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- PDB-6ezj: Imidazoleglycerol-phosphate dehydratase -

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Basic information

Entry
Database: PDB / ID: 6ezj
TitleImidazoleglycerol-phosphate dehydratase
ComponentsImidazoleglycerol-phosphate dehydratase 2, chloroplastic
KeywordsLYASE / Inhibitor / histidine biosynthesis / complex
Function / homology
Function and homology information


imidazoleglycerol-phosphate dehydratase / imidazoleglycerol-phosphate dehydratase activity / histidine biosynthetic process / chloroplast / metal ion binding
Similarity search - Function
Imidazole glycerol phosphate dehydratase; domain 1 / 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 / Ribosomal protein S5 domain 2-type fold / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-5LD / : / Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsRawson, S. / Bisson, C. / Hurdiss, D.L. / Muench, S.P.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust(009752/Z/12/Z) and (102572/B/13/Z) United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/I003703/1 United Kingdom
Medical Research Council (United Kingdom)G100567 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM.
Authors: Shaun Rawson / Claudine Bisson / Daniel L Hurdiss / Asif Fazal / Martin J McPhillie / Svetlana E Sedelnikova / Patrick J Baker / David W Rice / Stephen P Muench /
Abstract: Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate ...Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both (IGPD) and (IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against IGPD than IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the and IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the IGPD/C348 complex. The structure of _IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.
History
DepositionNov 15, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2018Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Feb 28, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
B: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
C: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
D: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
E: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
F: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
G: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
H: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
I: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
J: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
K: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
L: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
M: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
N: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
O: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
P: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
Q: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
R: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
S: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
T: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
U: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
V: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
W: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
X: Imidazoleglycerol-phosphate dehydratase 2, chloroplastic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)544,99596
Polymers537,38724
Non-polymers7,60872
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area77600 Å2
ΔGint-466 kcal/mol
Surface area142270 Å2
MethodPISA

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Components

#1: Protein ...
Imidazoleglycerol-phosphate dehydratase 2, chloroplastic / / IGPD 2 / Protein HISTIDINE BIOSYNTHESIS 5B


Mass: 22391.127 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HISN5B, At4g14910, dl3495c / Production host: Escherichia coli (E. coli)
References: UniProt: O23346, 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: Chemical...
ChemComp-5LD / [(2R)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl]phosphonic acid / (R)-C348


Mass: 207.124 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C5H10N3O4P / Feature type: SUBJECT OF INVESTIGATION

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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: Imidazoleglycerol-phosphate dehydratase (IGPD2) in complex with triazole-phosphonate inhibitor (C384)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.57 MDa
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 682
Image scansMovie frames/image: 32

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 110977
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55481 / Details: Relion1.4 auto-refine / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0234440
ELECTRON MICROSCOPYf_angle_d0.99146440
ELECTRON MICROSCOPYf_dihedral_angle_d5.79319944
ELECTRON MICROSCOPYf_chiral_restr0.0555424
ELECTRON MICROSCOPYf_plane_restr0.0076024

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