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- EMDB-40758: Isobutyryl-CoA mutase fused in the presence of GMPPCP -

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Basic information

Entry
Database: EMDB / ID: EMD-40758
TitleIsobutyryl-CoA mutase fused in the presence of GMPPCP
Map data
Sample
  • Complex: Supramolecular complexes of isobutyryl-CoA mutase fused
    • Protein or peptide: Isobutyryl-CoA mutase fused
Keywordssupramolecular complex / b12-binding / G-protein chaperone / mutase / 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
Fused isobutyryl-CoA mutase / Methylmalonyl Co-A mutase-associated GTPase MeaB / Methylmalonyl-CoA mutase, alpha chain, catalytic / Methylmalonyl-CoA mutase, alpha/beta chain, catalytic / Methylmalonyl-CoA mutase / Methylmalonyl-CoA mutase, C-terminal / Cobalamin (vitamin B12)-dependent enzyme, catalytic / B12 binding domain / Cobalamin-binding domain superfamily / B12-binding domain profile. ...Fused isobutyryl-CoA mutase / Methylmalonyl Co-A mutase-associated GTPase MeaB / Methylmalonyl-CoA mutase, alpha chain, catalytic / Methylmalonyl-CoA mutase, alpha/beta chain, catalytic / Methylmalonyl-CoA mutase / Methylmalonyl-CoA mutase, C-terminal / Cobalamin (vitamin B12)-dependent enzyme, catalytic / B12 binding domain / Cobalamin-binding domain superfamily / B12-binding domain profile. / Cobalamin (vitamin B12)-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Fused isobutyryl-CoA mutase
Similarity search - Component
Biological speciesCupriavidus metallidurans CH34 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsVaccaro FA / Drennan CL
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31 GM131648 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: 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
DepositionMay 9, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40758.png

Downloads & links

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Map

FileDownload / File: emd_40758.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.0143 Å
Density
Contour LevelBy AUTHOR: 2.36
Minimum - Maximum0.0 - 11.210789999999999
Average (Standard dev.)0.27255824 (±0.9556587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 257.8304 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40758_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40758_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Supramolecular complexes of isobutyryl-CoA mutase fused

EntireName: Supramolecular complexes of isobutyryl-CoA mutase fused
Components
  • Complex: Supramolecular complexes of isobutyryl-CoA mutase fused
    • Protein or peptide: Isobutyryl-CoA mutase fused

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Supramolecule #1: Supramolecular complexes of isobutyryl-CoA mutase fused

SupramoleculeName: Supramolecular complexes of isobutyryl-CoA mutase fused
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Cupriavidus metallidurans CH34 (bacteria)

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Macromolecule #1: Isobutyryl-CoA mutase fused

MacromoleculeName: Isobutyryl-CoA mutase fused / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: methylmalonyl-CoA mutase
Source (natural)Organism: Cupriavidus metallidurans CH34 (bacteria)
Molecular weightTheoretical: 122.927586 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 8
Details: 20 mM HEPES pH 8, 50 mM NaCl, 500 mM butyryl-CoA, 500 mM GMPPCP, 500 mM MgCl2
GridModel: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
DetailsIn the presence of the non-hydrolyzable GMPPCP, filamentous supramolecular complexes are formed

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm 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 beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 138956
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4xc6


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
DetailsInitial local fitting was done using ChimeraX and then Phenix real space refine was used for rigid body refinement of the defined fragments.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8sta:
Isobutyryl-CoA mutase fused in the presence of GMPPCP

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