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- EMDB-70238: (1-methylalkyl)succinate synthase alpha-beta-gamma-delta complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-70238
Title(1-methylalkyl)succinate synthase alpha-beta-gamma-delta complex with bound fumarate
Map data
Sample
  • Complex: Heterodimer complex of MASS alpha with beta, gamma, delta subunits
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit MasD
    • Protein or peptide: MasB protein
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit MasC
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit Mas E
  • Ligand: FUMARIC ACID
  • Ligand: 2,3-DIHYDROXY-1,4-DITHIOBUTANE
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE (III) ION
Keywordsglycyl radical enzyme / (1-methylalkyl)succinate synthase / X-succinate synthase / alkylsuccinate synthase / Hydrocarbon degradation / fumarate addition / LYASE
Function / homology
Function and homology information


lyase activity / cytosol
Similarity search - Function
Benzylsuccinate synthase gamma subunit / Benzylsuccinate synthase gamma subunit superfamily / BssC/TutF protein / : / Formate C-acetyltransferase glycine radical, conserved site / Glycine radical domain signature. / Pyruvate formate lyase domain / Pyruvate formate lyase-like / Pyruvate formate-lyase domain profile. / Glycine radical ...Benzylsuccinate synthase gamma subunit / Benzylsuccinate synthase gamma subunit superfamily / BssC/TutF protein / : / Formate C-acetyltransferase glycine radical, conserved site / Glycine radical domain signature. / Pyruvate formate lyase domain / Pyruvate formate lyase-like / Pyruvate formate-lyase domain profile. / Glycine radical / Glycine radical domain / Glycine radical domain profile.
Similarity search - Domain/homology
MasB protein / 1-methyl alkyl succinate synthase subunit MasC / 1-methyl alkyl succinate synthase subunit MasD / 1-methyl alkyl succinate synthase subunit Mas E
Similarity search - Component
Biological speciesAzoarcus sp. HxN1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsAndorfer MC / Drennan CL
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129882 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM145910 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for anaerobic alkane activation by a multisubunit glycyl radical enzyme.
Authors: Mary C Andorfer / Talya S Levitz / Jian Liu / Ankush Chakraborty / Devin T King-Roberts / Delight Nweneka / Christa N Imrich / Catherine L Drennan /
Abstract: X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, ...X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, the only structurally characterized XSS is benzylsuccinate synthase (BSS), which functionalizes toluene. A distinct subclass of XSSs acts on saturated hydrocarbons, which possess much stronger C(sp)-H bonds than toluene, suggesting mechanistic and structural differences from BSS. Here, we use cryogenic electron microscopy to determine the structure of one such enzyme, (1-methylalkyl)succinate synthase (MASS) from strain HxN1, which functionalizes -alkanes (C6-C8). The structure reveals an asymmetric dimer in which both sides contain a catalytic α-subunit and accessory γ-subunit. One α-subunit also binds two additional subunits, β and δ. The β-subunit binds a [4Fe-4S] cluster and adopts a fold similar to BSSβ. The β-subunit appears to regulate the flexibility of the α-subunit to enable opening of the active site, affording the binding of -alkane substrates. The δ-subunit, which lacks homology to known GRE subunits, adopts a rubredoxin-like fold that binds a single Fe ion, an architecture not previously reported for GREs. MASSδ occupies the same region of the α-subunit as the activating enzyme (AE) and may regulate the conformational changes required for glycyl radical installation. Structural comparisons between MASS and BSS reveal differences in how fumarate is bound and show amino acid substitutions that could account for the binding of alkanes versus toluene. Together, this structure offers insight into anaerobic alkane activation via fumarate addition.
History
DepositionApr 16, 2025-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateAug 20, 2025-
Current statusAug 20, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70238.png

Downloads & links

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Map

FileDownload / File: emd_70238.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 256 pix.
= 299.52 Å		1.17 Å/pix.
x 256 pix.
= 299.52 Å		1.17 Å/pix.
x 256 pix.
= 299.52 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.17 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0183
Minimum - Maximum-0.08370176 - 0.16106349
Average (Standard dev.)0.000095352036 (±0.002601549)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 299.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_70238_msk_1.map
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Additional map: #1

Fileemd_70238_additional_1.map
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Half map: #2

Fileemd_70238_half_map_1.map
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Half map: #1

Fileemd_70238_half_map_2.map
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Sample components

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Entire : Heterodimer complex of MASS alpha with beta, gamma, delta subunits

EntireName: Heterodimer complex of MASS alpha with beta, gamma, delta subunits
Components
  • Complex: Heterodimer complex of MASS alpha with beta, gamma, delta subunits
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit MasD
    • Protein or peptide: MasB protein
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit MasC
    • Protein or peptide: 1-methyl alkyl succinate synthase subunit Mas E
  • Ligand: FUMARIC ACID
  • Ligand: 2,3-DIHYDROXY-1,4-DITHIOBUTANE
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE (III) ION

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Supramolecule #1: Heterodimer complex of MASS alpha with beta, gamma, delta subunits

SupramoleculeName: Heterodimer complex of MASS alpha with beta, gamma, delta subunits
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: one alpha subunits binds beta, gamma, delta subunits, and the other alpha subunit bind only gamma subunits.
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Molecular weightTheoretical: 229 KDa

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Macromolecule #1: 1-methyl alkyl succinate synthase subunit MasD

MacromoleculeName: 1-methyl alkyl succinate synthase subunit MasD / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Molecular weightTheoretical: 97.053289 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQMTATSTLS KTDLKNCETV EELRENQQWW WLAERERSAR LDYLRKATWK KGALGGNYFD GIRLDLEYPT LFTEAWKKY PNDPSMLRRA KATAYVLDNI SIFITDSAQL VGYVGSAPHT IAWRVDGAST VNSEVYNEPG IHAEPEAESL K KVAEINSY ...String:
MGSSHHHHHH SQMTATSTLS KTDLKNCETV EELRENQQWW WLAERERSAR LDYLRKATWK KGALGGNYFD GIRLDLEYPT LFTEAWKKY PNDPSMLRRA KATAYVLDNI SIFITDSAQL VGYVGSAPHT IAWRVDGAST VNSEVYNEPG IHAEPEAESL K KVAEINSY WNGQTAVDKV GRLIDPEDAV KFFSGAIGWG TPSSAFGYSG KNFEYFMKGD RAFSQIIAEI DEKIDEAEEA TI GTPSPHI LPLYDKLNNW HAMKLVLEAA IRFAGRYARL ARVMAAKETD EQRKKELLRV AETCERVPAN PPRNLQESLQ YEH FVQVLA RYEAHEGAWP SRPDYYHGPL YAKDVEVEKN ITESEAIDLV GEYMIRCSEY GSFSPRYMRE GLQGVTGTFV WTLG GVNQD GTDACNGMTI ALLKAARLVR VANPTFGFRW HPKVSNEVLR ECFECIRQGL GYPTLRNDPV LIQNTMHWYG HPLEE ARTW VHMACMSPNP TTKHGTSPFR MASATMNSAK TIEYVLHNGY DRVVNMQMGP KTGDAREIKD FEDLFERWTV QLKWLM NLL VRTVNLGRFK DPEFFGRPFL SAITERAVEH GIDAVSPEGE RGNAWVTAFT WIENVDSMAA IKKLVFDDKK YTMSQLI DA LEAEWDGYEQ MRLDFVKNGP KWGNDDDYVD DIMLRCLSVA AEHSRNIQCT SGNCWPILPE NVSGNIHYAN IVGALPNG R RRGDALYDGG VSPGPGLDKA GPTAVLKSVG KIDHVNQGRS FLLNQRLSPT QLAGDKGFQL WNSYVRTWAE LGIDHIQFN VISDKVLRAA QNDPEGYQEV IVRVAGYSAH FIDISRKTQD NIIQRTVQGL GENLYFQG

UniProtKB: 1-methyl alkyl succinate synthase subunit MasD

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Macromolecule #2: MasB protein

MacromoleculeName: MasB protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Molecular weightTheoretical: 13.460091 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSRRDEWKKL QEEMTRDGGE IKSLETVPEQ ACGICLNFTD NAYGSDGRGS CNVLKAGSNI SLPDVIITRS GENGYITFFN SDAKYCPNF ERMKLIDTDG HECADPISRR VQRQLSSIKK S

UniProtKB: MasB protein

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Macromolecule #3: 1-methyl alkyl succinate synthase subunit MasC

MacromoleculeName: 1-methyl alkyl succinate synthase subunit MasC / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Molecular weightTheoretical: 6.894553 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
STCKECRNYF PINEEASRGD CVRRISDERQ SYYTARPTTE AAKCEGCSDY LENTRTAKAH

UniProtKB: 1-methyl alkyl succinate synthase subunit MasC

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Macromolecule #4: 1-methyl alkyl succinate synthase subunit Mas E

MacromoleculeName: 1-methyl alkyl succinate synthase subunit Mas E / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Molecular weightTheoretical: 8.033105 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKCTECGHEA EVMKFRYHYN PRIDASLSLR QCPECQAVVT VDELKREVLG RMHNGDDPWG KSAGIENLAE G

UniProtKB: 1-methyl alkyl succinate synthase subunit Mas E

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Macromolecule #5: FUMARIC ACID

MacromoleculeName: FUMARIC ACID / type: ligand / ID: 5 / Number of copies: 1 / Formula: FUM
Molecular weightTheoretical: 116.072 Da
Chemical component information

ChemComp-FUM:
FUMARIC ACID

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Macromolecule #6: 2,3-DIHYDROXY-1,4-DITHIOBUTANE

MacromoleculeName: 2,3-DIHYDROXY-1,4-DITHIOBUTANE / type: ligand / ID: 6 / Number of copies: 1 / Formula: DTT
Molecular weightTheoretical: 154.251 Da
Chemical component information

ChemComp-DTT:
2,3-DIHYDROXY-1,4-DITHIOBUTANE

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Macromolecule #7: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 3 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #8: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMHEPES
300.0 mMSodium Chloride
1.0 mMFumarate
1.0 mMDTT

Details: 50 mM HEPES pH 8.0, 300 mM NaCl, 1 mM Fumarate, 1 mM DTT
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.42 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 356678
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-9o8u:
(1-methylalkyl)succinate synthase alpha-beta-gamma-delta complex with bound fumarate

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