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- PDB-8p5r: Crystal structure of full-length, homohexameric 2-oxoglutarate de... -

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Basic information

Entry
Database: PDB / ID: 8p5r
TitleCrystal structure of full-length, homohexameric 2-oxoglutarate dehydrogenase KGD from Mycobacterium smegmatis in complex with GarA
Components
  • Glycogen accumulation regulator GarA
  • Multifunctional 2-oxoglutarate metabolism enzyme
KeywordsOXIDOREDUCTASE / 2-oxoglutarate dehydrogenase / ODH / KGD
Function / homology
Function and homology information


2-hydroxy-3-oxoadipate synthase / 2-oxoglutarate decarboxylase / 2-oxoglutarate decarboxylase activity / 2-hydroxy-3-oxoadipate synthase activity / oxoglutarate dehydrogenase (succinyl-transferring) / oxoglutarate dehydrogenase (succinyl-transferring) activity / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / oxoglutarate dehydrogenase complex / thiamine pyrophosphate binding ...2-hydroxy-3-oxoadipate synthase / 2-oxoglutarate decarboxylase / 2-oxoglutarate decarboxylase activity / 2-hydroxy-3-oxoadipate synthase activity / 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-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 / SMAD/FHA domain superfamily / Thiamin diphosphate-binding fold
Similarity search - Domain/homology
THIAMINE DIPHOSPHATE / Glycogen accumulation regulator GarA / Multifunctional 2-oxoglutarate metabolism enzyme
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4.562 Å
AuthorsWagner, T. / Mechaly, A.M. / Alzari, P.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: Multifunctional 2-oxoglutarate metabolism enzyme
B: Multifunctional 2-oxoglutarate metabolism enzyme
C: Multifunctional 2-oxoglutarate metabolism enzyme
D: Multifunctional 2-oxoglutarate metabolism enzyme
E: Multifunctional 2-oxoglutarate metabolism enzyme
F: Multifunctional 2-oxoglutarate metabolism enzyme
G: Glycogen accumulation regulator GarA
H: Glycogen accumulation regulator GarA
I: Glycogen accumulation regulator GarA
J: Glycogen accumulation regulator GarA
K: Glycogen accumulation regulator GarA
L: Glycogen accumulation regulator GarA
N: Multifunctional 2-oxoglutarate metabolism enzyme
O: Multifunctional 2-oxoglutarate metabolism enzyme
P: Multifunctional 2-oxoglutarate metabolism enzyme
Q: Multifunctional 2-oxoglutarate metabolism enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,488,09336
Polymers1,485,07516
Non-polymers3,01820
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)325.75, 325.75, 396.944
Angle α, β, γ (deg.)90, 90, 120
Int Tables number170
Space group name H-MP65

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Components

#1: Protein
Multifunctional 2-oxoglutarate metabolism enzyme / 2-hydroxy-3-oxoadipate synthase / HOA synthase / HOAS / 2-oxoglutarate carboxy-lyase / 2- ...2-hydroxy-3-oxoadipate synthase / HOA synthase / HOAS / 2-oxoglutarate carboxy-lyase / 2-oxoglutarate decarboxylase / Alpha-ketoglutarate decarboxylase / KG decarboxylase / KGD / Alpha-ketoglutarate-glyoxylate carboligase


Mass: 138548.078 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: kgd, sucA, MSMEG_5049, MSMEI_4922 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0R2B1, 2-hydroxy-3-oxoadipate synthase, 2-oxoglutarate decarboxylase, oxoglutarate dehydrogenase (succinyl-transferring), dihydrolipoyllysine-residue succinyltransferase
#2: Protein
Glycogen accumulation regulator GarA


Mass: 16599.041 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: garA, MSMEG_3647, MSMEI_3561 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0QYG2
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.09 Å3/Da / Density % sol: 69.95 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop
Details: 0.1 M bicine pH 8.0, 30% (v/v) PEG550MME, 0.2 M NaCl

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.9763 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Nov 21, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 4.562→282.108 Å / Num. obs: 121219 / % possible obs: 94.6 % / Redundancy: 11.45 %
Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last ...Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last updated 2020-04-13: http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v50.dic/Index/) and the actual quantities provided by MRFANA (https://github.com/githubgphl/MRFANA) from the autoPROC package (https://www.globalphasing.com/autoproc/). In general, the mmCIF categories here should provide items that are currently used in the PDB archive. If there are alternatives, the one recommended by the PDB developers has been selected. The distinction between *_all and *_obs quantities is not always clear: often only one version is actively used within the PDB archive (or is the one recommended by PDB developers). The intention of distinguishing between classes of reflections before and after some kind of observation criterion was applied, can in principle be useful - but such criteria change in various ways throughout the data processing steps (rejection of overloaded or too partial reflections, outlier/misfit rejections during scaling etc) and there is no retrospect computation of data scaling/merging statistics for the reflections used in the final refinement (where another observation criterion might have been applied). Typical data processing will usually only provide one version of statistics at various stages and these are given in the recommended item here, irrespective of the "_all" and "_obs" connotation, see e.g. the use of _reflns.pdbx_Rmerge_I_obs, _reflns.pdbx_Rrim_I_all and _reflns.pdbx_Rpim_I_all. Please note that all statistics related to "merged intensities" (or "merging") are based on inverse-variance weighting of the individual measurements making up a symmetry-unique reflection. This is standard for several decades now, even if some of the dictionary definitions seem to suggest that a simple "mean" or "average" intensity is being used instead. R-values are always given for all symmetry-equivalent reflections following Friedel's law, i.e. Bijvoet pairs are not treated separately (since we want to describe the overall mean intensity and not the mean I(+) and I(-) here). The Rrim metric is identical to the Rmeas R-value and only differs in name. _reflns.pdbx_number_measured_all is the number of measured intensities just before the final merging step (at which point no additional rejection takes place). _reflns.number_obs is the number of symmetry-unique observations, i.e. the result of merging those measurements via inverse-variance weighting. _reflns.pdbx_netI_over_sigmaI is based on the merged intensities (_reflns.number_obs) as expected. _reflns.pdbx_redundancy is synonymous with "multiplicity". The per-shell item _reflns_shell.number_measured_all corresponds to the overall value _reflns.pdbx_number_measured_all. The per-shell item _reflns_shell.number_unique_all corresponds to the overall value _reflns.number_obs. The per-shell item _reflns_shell.percent_possible_all corresponds to the overall value _reflns.percent_possible_obs. The per-shell item _reflns_shell.meanI_over_sigI_obs corresponds to the overall value given as _reflns.pdbx_netI_over_sigmaI. But be aware of the incorrect definition of the former in the current dictionary!
CC1/2: 0.998 / CC1/2 anomalous: -0.117 / Rmerge(I) obs: 0.245 / Rpim(I) all: 0.0758 / Rrim(I) all: 0.2565 / AbsDiff over sigma anomalous: 0.733 / Baniso tensor eigenvalue 1: 17.1186 Å2 / Baniso tensor eigenvalue 2: 17.1186 Å2 / Baniso tensor eigenvalue 3: 0 Å2 / Baniso tensor eigenvector 1 ortho1: 1 / Baniso tensor eigenvector 1 ortho2: 0 / Baniso tensor eigenvector 1 ortho3: 0 / Baniso tensor eigenvector 2 ortho1: 0 / Baniso tensor eigenvector 2 ortho2: 1 / Baniso tensor eigenvector 2 ortho3: 0 / Baniso tensor eigenvector 3 ortho1: 0 / Baniso tensor eigenvector 3 ortho2: 0 / Baniso tensor eigenvector 3 ortho3: 1 / Aniso diffraction limit 1: 4.713 Å / Aniso diffraction limit 2: 4.713 Å / Aniso diffraction limit 3: 4.562 Å / Aniso diffraction limit axis 1 ortho1: 1 / Aniso diffraction limit axis 1 ortho2: 0 / Aniso diffraction limit axis 1 ortho3: 0 / Aniso diffraction limit axis 2 ortho1: 0 / Aniso diffraction limit axis 2 ortho2: 1 / Aniso diffraction limit axis 2 ortho3: 0 / Aniso diffraction limit axis 3 ortho1: 0 / Aniso diffraction limit axis 3 ortho2: 0 / Aniso diffraction limit axis 3 ortho3: 1 / Net I/σ(I): 8.11 / Num. measured all: 1388278 / Observed signal threshold: 1.2 / Orthogonalization convention: pdb / % possible anomalous: 94.6 / % possible ellipsoidal: 94.6 / % possible ellipsoidal anomalous: 94.6 / % possible spherical: 89.6 / % possible spherical anomalous: 89.7 / Redundancy anomalous: 5.78 / Signal type: local
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. measured obsNum. unique allNum. unique obsCC1/2CC1/2 anomalousRpim(I) allRrim(I) allAbsDiff over sigma anomalous% possible anomalous% possible ellipsoidal% possible ellipsoidal anomalous% possible spherical% possible spherical anomalousRedundancy anomalous% possible all
12.91-282.10810.850.053828.086572165721605960590.999-0.2550.01710.05650.5499.799.999.799.999.75.5699.9
10.228-12.9110.780.06527.496539565395606460640.999-0.4120.02070.06820.5961001001001001005.47100
8.93-10.22810.630.084323.266444164441606060600.999-0.2870.02720.08860.6821001001001001005.38100
8.108-8.9310.990.116318.526660966609606260620.998-0.2350.03690.1220.7151001001001001005.55100
7.525-8.10811.290.169913.986840268402606060600.995-0.1490.05320.1780.751001001001001005.7100
7.078-7.52511.470.27149.66949269492606060600.989-0.0830.0840.28420.7611001001001001005.79100
6.723-7.07811.520.36697.366983669836606060600.979-0.0810.11320.3840.7491001001001001005.81100
6.429-6.72311.560.45765.947009670096606460640.971-0.0220.14090.47890.761001001001001005.83100
6.182-6.42911.590.64024.427023870238605860580.934-0.0380.19670.66980.7641001001001001005.84100
5.966-6.18211.610.83243.397031670316605960590.906-0.040.25550.87080.7561001001001001005.85100
5.78-5.96611.620.95337044470444606360630.8680.0040.29240.9970.7771001001001001005.85100
5.613-5.7811.631.15262.497051070510606260620.809-0.0190.35331.20560.7641001001001001005.86100
5.465-5.61311.631.2452.267047970479606060600.787-0.0260.38171.30230.7511001001001001005.86100
5.331-5.46511.651.32482.117060670606606160610.778-0.0240.40561.38560.75999.899.899.899.899.85.8699.8
5.208-5.33111.651.51731.847066270662606660660.758-0.0220.46481.5870.75198.898.698.898.698.85.8698.6
5.095-5.20811.661.49181.97067170671606060600.75-0.0080.45661.56030.7698.197.998.197.998.15.8697.9
4.988-5.09511.681.52341.857073070730605860580.74-0.0160.4661.59320.76595.995.695.995.695.95.8795.6
4.886-4.98811.721.74751.597102971029606160610.7150.010.53361.82720.76491.791.491.791.491.75.8991.4
4.778-4.88611.751.68621.697123671236606360630.726-0.0070.51411.76290.74579.679.279.679.279.65.979.2
4.562-4.77811.781.91451.487136571365605960590.701-0.0340.58282.00140.7395352.95334.8355.9152.9

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Processing

Software
NameVersionClassification
autoPROC1.0.5 20220608data processing
autoPROCJan 10, 2022data processing
Aimless0.7.7data scaling
autoPROC2.3.87data processing
BUSTER2.10.4 (8-JUN-2022)refinement
autoPROCdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 4.562→45.99 Å / Cor.coef. Fo:Fc: 0.944 / Cor.coef. Fo:Fc free: 0.928 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.707
RfactorNum. reflection% reflectionSelection details
Rfree0.2292 6055 -RANDOM
Rwork0.1983 ---
obs0.1998 121081 89.6 %-
Displacement parametersBiso mean: 236.11 Å2
Baniso -1Baniso -2Baniso -3
1--5.1547 Å20 Å20 Å2
2---5.1547 Å20 Å2
3---10.3094 Å2
Refine analyzeLuzzati coordinate error obs: 0.54 Å
Refinement stepCycle: LAST / Resolution: 4.562→45.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms56835 0 170 0 57005
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.00658151HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.7378847HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d26866SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes10192HARMONIC5
X-RAY DIFFRACTIONt_it57989HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion7539SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact46734SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.21
X-RAY DIFFRACTIONt_other_torsion2.83
LS refinement shellResolution: 4.562→4.69 Å
RfactorNum. reflection% reflection
Rfree0.3064 111 -
Rwork0.3096 --
obs--22.72 %

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