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- EMDB-42026: Human retinal variant phosphomimetic IMPDH1(546)-S477D filament b... -

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Basic information

Entry
Database: EMDB / ID: EMD-42026
TitleHuman retinal variant phosphomimetic IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+, octamer-centered
Map dataSharpened map from deepEMhancer
Sample
  • Complex: IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+
    • Protein or peptide: Inosine-5'-monophosphate dehydrogenase 1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: INOSINIC ACID
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Keywordsdehydrogenase / nucleotide synthesis / filament / phosphomimetic / OXIDOREDUCTASE
Function / homology
Function and homology information


Purine ribonucleoside monophosphate biosynthesis / IMP dehydrogenase activity / IMP dehydrogenase / GMP biosynthetic process / Azathioprine ADME / GTP biosynthetic process / azurophil granule lumen / secretory granule lumen / ficolin-1-rich granule lumen / Potential therapeutics for SARS ...Purine ribonucleoside monophosphate biosynthesis / IMP dehydrogenase activity / IMP dehydrogenase / GMP biosynthetic process / Azathioprine ADME / GTP biosynthetic process / azurophil granule lumen / secretory granule lumen / ficolin-1-rich granule lumen / Potential therapeutics for SARS / nucleic acid binding / nucleotide binding / Neutrophil degranulation / DNA binding / RNA binding / extracellular region / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
IMP dehydrogenase / GMP reductase domain / Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / GMP reductase, conserved site / IMP dehydrogenase / GMP reductase signature. / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
Inosine-5'-monophosphate dehydrogenase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsCalise SJ / Kollman JM
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM118396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM149542 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R21EY031546 United States
National Institutes of Health/Office of the DirectorS10OD032290 United States
Helen Hay Whitney Foundation United States
CitationJournal: J Cell Biol / Year: 2024
Title: Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.
Authors: S John Calise / Audrey G O'Neill / Anika L Burrell / Miles S Dickinson / Josephine Molfino / Charlie Clarke / Joel Quispe / David Sokolov / Rubén M Buey / Justin M Kollman /
Abstract: Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback ...Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.
History
DepositionSep 18, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42026.png

Downloads & links

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Map

FileDownload / File: emd_42026.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from deepEMhancer
Voxel sizeX=Y=Z: 0.843 Å
Density
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-0.0054131993 - 2.6195076
Average (Standard dev.)0.007583577 (±0.073512934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 337.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map from CryoSPARC

Fileemd_42026_additional_1.map
AnnotationSharpened map from CryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_42026_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_42026_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+

EntireName: IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+
Components
  • Complex: IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+
    • Protein or peptide: Inosine-5'-monophosphate dehydrogenase 1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: INOSINIC ACID
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+

SupramoleculeName: IMPDH1(546)-S477D filament bound by ATP, IMP, and NAD+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 468 KDa

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Macromolecule #1: Inosine-5'-monophosphate dehydrogenase 1

MacromoleculeName: Inosine-5'-monophosphate dehydrogenase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: IMP dehydrogenase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.542906 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSMADYLISG GTGYVPEDGL TAQQLFASAD GLTYNDFLIL PGFIDFIADE VDLTSALTRK ITLKTPLISS PMDTVTEADM AIAMALMGG IGFIHHNCTP EFQANEVRKV KKFEQGFITD PVVLSPSHTV GDVLEAKMRH GFSGIPITET GTMGSKLVGI V TSRDIDFL ...String:
GSMADYLISG GTGYVPEDGL TAQQLFASAD GLTYNDFLIL PGFIDFIADE VDLTSALTRK ITLKTPLISS PMDTVTEADM AIAMALMGG IGFIHHNCTP EFQANEVRKV KKFEQGFITD PVVLSPSHTV GDVLEAKMRH GFSGIPITET GTMGSKLVGI V TSRDIDFL AEKDHTTLLS EVMTPRIELV VAPAGVTLKE ANEILQRSKK GKLPIVNDCD ELVAIIARTD LKKNRDYPLA SK DSQKQLL CGAAVGTRED DKYRLDLLTQ AGVDVIVLDS SQGNSVYQIA MVHYIKQKYP HLQVIGGNVV TAAQAKNLID AGV DGLRVG MGCGSICITQ EVMACGRPQG TAVYKVAEYA RRFGVPIIAD GGIQTVGHVV KALALGASTV MMGSLLAATT EAPG EYFFS DGVRLKKYRG MGSLDAMEKS SSSQKRYFSE GDKVKIAQGV SGSIQDKGSI QKFVPYLIAG IQHGCQDIGA RSLDV LRSM MYSGELKFEK RTMSAQIEGG VHGLHSYTFL PFTKSGCTED SGGGRGGGGD APQCPLLGTA SLHN

UniProtKB: Inosine-5'-monophosphate dehydrogenase 1

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 16 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #3: INOSINIC ACID

MacromoleculeName: INOSINIC ACID / type: ligand / ID: 3 / Number of copies: 8 / Formula: IMP
Molecular weightTheoretical: 348.206 Da
Chemical component information

ChemComp-I:
INOSINIC ACID / Inosinic acid

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Macromolecule #4: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 8 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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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

BufferpH: 7
GridModel: C-flat-2/2 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number real images: 4632 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5836476
Startup modelType of model: EMDB MAP
EMDB ID:

24452

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1773701

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