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- EMDB-12938: Cryo-EM structure of Medicago truncatula HISN5 protein -

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Basic information

Entry
Database: EMDB / ID: EMD-12938
TitleCryo-EM structure of Medicago truncatula HISN5 protein
Map data
Sample
  • Complex: Medicago truncatula HISN5 protein
    • Protein or peptide: Imidazoleglycerol-phosphate dehydratase
  • Ligand: MANGANESE (II) ION
  • Ligand: water
Keywordshistidine biosynthesis / herbicide design / high-resolution Cryo-EM / LYASE
Function / homology
Function and homology information


imidazoleglycerol-phosphate dehydratase / imidazoleglycerol-phosphate dehydratase activity / L-histidine biosynthetic process
Similarity search - Function
Imidazoleglycerol-phosphate dehydratase signature 1. / Imidazoleglycerol-phosphate dehydratase / Imidazoleglycerol-phosphate dehydratase, conserved site / Imidazole glycerol phosphate dehydratase domain superfamily / Imidazoleglycerol-phosphate dehydratase / Imidazoleglycerol-phosphate dehydratase signature 2. / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
Imidazoleglycerol-phosphate dehydratase
Similarity search - Component
Biological speciesMedicago truncatula (barrel medic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsRuszkowski M / Witek W
Funding support Poland, 1 items
OrganizationGrant numberCountry
Polish National Science Centre2018/31/D/NZ1/03630 Poland
CitationJournal: Front Plant Sci / Year: 2024
Title: Targeting imidazole-glycerol phosphate dehydratase in plants: novel approach for structural and functional studies, and inhibitor blueprinting.
Authors: Wojciech Witek / Joanna Sliwiak / Michal Rawski / Milosz Ruszkowski /
Abstract: The histidine biosynthetic pathway (HBP) is targeted for herbicide design with preliminary success only regarding imidazole-glycerol phosphate dehydratase (IGPD, EC 4.2.1.19), or HISN5, as referred ...The histidine biosynthetic pathway (HBP) is targeted for herbicide design with preliminary success only regarding imidazole-glycerol phosphate dehydratase (IGPD, EC 4.2.1.19), or HISN5, as referred to in plants. HISN5 catalyzes the sixth step of the HBP, in which imidazole-glycerol phosphate (IGP) is dehydrated to imidazole-acetol phosphate. In this work, we present high-resolution cryoEM and crystal structures of HISN5 (HISN5) in complexes with an inactive IGP diastereoisomer and with various other ligands. HISN5 can serve as a new model for plant HISN5 structural studies, as it enables resolving protein-ligand interactions at high (2.2 Å) resolution using cryoEM. We identified ligand-binding hotspots and characterized the features of plant HISN5 enzymes in the context of the HISN5-targeted inhibitor design. Virtual screening performed against millions of small molecules not only revealed candidate molecules but also identified linkers for fragments that were experimentally confirmed to bind. Based on experimental and computational approaches, this study provides guidelines for designing symmetric HISN5 inhibitors that can reach two neighboring active sites. Finally, we conducted analyses of sequence similarity networks revealing that plant HISN5 enzymes derive from cyanobacteria. We also adopted a new approach to measure HISN5 enzymatic activity using isothermal titration calorimetry and enzymatically synthesized IGP.
History
DepositionMay 13, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.076
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.076
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oj5
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7oj5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12938.png

Downloads & links

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Map

FileDownload / File: emd_12938.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 352 pix.
= 302.72 Å		0.86 Å/pix.
x 352 pix.
= 302.72 Å		0.86 Å/pix.
x 352 pix.
= 302.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.076 / Movie #1: 0.076
Minimum - Maximum-0.27863917 - 0.49912426
Average (Standard dev.)0.000016332258 (±0.016990839)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 302.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z302.720302.720302.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ560560560
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.2790.4990.000

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Supplemental data

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Sample components

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Entire : Medicago truncatula HISN5 protein

EntireName: Medicago truncatula HISN5 protein
Components
  • Complex: Medicago truncatula HISN5 protein
    • Protein or peptide: Imidazoleglycerol-phosphate dehydratase
  • Ligand: MANGANESE (II) ION
  • Ligand: water

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Supramolecule #1: Medicago truncatula HISN5 protein

SupramoleculeName: Medicago truncatula HISN5 protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Imidazoleglycerol-phosphate dehydratase
Source (natural)Organism: Medicago truncatula (barrel medic)

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Macromolecule #1: Imidazoleglycerol-phosphate dehydratase

MacromoleculeName: Imidazoleglycerol-phosphate dehydratase / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: imidazoleglycerol-phosphate dehydratase
Source (natural)Organism: Medicago truncatula (barrel medic)
Molecular weightTheoretical: 22.692453 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSTFPIDSGA RIGEMKRVTK ETNVSVKINL DGTGVADNSS GIPFLDHMLD QLASHGLFDV HVKATGDTHI DDHHTNEDVA LAIGTALLQ ALGDRKGINR FGNFSAPLDE ALVHVSLDLS GRPHLGYDLN IPTQRVGKYD TQLVEHFFQS LVNTSGMTLH I RQFSGTNS ...String:
MSTFPIDSGA RIGEMKRVTK ETNVSVKINL DGTGVADNSS GIPFLDHMLD QLASHGLFDV HVKATGDTHI DDHHTNEDVA LAIGTALLQ ALGDRKGINR FGNFSAPLDE ALVHVSLDLS GRPHLGYDLN IPTQRVGKYD TQLVEHFFQS LVNTSGMTLH I RQFSGTNS HHIIEATFKA FARALRQATE YDTRRRGTIP SSKGVLSRS

UniProtKB: Imidazoleglycerol-phosphate dehydratase

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Macromolecule #2: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 2 / Number of copies: 48 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 931 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 1008 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 229366
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ezj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-7oj5:
Cryo-EM structure of Medicago truncatula HISN5 protein

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