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

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Basic information

Entry
Database: PDB / ID: 7oj5
TitleCryo-EM structure of Medicago truncatula HISN5 protein
ComponentsImidazoleglycerol-phosphate dehydratase
KeywordsLYASE / histidine biosynthesis / herbicide design / high-resolution Cryo-EM
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsRuszkowski, M. / Witek, W.
Funding support Poland, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update
Revision 1.2Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Imidazoleglycerol-phosphate dehydratase
B: Imidazoleglycerol-phosphate dehydratase
C: Imidazoleglycerol-phosphate dehydratase
D: Imidazoleglycerol-phosphate dehydratase
E: Imidazoleglycerol-phosphate dehydratase
F: Imidazoleglycerol-phosphate dehydratase
G: Imidazoleglycerol-phosphate dehydratase
H: Imidazoleglycerol-phosphate dehydratase
I: Imidazoleglycerol-phosphate dehydratase
J: Imidazoleglycerol-phosphate dehydratase
K: Imidazoleglycerol-phosphate dehydratase
L: Imidazoleglycerol-phosphate dehydratase
M: Imidazoleglycerol-phosphate dehydratase
N: Imidazoleglycerol-phosphate dehydratase
O: Imidazoleglycerol-phosphate dehydratase
P: Imidazoleglycerol-phosphate dehydratase
Q: Imidazoleglycerol-phosphate dehydratase
R: Imidazoleglycerol-phosphate dehydratase
S: Imidazoleglycerol-phosphate dehydratase
T: Imidazoleglycerol-phosphate dehydratase
V: Imidazoleglycerol-phosphate dehydratase
W: Imidazoleglycerol-phosphate dehydratase
X: Imidazoleglycerol-phosphate dehydratase
Y: Imidazoleglycerol-phosphate dehydratase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)547,25672
Polymers544,61924
Non-polymers2,63748
Water16,772931
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"
d_13ens_1chain "M"
d_14ens_1chain "N"
d_15ens_1chain "O"
d_16ens_1chain "P"
d_17ens_1chain "Q"
d_18ens_1chain "R"
d_19ens_1chain "S"
d_20ens_1chain "T"
d_21ens_1chain "V"
d_22ens_1chain "W"
d_23ens_1chain "X"
d_24ens_1chain "Y"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALAARGA1 - 184
d_12ens_1MNMNY
d_21ens_1ALAARGB1 - 184
d_22ens_1MNMNAA
d_31ens_1ALAARGC1 - 184
d_32ens_1MNMNCA
d_41ens_1ALAARGD1 - 184
d_42ens_1MNMNEA
d_51ens_1ALAARGE1 - 184
d_52ens_1MNMNGA
d_61ens_1ALAARGF1 - 184
d_62ens_1MNMNIA
d_71ens_1ALAARGG1 - 184
d_72ens_1MNMNKA
d_81ens_1ALAARGH1 - 184
d_82ens_1MNMNMA
d_91ens_1ALAARGI1 - 184
d_92ens_1MNMNOA
d_101ens_1ALAARGJ1 - 184
d_102ens_1MNMNQA
d_111ens_1ALAARGK1 - 184
d_112ens_1MNMNSA
d_121ens_1ALAARGL1 - 184
d_122ens_1MNMNUA
d_131ens_1ALAARGM1 - 184
d_132ens_1MNMNWA
d_141ens_1ALAARGN1 - 184
d_142ens_1MNMNYA
d_151ens_1ALAARGO1 - 184
d_152ens_1MNMNAB
d_161ens_1ALAARGP1 - 184
d_162ens_1MNMNCB
d_171ens_1ALAARGQ1 - 184
d_172ens_1MNMNEB
d_181ens_1ALAARGR1 - 184
d_182ens_1MNMNGB
d_191ens_1ALAARGS1 - 184
d_192ens_1MNMNIB
d_201ens_1ALAARGT1 - 184
d_202ens_1MNMNKB
d_211ens_1ALAARGU1 - 184
d_212ens_1MNMNMB
d_221ens_1ALAARGV1 - 184
d_222ens_1MNMNOB
d_231ens_1ALAARGW1 - 184
d_232ens_1MNMNQB
d_241ens_1ALAARGX1 - 184
d_242ens_1MNMNSB

NCS oper:
IDCodeMatrixVector
1given(1), (-1), (1)302.720001018
2given(-1), (-1), (1)302.719994019, 302.720002546
3given(-1), (1), (-1)302.719994506, 302.720012192
4given(-1), (1), (1)302.720000701
5given(-1), (-1), (-1)302.719994019, 302.72000251, 302.720002546
6given(1), (-1), (-1)302.720008705, 302.720001018
7given(-1), (1), (1)302.716601838
8given(1), (-1), (-1)302.719994019, 302.720002547
9given(1), (1), (-1)302.720012192
10given(1), (-1), (1)302.720012192
11given(1), (1), (1)
12given(-1), (-1), (-1)302.72000251, 302.720002546, 302.719994019
13given(-1), (-1), (1)302.720008705, 302.720001018
14given(1), (1), (1)
15given(-1), (1), (-1)302.720002546, 302.719994019
16given(-1), (1), (1)302.720001018
17given(1), (-1), (-1)302.720012192, 302.719994506
18given(1), (1), (-1)302.720000701
19given(-1), (1), (-1)302.720001018, 302.720008705
20given(-1), (-1), (-1)302.720002546, 302.719994019, 302.72000251
21given(1), (-1), (1)302.720000701
22given(1), (1), (-1)302.720001018
23given(-1), (-1), (1)302.720012192, 302.719994506

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Components

#1: Protein ...
Imidazoleglycerol-phosphate dehydratase


Mass: 22692.453 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Medicago truncatula (barrel medic) / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: I3SDM5, imidazoleglycerol-phosphate dehydratase
#2: Chemical...
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 48 / Source method: obtained synthetically / Formula: Mn
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 931 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Medicago truncatula HISN5 protein / Type: COMPLEX / Details: Imidazoleglycerol-phosphate dehydratase / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Medicago truncatula (barrel medic)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1008

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19_4092refinement
PHENIX1.19_4092refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selection
2EPUimage acquisition
4CTFFINDCTF correction
7PHENIX1.19-4092model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
13PHENIX1.19-4092model refinementreal-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229366 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6EZJ

6ezj


Accession code: 6EZJ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 18.17 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001934992
ELECTRON MICROSCOPYf_angle_d0.51447352
ELECTRON MICROSCOPYf_chiral_restr0.04265400
ELECTRON MICROSCOPYf_plane_restr0.00326240
ELECTRON MICROSCOPYf_dihedral_angle_d4.31224728
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints1.65706486177E-13
ens_1d_3AELECTRON MICROSCOPYNCS constraints3.72958914564E-5
ens_1d_4AELECTRON MICROSCOPYNCS constraints5.27564445605E-5
ens_1d_5AELECTRON MICROSCOPYNCS constraints3.7282915355E-5
ens_1d_6AELECTRON MICROSCOPYNCS constraints5.27564445594E-5
ens_1d_7AELECTRON MICROSCOPYNCS constraints3.72829153542E-5
ens_1d_8AELECTRON MICROSCOPYNCS constraints0.000706586252387
ens_1d_9AELECTRON MICROSCOPYNCS constraints3.72958914545E-5
ens_1d_10AELECTRON MICROSCOPYNCS constraints3.72958914545E-5
ens_1d_11AELECTRON MICROSCOPYNCS constraints3.72958914548E-5
ens_1d_12AELECTRON MICROSCOPYNCS constraints0
ens_1d_13AELECTRON MICROSCOPYNCS constraints5.27564445611E-5
ens_1d_14AELECTRON MICROSCOPYNCS constraints3.72829153544E-5
ens_1d_15AELECTRON MICROSCOPYNCS constraints0
ens_1d_16AELECTRON MICROSCOPYNCS constraints3.72958914564E-5
ens_1d_17AELECTRON MICROSCOPYNCS constraints1.86103091251E-13
ens_1d_18AELECTRON MICROSCOPYNCS constraints5.27564445611E-5
ens_1d_19AELECTRON MICROSCOPYNCS constraints3.72829153553E-5
ens_1d_20AELECTRON MICROSCOPYNCS constraints3.72829153553E-5
ens_1d_21AELECTRON MICROSCOPYNCS constraints5.27564445602E-5
ens_1d_22AELECTRON MICROSCOPYNCS constraints3.72829153542E-5
ens_1d_23AELECTRON MICROSCOPYNCS constraints2.11936877614E-13
ens_1d_24AELECTRON MICROSCOPYNCS constraints5.27564445595E-5

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