+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40751 | ||||||||||||
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Title | Isobutyryl-CoA mutase fused Q341A in the presence of GTP | ||||||||||||
Map data | |||||||||||||
Sample |
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Keywords | supramolecular complex / b12-binding / g-protein chaperone / ISOMERASE | ||||||||||||
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 | single particle reconstruction / cryo EM / Resolution: 4.6 Å | ||||||||||||
Authors | Vaccaro FA / Drennan CL | ||||||||||||
Funding support | United States, 3 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40751.map.gz | 4.2 MB | EMDB map data format | |
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Header (meta data) | emd-40751-v30.xml emd-40751.xml | 17.7 KB 17.7 KB | Display Display | EMDB header |
Images | emd_40751.png | 71.2 KB | ||
Filedesc metadata | emd-40751.cif.gz | 6.7 KB | ||
Others | emd_40751_half_map_1.map.gz emd_40751_half_map_2.map.gz | 26.7 MB 26.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40751 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40751 | HTTPS FTP |
-Related structure data
Related structure data | 8sslMC 8staC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_40751.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.5998 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_40751_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_40751_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Supramolecular complex of isobutyryl-CoA mutase fused
Entire | Name: Supramolecular complex of isobutyryl-CoA mutase fused |
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Components |
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-Supramolecule #1: Supramolecular complex of isobutyryl-CoA mutase fused
Supramolecule | Name: Supramolecular complex of isobutyryl-CoA mutase fused / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Cupriavidus metallidurans CH34 (bacteria) |
-Macromolecule #1: Fused isobutyryl-CoA mutase
Macromolecule | Name: Fused isobutyryl-CoA mutase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: isobutyryl-CoA mutase |
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Source (natural) | Organism: Cupriavidus metallidurans CH34 (bacteria) |
Molecular weight | Theoretical: 122.870523 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
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 GAASALEKID 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 |
-Macromolecule #2: GUANOSINE-5'-DIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: GDP |
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Molecular weight | Theoretical: 443.201 Da |
Chemical component information | ChemComp-GDP: |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-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 GTP, 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: 60 sec. / Pretreatment - Pressure: 3.9 kPa |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 3s. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.2 µm |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 673 / Average exposure time: 3.99 sec. / Average electron dose: 58.71 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 180271 |
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Startup model | Type of model: NONE |
Initial angle assignment | Type: NOT APPLICABLE |
Final 3D classification | Number classes: 3 |
Final angle assignment | Type: NOT APPLICABLE |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 59335 |
-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: Docked the model as two different fragments: resi 22-442 and resi 443-1093 |
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Refinement | Protocol: RIGID BODY FIT |
Output model | PDB-8ssl: |