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- PDB-8g3h: Structure of cobalamin-dependent methionine synthase (MetH) in a ... -

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Basic information

Entry
Database: PDB / ID: 8g3h
TitleStructure of cobalamin-dependent methionine synthase (MetH) in a resting state
ComponentsMethionine synthase
KeywordsTRANSFERASE / Methyltransferase / Amino-acid biosynthesis / Methionine biosynthesis
Function / homology
Function and homology information


pteridine-containing compound metabolic process / methionine synthase / methionine synthase activity / cobalamin binding / methylation / zinc ion binding
Similarity search - Function
Vitamin B12-dependent methionine synthase, activation domain / Vitamin B12 dependent methionine synthase, activation domain / AdoMet activation domain profile. / Cobalamin-dependent methionine synthase / Methionine synthase, B12-binding domain / Vitamin B12-dependent methionine synthase, activation domain superfamily / B12-binding N-terminal domain profile. / B12 binding domain / Homocysteine-binding domain / Homocysteine-binding domain superfamily ...Vitamin B12-dependent methionine synthase, activation domain / Vitamin B12 dependent methionine synthase, activation domain / AdoMet activation domain profile. / Cobalamin-dependent methionine synthase / Methionine synthase, B12-binding domain / Vitamin B12-dependent methionine synthase, activation domain superfamily / B12-binding N-terminal domain profile. / B12 binding domain / Homocysteine-binding domain / Homocysteine-binding domain superfamily / Homocysteine S-methyltransferase / Homocysteine-binding domain profile. / Cobalamin (vitamin B12)-binding module, cap domain / B12 binding domain / Methionine synthase domain / B12 binding domain / Cobalamin-binding domain superfamily / B12-binding domain profile. / Cobalamin (vitamin B12)-binding domain / Pterin-binding domain / Pterin binding enzyme / Pterin-binding domain profile. / Dihydropteroate synthase-like
Similarity search - Domain/homology
COBALAMIN / Methionine synthase
Similarity search - Component
Biological speciesThermus filiformis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWatkins, M.B. / Ando, N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM100008 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124847 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy.
Authors: Maxwell B Watkins / Haoyue Wang / Audrey Burnim / Nozomi Ando /
Abstract: Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH-Hfolate) using the unique chemistry of its cofactor. In doing ...Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH-Hfolate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of -adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on  MetH have shown that this flexible, multidomain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a "divide and conquer" approach. In this study, we investigate MetH and a thermophilic homolog from using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH-Hfolate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.
History
DepositionFeb 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Methionine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,7083
Polymers131,3121
Non-polymers1,3962
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Methionine synthase / 5-methyltetrahydrofolate--homocysteine methyltransferase


Mass: 131312.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus filiformis (bacteria) / Gene: THFILI_06775 / Plasmid: pET-28c+ / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A0A2XCD7, methionine synthase
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-B12 / COBALAMIN


Mass: 1330.356 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C62H89CoN13O14P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cobalamin-dependent methionine synthase holoprotein / Type: COMPLEX
Details: Cobalamin-dependent methionine synthase (MetH) in complex with B12 and Zinc
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.132539 MDa / Experimental value: NO
Source (natural)Organism: Thermus filiformis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pET-28c+
Buffer solutionpH: 7.6 / Details: 50 mM HEPES, 150 mM NaCl, 2.5 mM DTT pH 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hrazineethanesulfonic acidC8H18N2O4S1
2150 mMsodium chlorideNaCl1
32.5 mMDithiothreitolC4H10O2S21
SpecimenConc.: 0.264 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was mixed with equimolar commercial horse spleen apoferritin for freezing.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12768
Details: Images were collected in movie mode with a total of 50 frames over 2.5 seconds.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 30 eV
Image scansWidth: 5760 / Height: 5092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2particle selectionTemplates from screening dataset reconstruction
2Leginonimage acquisition
4cryoSPARC3.2CTF correction
7UCSF Chimera1.15model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstructionNon-uniform refinement in cryosparc (without per-particle defocus or CTF refinement)
13ISOLDE1.3model refinement
14PHENIX1.20.1-4487model refinement
CTF correctionDetails: cryoSPARC patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9994870
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 257706 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial model used was the 3 N-terminal domains of AlphaFold database model A0A1Q9SZ17. Each domain was fit individually by rigid-body fitting in Chimera. The cobalamin ligand was initially ...Details: Initial model used was the 3 N-terminal domains of AlphaFold database model A0A1Q9SZ17. Each domain was fit individually by rigid-body fitting in Chimera. The cobalamin ligand was initially fit by alignment of the 1BMT crystal structure to the appropriate binding region.
Atomic model buildingAccession code: A0A1Q9SZ17 / Chain residue range: 2-896 / Source name: AlphaFold / Type: in silico model

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