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- EMDB-27295: Human PRPS1-E307A engineered mutation with ADP; Symmetry-expanded... -

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Basic information

Entry
Database: EMDB / ID: EMD-27295
TitleHuman PRPS1-E307A engineered mutation with ADP; Symmetry-expanded Hexamer 2
Map dataHuman PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer Centered
Sample
  • Complex: Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engineered mutation with ADP
    • Protein or peptide: Human Phosphoribosyl Pyrophosphate Synthetase 1 E307A Mutation
Keywordsphosphoribosyl pyrophosphate synthetase / ADP inhibitor / Filament-breaking mutation / TRANSFERASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsHvorecny KL / Kollman JM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F32AI145111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM118396 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Human PRPS1 filaments stabilize allosteric sites to regulate activity.
Authors: Kelli L Hvorecny / Kenzee Hargett / Joel D Quispe / Justin M Kollman /
Abstract: The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and ...The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here we determine structures of human PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.
History
DepositionJun 14, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27295.png

Downloads & links

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Map

FileDownload / File: emd_27295.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer Centered
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 1.75
Minimum - Maximum-10.613586 - 16.365684999999999
Average (Standard dev.)0.000000000001354 (±0.34671462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Human PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer...

Fileemd_27295_half_map_1.map
AnnotationHuman PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer Centered half 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Human PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer...

Fileemd_27295_half_map_2.map
AnnotationHuman PRPS1-E307A engineered mutation with ADP, C-terminus, Hexamer Centered half 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engine...

EntireName: Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engineered mutation with ADP
Components
  • Complex: Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engineered mutation with ADP
    • Protein or peptide: Human Phosphoribosyl Pyrophosphate Synthetase 1 E307A Mutation

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Supramolecule #1: Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engine...

SupramoleculeName: Hexamer of phosphoribosyl pyrophosphate synthetase 1 E307A engineered mutation with ADP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Six copies of PRPS1-E307A assemble to form a hexamer.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Human Phosphoribosyl Pyrophosphate Synthetase 1 E307A Mutation

MacromoleculeName: Human Phosphoribosyl Pyrophosphate Synthetase 1 E307A Mutation
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPNIKIFSGS SHQDLSQKIA DRLGLELGKV VTKKFSNQET CVEIGESVRG EDVYIVQSGC GEINDNLME LLIMINACKI ASASRVTAVI PCFPYARQDK KDKSRAPISA KLVANMLSVA G ADHIITMD LHASQIQGFF DIPVDNLYAE PAVLKWIREN ISEWRNCTIV ...String:
MPNIKIFSGS SHQDLSQKIA DRLGLELGKV VTKKFSNQET CVEIGESVRG EDVYIVQSGC GEINDNLME LLIMINACKI ASASRVTAVI PCFPYARQDK KDKSRAPISA KLVANMLSVA G ADHIITMD LHASQIQGFF DIPVDNLYAE PAVLKWIREN ISEWRNCTIV SPDAGGAKRV TS IADRLNV DFALIHKERK KANEVDRMVL VGDVKDRVAI LVDDMADTCG TICHAADKLL SAG ATRVYA ILTHGIFSGP AISRINNACF EAVVVTNTIP QEDKMKHCSK IQVIDISMIL AEAI RRTHN GASVSYLFSH VPL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.6
GridModel: C-flat / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.75 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 90.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PHENIX (ver. 1.18) / Number images used: 851443

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT

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