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- EMDB-18299: MTHFR + SAH asymmetric dis-inhibited state -

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Basic information

Entry
Database: EMDB / ID: EMD-18299
TitleMTHFR + SAH asymmetric dis-inhibited state
Map dataMain local filtered
Sample
  • Complex: Human 5,10-methylenetetrahydrofolate reductase in complex with S-Adenosyl-L-homocysteine, regulatory domains with one catalytic domain
    • Protein or peptide: Methylenetetrahydrofolate reductase (NADPH)
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
KeywordsDis-inhibited / allosteric / folate / S-adenosylhomocysteine / FLAVOPROTEIN
Function / homology
Function and homology information


methylenetetrahydrofolate reductase (NADPH) / response to vitamin B2 / methylenetetrahydrofolate reductase (NADPH) activity / methylenetetrahydrofolate reductase (NAD(P)H) activity / methionine metabolic process / modified amino acid binding / homocysteine metabolic process / heterochromatin organization / S-adenosylmethionine metabolic process / methionine biosynthetic process ...methylenetetrahydrofolate reductase (NADPH) / response to vitamin B2 / methylenetetrahydrofolate reductase (NADPH) activity / methylenetetrahydrofolate reductase (NAD(P)H) activity / methionine metabolic process / modified amino acid binding / homocysteine metabolic process / heterochromatin organization / S-adenosylmethionine metabolic process / methionine biosynthetic process / Metabolism of folate and pterines / response to folic acid / tetrahydrofolate interconversion / response to amino acid / FAD binding / response to interleukin-1 / neural tube closure / flavin adenine dinucleotide binding / NADP binding / response to hypoxia / response to xenobiotic stimulus / protein-containing complex binding / cytosol
Similarity search - Function
Eukaryotic-type methylenetetrahydrofolate reductase / Methylenetetrahydrofolate reductase-like / Methylenetetrahydrofolate reductase / FAD-linked oxidoreductase-like
Similarity search - Domain/homology
Methylenetetrahydrofolate reductase (NADPH)
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsBlomgren LKM / Yue WW / Froese DS / McCorvie TJ
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_192505 Switzerland
CitationJournal: Nat Commun / Year: 2024
Title: Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase.
Authors: Linnea K M Blomgren / Melanie Huber / Sabrina R Mackinnon / Céline Bürer / Arnaud Baslé / Wyatt W Yue / D Sean Froese / Thomas J McCorvie /
Abstract: 5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved ...5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.
History
DepositionAug 22, 2023-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18299.png

Downloads & links

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Map

FileDownload / File: emd_18299.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain local filtered
Voxel sizeX=Y=Z: 0.574 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.39427426 - 0.70700014
Average (Standard dev.)0.00032612748 (±0.008330533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 229.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18299_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Sharpened

Fileemd_18299_additional_1.map
AnnotationSharpened
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Additional map: Non-sharpened

Fileemd_18299_additional_2.map
AnnotationNon-sharpened
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_18299_half_map_1.map
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Half map: #1

Fileemd_18299_half_map_2.map
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Sample components

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Entire : Human 5,10-methylenetetrahydrofolate reductase in complex with S-...

EntireName: Human 5,10-methylenetetrahydrofolate reductase in complex with S-Adenosyl-L-homocysteine, regulatory domains with one catalytic domain
Components
  • Complex: Human 5,10-methylenetetrahydrofolate reductase in complex with S-Adenosyl-L-homocysteine, regulatory domains with one catalytic domain
    • Protein or peptide: Methylenetetrahydrofolate reductase (NADPH)
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE

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Supramolecule #1: Human 5,10-methylenetetrahydrofolate reductase in complex with S-...

SupramoleculeName: Human 5,10-methylenetetrahydrofolate reductase in complex with S-Adenosyl-L-homocysteine, regulatory domains with one catalytic domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Methylenetetrahydrofolate reductase (NADPH)

MacromoleculeName: Methylenetetrahydrofolate reductase (NADPH) / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 75.461195 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVNEARGNSS LNPCLEGSAS SGSESSKDSS RCSTPGLDPE RHERLREKMR RRLESGDKWF SLEFFPPRTA EGAVNLISRF DRMAAGGPL YIDVTWHPAG DPGSDKETSS MMIASTAVNY CGLETILHMT CCRQRLEEIT GHLHKAKQLG LKNIMALRGD P IGDQWEEE ...String:
MVNEARGNSS LNPCLEGSAS SGSESSKDSS RCSTPGLDPE RHERLREKMR RRLESGDKWF SLEFFPPRTA EGAVNLISRF DRMAAGGPL YIDVTWHPAG DPGSDKETSS MMIASTAVNY CGLETILHMT CCRQRLEEIT GHLHKAKQLG LKNIMALRGD P IGDQWEEE EGGFNYAVDL VKHIRSEFGD YFDICVAGYP KGHPEAGSFE ADLKHLKEKV SAGADFIITQ LFFEADTFFR FV KACTDMG ITCPIVPGIF PIQGYHSLRQ LVKLSKLEVP QEIKDVIEPI KDNDAAIRNY GIELAVSLCQ ELLASGLVPG LHF YTLNRE MATTEVLKRL GMWTEDPRRP LPWALSAHPK RREEDVRPIF WASRPKSYIY RTQEWDEFPN GRWGNSSSPA FGEL KDYYL FYLKSKSPKE ELLKMWGEEL TSEASVFEVF VLYLSGEPNR NGHKVTCLPW NDEPLAAETS LLKEELLRVN RQGIL TINS QPNINGKPSS DPIVGWGPSG GYVFQKAYLE FFTSRETAEA LLQVLKKYEL RVNYHLVNVK GENITNAPEL QPNAVT WGI FPGREIIQPT VVDPVSFMFW KDEAFALWIE QWGKLYEEES PSRTIIQYIH DNYFLVNLVD NDFPLDNCLW QVVEDTL EL LNRPTQNARE TEAPAENLYF Q

UniProtKB: Methylenetetrahydrofolate reductase (NADPH)

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Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 1 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM / Flavin adenine dinucleotide

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Macromolecule #3: S-ADENOSYL-L-HOMOCYSTEINE

MacromoleculeName: S-ADENOSYL-L-HOMOCYSTEINE / type: ligand / ID: 3 / Number of copies: 2 / Formula: SAH
Molecular weightTheoretical: 384.411 Da
Chemical component information

ChemComp-SAH:
S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.0025% Tween20, 1 mM S-Adenosyl-L-homocysteine, filter sterilised
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 240000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 2 / Number real images: 5606 / Average exposure time: 5.18 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 1900000
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 104101
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6fcx


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 86.3 / Target criteria: Cross-correlation coeficient
Output model

PDB-8qa5:
MTHFR + SAH asymmetric dis-inhibited state

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