+Open data
-Basic information
Entry | Database: PDB / ID: 8ssl | ||||||||||||
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Title | Isobutyryl-CoA mutase fused Q341A in the presence of GTP | ||||||||||||
Components | Fused isobutyryl-CoA mutase | ||||||||||||
Keywords | ISOMERASE / supramolecular complex / b12-binding / g-protein chaperone | ||||||||||||
Function / homology | Function and homology information isobutyryl-CoA mutase / pivalyl-CoA mutase activity / isobutyryl-CoA mutase activity / methylmalonyl-CoA mutase activity / acyl-CoA metabolic process / cobalamin binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / GTPase activity / GTP binding / magnesium ion binding Similarity search - Function | ||||||||||||
Biological species | Cupriavidus metallidurans CH34 (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å | ||||||||||||
Authors | Vaccaro, F.A. / Drennan, C.L. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: J Biol Chem / Year: 2023 Title: Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase. Authors: Francesca A Vaccaro / Daphne A Faber / Gisele A Andree / David A Born / Gyunghoon Kang / Dallas R Fonseca / Marco Jost / Catherine L Drennan / Abstract: G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the ...G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(β,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ssl.cif.gz | 1.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8ssl.ent.gz | 1022.4 KB | Display | PDB format |
PDBx/mmJSON format | 8ssl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ss/8ssl ftp://data.pdbj.org/pub/pdb/validation_reports/ss/8ssl | HTTPS FTP |
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-Related structure data
Related structure data | 40751MC 8staC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 122870.523 Da / Num. of mol.: 3 / Mutation: Q341A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Cupriavidus metallidurans CH34 (bacteria) Gene: icmF, sbm, Rmet_0210 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: Q1LRY0, isobutyryl-CoA mutase, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement #2: Chemical | #3: Chemical | ChemComp-MG / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Supramolecular complex of isobutyryl-CoA mutase fused / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Cupriavidus metallidurans CH34 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 8 Details: 20 mM HEPES pH 8, 50 mM NaCl, 500 mM GTP, 500 mM MgCl2 |
Specimen | Conc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281.15 K / Details: Blot for 3s |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3100 nm / Nominal defocus min: 1200 nm |
Image recording | Average exposure time: 3.99 sec. / Electron dose: 58.71 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 673 |
-Processing
EM software | Name: PHENIX / Version: 1.20.1_4487: / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 180271 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59335 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 4XC6 Pdb chain-ID: B / Accession code: 4XC6 / Chain residue range: 22-1093 Details: Docked the model as two different fragments: resi 22-442 and resi 443-1093 Pdb chain residue range: 22-1093 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
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