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Open data
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Basic information
Entry | Database: PDB / ID: 8sta | ||||||||||||
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Title | Isobutyryl-CoA mutase fused in the presence of GMPPCP | ||||||||||||
![]() | Isobutyryl-CoA mutase fused | ||||||||||||
![]() | ISOMERASE / supramolecular complex / b12-binding / G-protein chaperone / mutase | ||||||||||||
Function / homology | ![]() 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 | ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 Å | ||||||||||||
![]() | Vaccaro, F.A. / Drennan, C.L. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 1.6 MB | Display | ![]() |
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PDB format | ![]() | 1.3 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 123.1 KB | Display | |
Data in CIF | ![]() | 182.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 40758MC ![]() 8sslC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 122927.586 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: sbm, Rmet_0210 / Production host: ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Supramolecular complexes of isobutyryl-CoA mutase fused Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 Details: 20 mM HEPES pH 8, 50 mM NaCl, 500 mM butyryl-CoA, 500 mM GMPPCP, 500 mM MgCl2 |
Specimen | Conc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: In the presence of the non-hydrolyzable GMPPCP, filamentous supramolecular complexes are formed |
Specimen support | Details: After glow discharge, the grid was coated with graphene oxide prior to use. 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 |
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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: ![]() |
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: 42.81 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 602 |
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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 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138956 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL Details: Initial local fitting was done using ChimeraX and then Phenix real space refine was used for rigid body refinement of the defined fragments. | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 4XC6 Pdb chain-ID: B / Accession code: 4XC6 / Chain residue range: 22-1093 Details: The initial model consisted of 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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