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

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Basic information

Entry
Database: PDB / ID: 8rth
TitleTrypanosoma brucei 3-methylcrotonyl-CoA carboxylase
Components
  • 3-methylcrotonyl-CoA carboxylase, putative
  • methylcrotonoyl-CoA carboxylaseMethylcrotonyl-CoA carboxylase
KeywordsTRANSFERASE / carboxylase / trypanosoma brucei / BIOSYNTHETIC PROTEIN
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 / Rudiment single hybrid motif / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / 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
Chem-BTI / methylcrotonoyl-CoA carboxylase / 3-methylcrotonyl-CoA carboxylase, putative
Similarity search - Component
Biological speciesTrypanosoma brucei (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsRuiz, F.M. / Plaza-Pegueroles, A. / Fernandez-Tornero, C.
Funding support Spain, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Database references / Category: citation / Item: _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: 3-methylcrotonyl-CoA carboxylase, putative
B: methylcrotonoyl-CoA carboxylase
C: 3-methylcrotonyl-CoA carboxylase, putative
D: methylcrotonoyl-CoA carboxylase
E: 3-methylcrotonyl-CoA carboxylase, putative
F: methylcrotonoyl-CoA carboxylase
G: 3-methylcrotonyl-CoA carboxylase, putative
H: methylcrotonoyl-CoA carboxylase
I: 3-methylcrotonyl-CoA carboxylase, putative
J: methylcrotonoyl-CoA carboxylase
K: 3-methylcrotonyl-CoA carboxylase, putative
L: methylcrotonoyl-CoA carboxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)844,98018
Polymers843,61012
Non-polymers1,3706
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 73987.648 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Trypanosoma brucei (eukaryote) / References: UniProt: Q57YQ4
#2: Protein
methylcrotonoyl-CoA carboxylase / Methylcrotonyl-CoA carboxylase / 3-methylcrotonyl-CoA carboxylase 2 / 3-methylcrotonyl-CoA:carbon dioxide ligase subunit beta


Mass: 66613.969 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Trypanosoma brucei (eukaryote)
References: UniProt: Q385A6, methylcrotonoyl-CoA carboxylase
#3: Chemical
ChemComp-BTI / 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL


Mass: 228.311 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N2O2S / 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: 3-methylcrotonyl-CoA carboxylase / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Trypanosoma brucei (eukaryote)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMammonium sulfate(NH4)2SO41
35 mMmagnesium chlorideMgCl21
45 % v/vglycerolC3H8O31
52 mM2-MercaptoethanolHOCH2CH2SH1
SpecimenConc.: 0.07 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 38.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
7UCSF Chimeramodel fitting
12cryoSPARC4.43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 490000
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126391 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
ID 3D fitting-IDChain-IDSource nameType
11AAlphaFoldin silico model
21BAlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00356604
ELECTRON MICROSCOPYf_angle_d0.56676668
ELECTRON MICROSCOPYf_dihedral_angle_d4.2627920
ELECTRON MICROSCOPYf_chiral_restr0.0438598
ELECTRON MICROSCOPYf_plane_restr0.00410086

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