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- PDB-7xn3: E.coli phosphoribosylpyrophosphate (PRPP) synthetase type B filam... -

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Basic information

Entry
Database: PDB / ID: 7xn3
TitleE.coli phosphoribosylpyrophosphate (PRPP) synthetase type B filament bound with Pi
ComponentsRibose-phosphate pyrophosphokinase
KeywordsBIOSYNTHETIC PROTEIN / Allosteric enzyme / Kinase / Transferase / Nucleotide biosynthesis / ATP-binding / Magnesium / Manganese / Metal-binding / Nucleotide-binding
Function / homology
Function and homology information


ribose phosphate diphosphokinase complex / ribonucleoside monophosphate biosynthetic process / ribose-phosphate diphosphokinase / ribose phosphate diphosphokinase activity / 5-phosphoribose 1-diphosphate biosynthetic process / purine nucleotide biosynthetic process / protein hexamerization / phosphate ion binding / ADP binding / kinase activity ...ribose phosphate diphosphokinase complex / ribonucleoside monophosphate biosynthetic process / ribose-phosphate diphosphokinase / ribose phosphate diphosphokinase activity / 5-phosphoribose 1-diphosphate biosynthetic process / purine nucleotide biosynthetic process / protein hexamerization / phosphate ion binding / ADP binding / kinase activity / magnesium ion binding / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Phosphoribosyl pyrophosphate synthetase, conserved site / Phosphoribosyl pyrophosphate synthase signature. / Ribose-phosphate pyrophosphokinase, bacterial-type / Phosphoribosyl synthetase-associated domain / N-terminal domain of ribose phosphate pyrophosphokinase / Ribose-phosphate pyrophosphokinase / Ribose-phosphate pyrophosphokinase, N-terminal domain / N-terminal domain of ribose phosphate pyrophosphokinase / Phosphoribosyltransferase-like / Phosphoribosyltransferase domain
Similarity search - Domain/homology
PHOSPHATE ION / Ribose-phosphate pyrophosphokinase
Similarity search - Component
Biological speciesEscherichia coli str. K-12 substr. MG1655 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHu, H.H. / Lu, G.M. / Chang, C.C. / Liu, J.L.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)No. 2021YFA0804701-4 China
National Natural Science Foundation of China (NSFC)No. 31771490 China
CitationJournal: Elife / Year: 2022
Title: Filamentation modulates allosteric regulation of PRPS.
Authors: Huan-Huan Hu / Guang-Ming Lu / Chia-Chun Chang / Yilan Li / Jiale Zhong / Chen-Jun Guo / Xian Zhou / Boqi Yin / Tianyi Zhang / Ji-Long Liu /
Abstract: Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP ...Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.
History
DepositionApr 27, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year
Revision 1.2Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ribose-phosphate pyrophosphokinase
B: Ribose-phosphate pyrophosphokinase
F: Ribose-phosphate pyrophosphokinase
E: Ribose-phosphate pyrophosphokinase
C: Ribose-phosphate pyrophosphokinase
D: Ribose-phosphate pyrophosphokinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)211,64418
Polymers210,5056
Non-polymers1,14012
Water97354
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "E"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "B"
d_6ens_1chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASPHISE1 - 305
d_12ens_1PO4PO4E1001
d_13ens_1PO4PO4E2601
d_21ens_1ASPHISA1 - 305
d_22ens_1PO4PO4A1001
d_23ens_1PO4PO4A2601
d_31ens_1ASPHISC1 - 305
d_32ens_1PO4PO4C1001
d_33ens_1PO4PO4C2601
d_41ens_1ASPHISD1 - 305
d_42ens_1PO4PO4D1001
d_43ens_1PO4PO4D2601
d_51ens_1ASPHISB1 - 305
d_52ens_1PO4PO4B1001
d_53ens_1PO4PO4B2601
d_61ens_1ASPHISF1 - 305
d_62ens_1PO4PO4F1001
d_63ens_1PO4PO4F2601

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Components

#1: Protein
Ribose-phosphate pyrophosphokinase / RPPK / 5-phospho-D-ribosyl alpha-1-diphosphate / Phosphoribosyl diphosphate synthase / ...RPPK / 5-phospho-D-ribosyl alpha-1-diphosphate / Phosphoribosyl diphosphate synthase / Phosphoribosyl pyrophosphate synthase / P-Rib-PP synthase / PRPP synthase / PRPPase


Mass: 35084.090 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli str. K-12 substr. MG1655 (bacteria)
Gene: prs, prsA, b1207, JW1198
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A717, ribose-phosphate diphosphokinase
#2: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 54 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: E.coli PRPP syhthetase type B filament structure complex with Pi
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chloeideNaCl1
250 mMsodium dihydrogen phosphate anhydrousNa2HPO41
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The purifide monomer or oligomer protein was incubated with Pi to form this type B filament.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: blot for 3.5 seconds with blot force of -1 before plunge-freezing

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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 / Nominal magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3131

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimera1.14model fitting
9Cootmodel refinement
10PHENIXmodel refinement
13RELIONclassification
14RELION3.1-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1186879
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 168218 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4S2U

4s2u


Pdb chain-ID: A / Accession code: 4S2U / Pdb chain residue range: 2-309 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.63 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414118
ELECTRON MICROSCOPYf_angle_d0.59219146
ELECTRON MICROSCOPYf_dihedral_angle_d26.6791980
ELECTRON MICROSCOPYf_chiral_restr0.0482298
ELECTRON MICROSCOPYf_plane_restr0.0042514
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000706473322223
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000705817048413
ens_1d_4DELECTRON MICROSCOPYNCS constraints0.0007034895201
ens_1d_5BELECTRON MICROSCOPYNCS constraints0.000705358949613
ens_1d_6FELECTRON MICROSCOPYNCS constraints0.000709981494244

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