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Open data
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Basic information
Entry | ![]() | ||||||||||||
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Title | Isobutyryl-CoA mutase fused in the presence of GMPPCP | ||||||||||||
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![]() | supramolecular complex / b12-binding / G-protein chaperone / mutase / ISOMERASE | ||||||||||||
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 | single particle reconstruction / cryo EM / Resolution: 7.3 Å | ||||||||||||
![]() | Vaccaro FA / Drennan CL | ||||||||||||
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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 427.5 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 17.2 KB 17.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 4.2 KB | Display | ![]() |
Images | ![]() | 48.7 KB | ||
Others | ![]() ![]() | 7.3 MB 7.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 651.3 KB | Display | ![]() |
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Full document | ![]() | 650.9 KB | Display | |
Data in XML | ![]() | 11.2 KB | Display | |
Data in CIF | ![]() | 14 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8staMC ![]() 8sslC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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Voxel size | X=Y=Z: 2.0143 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_40758_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_40758_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Supramolecular complexes of isobutyryl-CoA mutase fused
Entire | Name: Supramolecular complexes of isobutyryl-CoA mutase fused |
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Components |
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-Supramolecule #1: Supramolecular complexes of isobutyryl-CoA mutase fused
Supramolecule | Name: Supramolecular complexes of isobutyryl-CoA mutase fused type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Isobutyryl-CoA mutase fused
Macromolecule | Name: Isobutyryl-CoA mutase fused / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: methylmalonyl-CoA mutase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 122.927586 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MGSSHHHHHH SSGLVPRGSH MTDLSDVSRT AAAKPPAVPG RGPANKVRFV TAASLFDGHD ASINIMRRIL QSQGCEVIHL GHNRSVQEV VTAALQEDVQ GIAISSYQGG HVEYFKYMID LLREHGGEHI QVFGGGGGVI VPDEIRELQA YGVARIYSPE D GQRMGLAG ...String: MGSSHHHHHH SSGLVPRGSH MTDLSDVSRT AAAKPPAVPG RGPANKVRFV TAASLFDGHD ASINIMRRIL QSQGCEVIHL GHNRSVQEV VTAALQEDVQ GIAISSYQGG HVEYFKYMID LLREHGGEHI QVFGGGGGVI VPDEIRELQA YGVARIYSPE D GQRMGLAG MITDMAQRCD IDLTRYAPTT LDTVVAGDRR ALAQLITALE NGKADPELVS ALHAQAKAAA VPVLGITGTG GA GKSSLTD ELIRRFRLDQ DDALSIAVIS IDPSRRKSGG ALLGDRIRMN AINHPNIFMR SLATREAGSE ISQALPDVIA ACK AARFDL VIVETSGIGQ GDAAIVPHVD LSLYVMTPEF GAASQLEKID MLDFADFVAI NKFDRKGAQD AWRDVAKQVQ RNRE QWHSR AEDMPVYGTQ ASRFNDDGVT MLYQGLVGAL GARGMSLKPG TLPNLEGRIS TGQNVIVPPA RSRYLAELAD TVRAY HRRV VAQSKLARER QQLRAAHDML QGAGHESAAL ETLASERDVS LGAVERKLLA MWPQMQQAYS GDEYVVKIRD KEIRTG LIS TTLSGTKIRK VVLPRFEDEG EILKWLMREN VPGSFPYTAG VFAFKREGED PTRMFAGEGD AFRTNRRFKL VSEGMEA KR LSTAFDSVTL YGEDPHERPD IYGKVGNSGV SIATLEDMKV LYDGFDLTNP STSVSMTING PAPTILAMFM NTAIDQQI D RFRADNGRDP TADEEAKIRA WVLQNVRGTV QADILKEDQG QNTCIFSTEF SLKVMGDIQE YFVHHQVRNF YSVSISGYH IAEAGANPIS QLAFTLANGF TYVEAYLARG MHIDDFAPNL SFFFSNGMDP EYSVLGRVAR RIWAVTMRDK YGANDRSQKL KYHIQTSGR SLHAQEIDFN DIRTTLQALI AIYDNCNSLH TNAYDEAITT PTAESVRRAL AIQLIINREW GVAKCENPNQ G SFLIEELT DLVEEAVLQE FERIAERGGV LGAMETGYQR GKIQEESLYY EQLKHDGTLP IIGVNTFRNP NGDPTPQTLE LA RSSEDEK QSQLHRLTEF HGAHQADAEA MLARLRQAVI DNRNVFAVLM DAVRVCSLGQ ITHALFEVGG QYRRNM UniProtKB: Fused isobutyryl-CoA mutase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 0.25 mg/mL |
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Buffer | pH: 8 Details: 20 mM HEPES pH 8, 50 mM NaCl, 500 mM butyryl-CoA, 500 mM GMPPCP, 500 mM MgCl2 |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Pressure: 1.0 kPa Details: After glow discharge, the grid was coated with graphene oxide prior to use. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV |
Details | In the presence of the non-hydrolyzable GMPPCP, filamentous supramolecular complexes are formed |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 602 / Average exposure time: 3.99 sec. / Average electron dose: 42.81 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.2 µm |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: B / Chain - Residue range: 22-1093 / Chain - Source name: PDB / Chain - Initial model type: experimental model Details: The initial model consisted of two different fragments: resi 22-442 and resi 443-1093 |
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Details | Initial local fitting was done using ChimeraX and then Phenix real space refine was used for rigid body refinement of the defined fragments. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | ![]() PDB-8sta: |