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- EMDB-19492: Trypanosoma brucei 3-methylcrotonyl-CoA carboxylase -

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Basic information

Entry
Database: EMDB / ID: EMD-19492
TitleTrypanosoma brucei 3-methylcrotonyl-CoA carboxylase
Map datavolume map sharp
Sample
  • Complex: 3-methylcrotonyl-CoA carboxylase
    • Protein or peptide: 3-methylcrotonyl-CoA carboxylase, putative
    • Protein or peptide: methylcrotonoyl-CoA carboxylaseMethylcrotonyl-CoA carboxylase
  • Ligand: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL
Keywordscarboxylase / trypanosoma brucei / BIOSYNTHETIC PROTEIN / TRANSFERASE
Function / homology
Function and homology information


methylcrotonoyl-CoA carboxylase / methylcrotonoyl-CoA carboxylase activity / methylcrotonoyl-CoA carboxylase complex / L-leucine catabolic process / pyrimidine nucleobase biosynthetic process / biotin binding / cilium / mitochondrion / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Methylcrotonoyl-CoA carboxylase beta chain MCCB/AccD1-like / Acetyl-coenzyme A carboxyltransferase, C-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase C-terminal domain profile. / Acetyl-coenzyme A carboxyltransferase, N-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase N-terminal domain profile. / Acetyl-CoA carboxylase / Carboxyl transferase domain / Biotin-binding site / Biotin-requiring enzymes attachment site. / Biotin carboxylase-like, N-terminal domain ...Methylcrotonoyl-CoA carboxylase beta chain MCCB/AccD1-like / Acetyl-coenzyme A carboxyltransferase, C-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase C-terminal domain profile. / Acetyl-coenzyme A carboxyltransferase, N-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase N-terminal domain profile. / Acetyl-CoA carboxylase / Carboxyl transferase domain / Biotin-binding site / Biotin-requiring enzymes attachment site. / Biotin carboxylase-like, N-terminal domain / Biotin carboxylase, C-terminal / Biotin carboxylation domain / Biotin carboxylase, N-terminal domain / Biotin carboxylase C-terminal domain / Biotin carboxylation domain profile. / Biotin carboxylase C-terminal domain / Carbamoyl-phosphate synthetase large subunit-like, ATP-binding domain / Carbamoyl-phosphate synthase L chain, ATP binding domain / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Rudiment single hybrid motif / Single hybrid motif / Pre-ATP-grasp domain superfamily / ATP-grasp fold / ATP-grasp fold profile. / ClpP/crotonase-like domain superfamily / Carbamoyl-phosphate synthase subdomain signature 2.
Similarity search - Domain/homology
methylcrotonoyl-CoA carboxylase / 3-methylcrotonyl-CoA carboxylase, putative
Similarity search - Component
Biological speciesTrypanosoma brucei (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsRuiz FM / Plaza-Pegueroles A / Fernandez-Tornero C
Funding support Spain, 1 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and Universities Spain
CitationJournal: Structure / Year: 2024
Title: The cryo-EM structure of trypanosome 3-methylcrotonyl-CoA carboxylase provides mechanistic and dynamic insights into its enzymatic function.
Authors: Adrián Plaza-Pegueroles / Inna Aphasizheva / Ruslan Aphasizhev / Carlos Fernández-Tornero / Federico M Ruiz /
Abstract: 3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic ...3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and β-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2.4 Å resolution. Biotin is covalently bound to the biotin carboxyl carrier protein domain of α-subunits and positioned in a non-canonical pocket near the active site of neighboring β-subunit dimers. Moreover, flexibility of key residues at α-subunit interfaces and loops enables pivoting of α-subunit trimers to partly reduce the distance between α- and β-subunit active sites, required for MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and to assist drug discovery against trypanosome infections.
History
DepositionJan 26, 2024-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19492.png

Downloads & links

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Map

FileDownload / File: emd_19492.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationvolume map sharp
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.82 Å/pix.
x 540 pix.
= 444.852 Å		0.82 Å/pix.
x 540 pix.
= 444.852 Å		0.82 Å/pix.
x 540 pix.
= 444.852 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8238 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.0060425 - 1.9122305
Average (Standard dev.)0.00046380475 (±0.042672075)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 444.85202 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19492_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_19492_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_19492_half_map_2.map
Projections & Slices
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Sample components

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Entire : 3-methylcrotonyl-CoA carboxylase

EntireName: 3-methylcrotonyl-CoA carboxylase
Components
  • Complex: 3-methylcrotonyl-CoA carboxylase
    • Protein or peptide: 3-methylcrotonyl-CoA carboxylase, putative
    • Protein or peptide: methylcrotonoyl-CoA carboxylaseMethylcrotonyl-CoA carboxylase
  • Ligand: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL

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Supramolecule #1: 3-methylcrotonyl-CoA carboxylase

SupramoleculeName: 3-methylcrotonyl-CoA carboxylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Trypanosoma brucei (eukaryote)

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Macromolecule #1: 3-methylcrotonyl-CoA carboxylase, putative

MacromoleculeName: 3-methylcrotonyl-CoA carboxylase, putative / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei (eukaryote)
Molecular weightTheoretical: 73.987648 KDa
SequenceString: MLRYNVFYHG DFKKVLVANR GEIACRVFRT CREMNIRTVA VCCEGEPNAK HVLEADEAFV LGPPPASTSY LRGDRIICAA KKLQADAVH PGYGFLSENA EFASAVLAAG LKFVGPPPAA MLSMGSKSES KRIMEAAGVP IVPGYYGEDQ NPDRLLHEAK T IGFPVLIK ...String:
MLRYNVFYHG DFKKVLVANR GEIACRVFRT CREMNIRTVA VCCEGEPNAK HVLEADEAFV LGPPPASTSY LRGDRIICAA KKLQADAVH PGYGFLSENA EFASAVLAAG LKFVGPPPAA MLSMGSKSES KRIMEAAGVP IVPGYYGEDQ NPDRLLHEAK T IGFPVLIK AVSGGGGKGM KIVMEETEFH LMLESAKREA INFFKDDRVI LERYVMHPRH IECQIFFDSF GNGVFFFERD CS VQRRHQK VIEEAPAPGL SVDMRRRIGD VALTAARAVG YVGAGTVEFI FDTEKDEFFF MEMNTRLQVE HPVTEQVCQV RGR PLDLVR LQLQTAMGLP LGFRQEDISM SGASVEARIY AESPRNGFLP VGGRLRYLKE PPQGNRGTVK VRLDTGFRAG DDVL VHYDP MIAKLVVWGD NRATALEGLR TALASYHIVG VETNIDFLQC CLSNPGFVEG GVTTRFIEDN SVNLLQPREI PNNVL ALAA VSYLCSQRGT STLFWPNRQI SQGVCFTVGG NPVVVRVTVS TKMCFTCDFD SSSVTVYVES TTNMPDSSTF IRVTVD GET RFGFTSFVTD SEVAVALPQG FYTLALQPLA TDFGSTSAQA NGSASVLSPM PGKVTKLLVA DGTLVQQGQA ILILEAM KM EHVVKASCDG EVKFCVHADG IVGGSTLLAH IASAAV

UniProtKB: 3-methylcrotonyl-CoA carboxylase, putative

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Macromolecule #2: methylcrotonoyl-CoA carboxylase

MacromoleculeName: methylcrotonoyl-CoA carboxylase / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: methylcrotonoyl-CoA carboxylase
Source (natural)Organism: Trypanosoma brucei (eukaryote)
Molecular weightTheoretical: 66.613969 KDa
SequenceString: MKSFCRLGKV CGCSVSVVFS HRVFALGPRR DYSTSEVPLG SSQVPKGDPR KEQKGGNMSE VYLFHPAQYE SAPATTRPNV LHYPAESTN PEFKANTERM KALTAELRRR VQVIVDGDSE ADKRARDRHI SRGKLLVHQR IEKLVDPMSP FLELSQLAGG D LYPGEACH ...String:
MKSFCRLGKV CGCSVSVVFS HRVFALGPRR DYSTSEVPLG SSQVPKGDPR KEQKGGNMSE VYLFHPAQYE SAPATTRPNV LHYPAESTN PEFKANTERM KALTAELRRR VQVIVDGDSE ADKRARDRHI SRGKLLVHQR IEKLVDPMSP FLELSQLAGG D LYPGEACH RGGILTGIGV VHGMRVMIVA NDATVKGGTY YPITVKKHLR AQRIAEENRL PCIYLVDSGG ANLGMQGDVF PD EQHFGRI FFNQANMSAK GIAQIATVMG SCTAGGAYVP AMSDESIIVK GNGTIFLGGP PLVFAATGEE VTPEELGGAD VHC RASGVT DYFATDDLHA LYLTRRIVAN LNRNDCERPC RGREFTPPLY DPSEIGGFIP DMGADVVKGF DVRAVIARLV DGSE FDEFK KLYGDTLVCG FARFEGMLVG IVANNGILYS ESALKGAHFV ELCSHRNIPL LFLQNITGFM VGKTYEEGGI AKNGA KLVT AVSTTHVPKI TIIIGGSYGA GNYGMCGRAF GPRFLFMWPN ARISVMGGNQ AATVLALTNS KLRENEVQDF KAKVRS KYE YEGSCYYSTA RLWDDGVIAP EDTRAVVVQA LLSTLSAPCG ETKFGVFRM

UniProtKB: methylcrotonoyl-CoA carboxylase

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Macromolecule #3: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL

MacromoleculeName: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL
type: ligand / ID: 3 / Number of copies: 6 / Formula: BTI
Molecular weightTheoretical: 228.311 Da
Chemical component information

ChemComp-BTI:
5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL

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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

Concentration0.07 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mM(NH4)2SO4ammonium sulfate
5.0 mMMgCl2magnesium chloride
5.0 % v/vC3H8O3glycerol
2.0 mMHOCH2CH2SH2-Mercaptoethanol
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: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 38.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 490000
Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 126391
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(Chain: A, AlphaFold, in silico model, B, AlphaFold, in silico model)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8rth:
Trypanosoma brucei 3-methylcrotonyl-CoA carboxylase

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