.01553239
ELECTRON MICROSCOPYr_angle_refined_deg1.4411.64476597
ELECTRON MICROSCOPYr_angle_other_deg1.1221.581122352
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.11357086
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.30422.0093101
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.946159370
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.33315432
ELECTRON MICROSCOPYr_chiral_restr0.0710.27290
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0265112
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0213096
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_it5.4464.21728416
ELECTRON MICROSCOPYr_mcbond_other5.4464.21728415
ELECTRON MICROSCOPYr_mcangle_it8.0976.39435478
ELECTRON MICROSCOPYr_mcangle_other8.0976.39435479
ELECTRON MICROSCOPYr_scbond_it6.1724.9128084
ELECTRON MICROSCOPYr_scbond_other6.1724.9128085
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.7227.13241120
ELECTRON MICROSCOPYr_long_range_B_refined13.45349.56956577
ELECTRON MICROSCOPYr_long_range_B_other13.45349.57156578
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A630440.01
12B630440.01
21A630520.01
22C630520.01
31A630120.01
32D630120.01
41A630920.01
42E630920.01
51A630320.01
52F630320.01
61B630120
62C630120
71B630560.01
72D630560.01
81B630600
82E630600
91B630360
92F630360
101C629940.01
102D629940.01
111C630800
112E630800
121C630280
122F630280
131D630440.01
132E630440.01
141D630140.01
142F630140.01
151E630580
152F630580
161G55220
162H55220
171G55200
172I55200
181G55220
182J55220
191G55200
192K55200
201G55220
202L55220
211H55240
212I55240
221H55220
222J55220
231H55220
232K55220
241H55200
242L55200
251I55200
252J55200
261I55200
262K55200
271I55200
272L55200
281J55180
282K55180
291J55200
292L55200
301K55200
302L55200
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.469 125688 -
obs--100 %

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