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- EMDB-29699: Structure of cobalamin-dependent methionine synthase (MetH) in a ... -

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Basic information

Entry
Database: EMDB / ID: EMD-29699
TitleStructure of cobalamin-dependent methionine synthase (MetH) in a resting state
Map data
Sample
  • Complex: Cobalamin-dependent methionine synthase holoprotein
    • Protein or peptide: Methionine synthase
  • Ligand: ZINC ION
  • Ligand: COBALAMIN
KeywordsMethyltransferase / Transferase / Amino-acid biosynthesis / Methionine biosynthesis
Function / homology
Function and homology information


methionine synthase / methionine synthase activity / homocysteine metabolic process / cobalamin binding / tetrahydrofolate metabolic process / methylation / zinc ion binding / cytosol
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
Biological speciesThermus filiformis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWatkins MB / Ando N
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29699.png

Downloads & links

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Map

FileDownload / File: emd_29699.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 410.534 Å		1.07 Å/pix.
x 384 pix.
= 410.534 Å		1.07 Å/pix.
x 384 pix.
= 410.534 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0691 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-1.3906168 - 2.115265
Average (Standard dev.)-0.0003941553 (±0.019069694)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 410.53442 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29699_msk_1.map
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Half map: #2

Fileemd_29699_half_map_1.map
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Half map: #1

Fileemd_29699_half_map_2.map
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Sample components

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Entire : Cobalamin-dependent methionine synthase holoprotein

EntireName: Cobalamin-dependent methionine synthase holoprotein
Components
  • Complex: Cobalamin-dependent methionine synthase holoprotein
    • Protein or peptide: Methionine synthase
  • Ligand: ZINC ION
  • Ligand: COBALAMIN

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Supramolecule #1: Cobalamin-dependent methionine synthase holoprotein

SupramoleculeName: Cobalamin-dependent methionine synthase holoprotein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Cobalamin-dependent methionine synthase (MetH) in complex with B12 and Zinc
Source (natural)Organism: Thermus filiformis (bacteria)
Molecular weightTheoretical: 132.539 KDa

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Macromolecule #1: Methionine synthase

MacromoleculeName: Methionine synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: methionine synthase
Source (natural)Organism: Thermus filiformis (bacteria)
Molecular weightTheoretical: 131.312266 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MVEVHTCSPG CRHHLGGAGW GDAPLLRLGY NKEARARRFP YLRALSQRVL VFDGAMGTEL QKRDLTPEDY GEEAYYGCPE VLNRTRPDV VREIHLSYLE AGAEVIETNS FGALRHVLAE YGLGEEAEEL AYRAARIAKE AAEPYGAFVA GALGPGTKLV S LGQISWEE ...String:
MVEVHTCSPG CRHHLGGAGW GDAPLLRLGY NKEARARRFP YLRALSQRVL VFDGAMGTEL QKRDLTPEDY GEEAYYGCPE VLNRTRPDV VREIHLSYLE AGAEVIETNS FGALRHVLAE YGLGEEAEEL AYRAARIAKE AAEPYGAFVA GALGPGTKLV S LGQISWEE LFSAYKEAVR GLVRGGVDLI LLETAQDILQ VRCAVLAARE AMAELGVELP LQVQVTFEAT GTMLVGTDEQ AA LAALESL PIDVVGMNCA TGPDLMDAKI RYFAQESTRF VACLPNAGLP RNEGGRVVYD LTPEELARWH LKFVTEYGVN AVG GCCGTG PEHIRKVAEA VGGRPSPVHK TAFPPQVASL YQAVPLRQET SLLLVGERLN ATGSKRFREL LFAGDLEGIL ALAQ EQVAE GAHVLDLSVA WTGRDELEDM KRVLSKLATG VTVPFMVDST SPEVMEEALK RLPGRAILNS ANLEDGLEKF DRVAS LAKA HGAALVALAI DEEGMAKTRV KKVEVALRMY ERLTEHHGLR PEDLLFDLLT FPITQGDEET RPLARETLLA LEELRE RLP GVGFVLGVSN VSFGLKPKAR RVLNSVFLDE ARKKGLTAAI VDAGKILPIS QIPEEAYALA LDLIYDRREG QDPLFAF IR FFEEHKEVLA EDKEAFQALP VEERLRRRVL EGKRVGLEED LAEALGRMRP LEIINGPLLE AMKEVGELFG AGKMQLPF V LQAAEVMKQA VAYLEPHMEK KGEGKGKLVL ATVKGDVHDI GKNLVDIILT NNGYTVYNLG IKVPIEEMLK AVDEVKPHA LGMSGLLVKS TQVMKENLEY MRARGYTLPV ILGGAALTRA YVEELRSIYP EVYYAEDAFE GLALMEELTG HRPKDLTLRR ARTTRKEAP APRSKPVSPA PDLPRPPFFG VRVEEGLDLA TIAHYVNRLA LYRGQWGFSR GGLSREEWEA YVKREADPVF R RLLAEAMA EGWLRPRVLY GFFPVAREGE ELWVYSPEGE LLEKLRFPRQ RGGGISLVDY FRPRFAEPLS DEADWLPGYE EG ARDVLGV QLVTMGEEPK RKAEALYREG RYQDYLFVHG FAVEMTEALA EYWHKRMRQM WGIAGQDAPE IRRLFQQEYR GAR YSFGYP ACPDLADQAK LDRLMDFSRI GVRLTENYQL DPEHSTSALV VHHPEARYFS VD

UniProtKB: Methionine synthase

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: COBALAMIN

MacromoleculeName: COBALAMIN / type: ligand / ID: 3 / Number of copies: 1 / Formula: B12
Molecular weightTheoretical: 1.330356 KDa
Chemical component information

ChemComp-B12:
COBALAMIN

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration0.264 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4S4-(2-hrazineethanesulfonic acid
150.0 mMNaClsodium chloride
2.5 mMC4H10O2S2Dithiothreitol

Details: 50 mM HEPES, 150 mM NaCl, 2.5 mM DTT pH 7.6
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was mixed with equimolar commercial horse spleen apoferritin for freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 5092 pixel / Number grids imaged: 2 / Number real images: 12768 / Average exposure time: 2.5 sec. / Average electron dose: 65.0 e/Å2
Details: Images were collected in movie mode with a total of 50 frames over 2.5 seconds.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9994870
Startup modelType of model: INSILICO MODEL
In silico model: cryoSPARC ab-initio model generation (with 3 models, followed by 3D classification)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Software - details: Non-uniform refinement in cryosparc (without per-particle defocus or CTF refinement)
Number images used: 257706
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 200000 / Software - Name: cryoSPARC (ver. 3.2)
Details: Initial 3D classification of particles into class of interest and two junk classes, final classification of particles between 2-domain (incomplete) and 3-domain (complete) classes
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

sz17


Chain - Chain ID: A / Chain - Residue range: 2-896 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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