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- EMDB-17455: Single particle cryo-EM structure of homohexameric 2-oxoglutarate... -

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Basic information

Entry
Database: EMDB / ID: EMD-17455
TitleSingle particle cryo-EM structure of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum following reaction with the 2-oxoglutarate analogue succinyl phosphonate
Map dataOdhA_SP main_map_dens_mod
Sample
  • Complex: Homohexameric 2-oxoglutarate dehydrogenase
    • Protein or peptide: 2-oxoglutarate dehydrogenase E1/E2 component
  • Ligand: MAGNESIUM ION
  • Ligand: ACETYL COENZYME *A
  • Ligand: (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
Keywords2-oxoglutarate dehydrogenase / ODH / OXIDOREDUCTASE
Function / homology
Function and homology information


oxoglutarate dehydrogenase (succinyl-transferring) / oxoglutarate dehydrogenase (succinyl-transferring) activity / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / thiamine pyrophosphate binding / tricarboxylic acid cycle / magnesium ion binding
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
2-oxoglutarate dehydrogenase E1/E2 component
Similarity search - Component
Biological speciesCorynebacterium glutamicum ATCC 13032 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.26 Å
AuthorsYang L / Mechaly AM / Bellinzoni M
Funding support France, 2 items
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 EM / Megrian D / Gubellini F / Alzari PM / Bellinzoni M
History
DepositionMay 24, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17455.png

Downloads & links

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Map

FileDownload / File: emd_17455.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOdhA_SP main_map_dens_mod
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 448 pix.
= 385.28 Å		0.86 Å/pix.
x 448 pix.
= 385.28 Å		0.86 Å/pix.
x 448 pix.
= 385.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-1.748818 - 66.571915000000004
Average (Standard dev.)0.012396597 (±1.183449)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: OdhA SP half map 1 no mask cryoSPARC

Fileemd_17455_half_map_1.map
AnnotationOdhA_SP half_map_1_no_mask_cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: OdhA SP half map 2 no mask cryoSPARC

Fileemd_17455_half_map_2.map
AnnotationOdhA_SP half_map_2_no_mask_cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homohexameric 2-oxoglutarate dehydrogenase

EntireName: Homohexameric 2-oxoglutarate dehydrogenase
Components
  • Complex: Homohexameric 2-oxoglutarate dehydrogenase
    • Protein or peptide: 2-oxoglutarate dehydrogenase E1/E2 component
  • Ligand: MAGNESIUM ION
  • Ligand: ACETYL COENZYME *A
  • Ligand: (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

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Supramolecule #1: Homohexameric 2-oxoglutarate dehydrogenase

SupramoleculeName: Homohexameric 2-oxoglutarate dehydrogenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Corynebacterium glutamicum ATCC 13032 (bacteria)
Molecular weightTheoretical: 808 KDa

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Macromolecule #1: 2-oxoglutarate dehydrogenase E1/E2 component

MacromoleculeName: 2-oxoglutarate dehydrogenase E1/E2 component / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: oxoglutarate dehydrogenase (succinyl-transferring)
Source (natural)Organism: Corynebacterium glutamicum ATCC 13032 (bacteria)
Molecular weightTheoretical: 134.972078 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSMSSASTFG QNAWLVDEMF QQFQKDPKSV DKEWRELFEA QGGPNTTPAT TEAQPSAPKE SAKPAPKAAP AAKAAPRVET KPADKTAPK AKESSVPQQP KLPEPGQTPI RGIFKSIAKN MDISLEIPTA TSVRDMPARL MFENRAMVND QLKRTRGGKI S FTHIIGYA ...String:
GSMSSASTFG QNAWLVDEMF QQFQKDPKSV DKEWRELFEA QGGPNTTPAT TEAQPSAPKE SAKPAPKAAP AAKAAPRVET KPADKTAPK AKESSVPQQP KLPEPGQTPI RGIFKSIAKN MDISLEIPTA TSVRDMPARL MFENRAMVND QLKRTRGGKI S FTHIIGYA MVKAVMAHPD MNNSYDVIDG KPTLIVPEHI NLGLAIDLPQ KDGSRALVVA AIKETEKMNF SEFLAAYEDI VA RSRKGKL TMDDYQGVTV SLTNPGGIGT RHSVPRLTKG QGTIIGVGSM DYPAEFQGAS EDRLAELGVG KLVTITSTYD HRV IQGAVS GEFLRTMSRL LTDDSFWDEI FDAMNVPYTP MRWAQDVPNT GVDKNTRVMQ LIEAYRSRGH LIADTNPLSW VQPG MPVPD HRDLDIETHN LTIWDLDRTF NVGGFGGKET MTLREVLSRL RAAYTLKVGS EYTHILDRDE RTWLQDRLEA GMPKP TQAE QKYILQKLNA AEAFENFLQT KYVGQKRFSL EGAEALIPLM DSAIDTAAGQ GLDEVVIGMP HRGRLNVLFN IVGKPL ASI FNEFEGQMEQ GQIGGSGDVK YHLGSEGQHL QMFGDGEIKV SLTANPSHLE AVNPVMEGIV RAKQDYLDKG VDGKTVV PL LLHGDAAFAG LGIVPETINL AKLRGYDVGG TIHIVVNNQI GFTTTPDSSR SMHYATDYAK AFGCPVFHVN GDDPEAVV W VGQLATEYRR RFGKDVFIDL VCYRLRGHNE ADDPSMTQPK MYELITGRET VRAQYTEDLL GRGDLSNEDA EAVVRDFHD QMESVFNEVK EGGKKQAEAQ TGITGSQKLP HGLETNISRE ELLELGQAFA NTPEGFNYHP RVAPVAKKRV SSVTEGGIDW AWGELLAFG SLANSGRLVR LAGEDSRRGT FTQRHAVAID PATAEEFNPL HELAQSKGNN GKFLVYNSAL TEYAGMGFEY G YSVGNEDS IVAWEAQFGD FANGAQTIID EYVSSGEAKW GQTSKLILLL PHGYEGQGPD HSSARIERFL QLCAEGSMTV AQ PSTPANH FHLLRRHALS DLKRPLVIFT PKSMLRNKAA ASAPEDFTEV TKFQSVINDP NVADAAKVKK VMLVSGKLYY ELA KRKEKD GRDDIAIVRI EMLHPIPFNR ISEALAGYPN AEEVLFVQDE PANQGPWPFY QEHLPELIPN MPKMRRVSRR AQSS TATGV AKVHQLEEKQ LIDEAFEA

UniProtKB: 2-oxoglutarate dehydrogenase E1/E2 component

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ACETYL COENZYME *A

MacromoleculeName: ACETYL COENZYME *A / type: ligand / ID: 3 / Number of copies: 6 / Formula: ACO
Molecular weightTheoretical: 809.571 Da
Chemical component information

ChemComp-ACO:
ACETYL COENZYME *A

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Macromolecule #4: (4~{S})-4-[(2~{R})-3-[(4-azanyl-2-methyl-pyrimidin-5-yl)methyl]-4...

MacromoleculeName: (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
type: ligand / ID: 4 / Number of copies: 6 / Formula: QSP
Molecular weightTheoretical: 608.391 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration12.0 mg/mL
BufferpH: 7.5 / Component:
ConcentrationName
20.0 mMHEPES-Na
500.0 mMNaCl
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 16647 / Average exposure time: 1.4 sec. / Average electron dose: 40.0 e/Å2 / Details: Each image was composed by 60 frames
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model from cryoSPARC
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 646352
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8p5t


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8p5w:
Single particle cryo-EM structure of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum following reaction with the 2-oxoglutarate analogue succinyl phosphonate

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