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- EMDB-50149: Myo-inositol-1-phosphate synthase from Thermochaetoides thermophi... -

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Basic information

Entry
Database: EMDB / ID: EMD-50149
TitleMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila in complex with NAD
Map dataMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 1
Sample
  • Complex: Native homotetramer of the Myo-inositol-1-phosphate synthase
    • Protein or peptide: inositol-3-phosphate synthase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Keywordsinositol metabolism / endogenous / conformational selection / ISOMERASE
Function / homology
Function and homology information


inositol-3-phosphate synthase / inositol-3-phosphate synthase activity / inositol biosynthetic process / phospholipid biosynthetic process / cytoplasm
Similarity search - Function
Myo-inositol-1-phosphate synthase / Myo-inositol-1-phosphate synthase / Myo-inositol-1-phosphate synthase, GAPDH-like / Myo-inositol-1-phosphate synthase / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
inositol-3-phosphate synthase
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsTraeger TK / Kyrilis FL / Hamdi F / Kastritis PL
Funding supportEuropean Union, Germany, 4 items
OrganizationGrant numberCountry
European Union (EU)101086665European Union
German Federal Ministry for Education and Research03Z22HN23, 03Z22HI2 and 03COV04 Germany
European Regional Development FundZS/2016/04/78115European Union
German Research Foundation (DFG)391498659 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway.
Authors: Toni K Träger / Fotis L Kyrilis / Farzad Hamdi / Christian Tüting / Marie Alfes / Tommy Hofmann / Carla Schmidt / Panagiotis L Kastritis /
Abstract: Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol ...Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus . By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.
History
DepositionApr 23, 2024-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 28, 2024-
Current statusAug 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50149.png

Downloads & links

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Map

FileDownload / File: emd_50149.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.59 Å/pix.
x 512 pix.
= 302.08 Å		0.59 Å/pix.
x 512 pix.
= 302.08 Å		0.59 Å/pix.
x 512 pix.
= 302.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.59 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.152
Minimum - Maximum-0.9780574 - 1.7541344
Average (Standard dev.)-0.0013905324 (±0.035171174)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 302.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50149_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Myo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 3...

Fileemd_50149_additional_1.map
AnnotationMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Myo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 2...

Fileemd_50149_additional_2.map
AnnotationMyo-inositol-1-phosphate synthase from Thermochaetoides thermophila; state 2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Halfmap 5; state1

Fileemd_50149_half_map_1.map
AnnotationHalfmap 5; state1
Projections & Slices
AxesZYX

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Half map: Halfmap A; state1

Fileemd_50149_half_map_2.map
AnnotationHalfmap A; state1
Projections & Slices
AxesZYX

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Sample components

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Entire : Native homotetramer of the Myo-inositol-1-phosphate synthase

EntireName: Native homotetramer of the Myo-inositol-1-phosphate synthase
Components
  • Complex: Native homotetramer of the Myo-inositol-1-phosphate synthase
    • Protein or peptide: inositol-3-phosphate synthase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: Native homotetramer of the Myo-inositol-1-phosphate synthase

SupramoleculeName: Native homotetramer of the Myo-inositol-1-phosphate synthase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: inositol-3-phosphate synthase

MacromoleculeName: inositol-3-phosphate synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: inositol-3-phosphate synthase
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 56.735477 KDa
SequenceString: IFKVNSPNVV YTDDEIRSKY VYRTTEVTTA EDGSLIATPR ETVYDFKVDR KLPKLGVMLV GWGGNNGSTI TAGIIANRRG LVWETRNGK QEANYYGSVI MGSTIKLGTD AKTHKDINIP FHSVLPMVHP NDIVIGGWDI SGLNLADAMD RAQVLEPSLK A LVRKEMAS ...String:
IFKVNSPNVV YTDDEIRSKY VYRTTEVTTA EDGSLIATPR ETVYDFKVDR KLPKLGVMLV GWGGNNGSTI TAGIIANRRG LVWETRNGK QEANYYGSVI MGSTIKLGTD AKTHKDINIP FHSVLPMVHP NDIVIGGWDI SGLNLADAMD RAQVLEPSLK A LVRKEMAS MKPLPSIYYP DFIAANQEDR ADNILPGNKK CWEHVEEIRK NIRDFKAANG LDKVIVLWTA NTERYASIIE GV NDTADNL LNAIKNGHEE VSPSTVFAVS SILEGVPFIN GSPQNTFVPG CIELAERHGA FIGGDDFKSG QTKMKSALVD FLI NAGIKL TSIASYNHLG NNDGKNLSSQ RQFRSKEISK SNVVDDMVEA NTVLYKPGEH PDHIVVIKYV PAVGDSKRAM DEYH GEIFL GGHQTISIAN VCEDSLLASP LIIDLVIVAE LMTRIQWRLH KEDATEADWK YFHSVLSILS YMLKAPMTPP GTPVV NALA KQRAAMANIF RACLGLDPEN DMTLEHKLF

UniProtKB: inositol-3-phosphate synthase

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Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 1 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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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

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
100.0 mMHEPES
95.0 mMNaCl
5.0 mMKCl
1.0 mMMgCl2
0.5 mMEDTA
5.0 %Glycerol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 400.0 kPa / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 6 s blot time with a blot force of -1.

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Electron microscopy

MicroscopeTFS GLACIOS
TemperatureMin: 77.15 K / Max: 103.15 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 2 / Number real images: 6261 / Average electron dose: 28.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 240000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 309043
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Software - details: Local refinement / Number images used: 255354
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Ab-Initio Reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Non uniform Refinement
Final 3D classificationNumber classes: 5 / Avg.num./class: 70000 / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: 3D classification
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: Local Installation
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9f2k:
Myo-inositol-1-phosphate synthase from Thermochaetoides thermophila in complex with NAD

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Atomic model buiding 2

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: Local Installation
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9f2k:
Myo-inositol-1-phosphate synthase from Thermochaetoides thermophila in complex with NAD

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