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- EMDB-33306: E.coli phosphoribosylpyrophosphate (PRPP) synthetase type A(AMP/A... -

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Basic information

Entry
Database: EMDB / ID: EMD-33306
TitleE.coli phosphoribosylpyrophosphate (PRPP) synthetase type A(AMP/ADP) filament bound with ADP, AMP and R5P
Map data
Sample
  • Organelle or cellular component: E.coli PRPP syhthetase filament structure complex with ADP,AMP, Mg2+ and R5P
    • Protein or peptide: Ribose-phosphate pyrophosphokinase
  • Ligand: 5-O-phosphono-alpha-D-ribofuranose
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: water
KeywordsAllosteric enzyme / Kinase / Transferase / Nucleotide biosynthesis / ATP-binding / Magnesium / Manganese / Metal-binding / Nucleotide-binding / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


ribonucleoside monophosphate biosynthetic process / ribose phosphate diphosphokinase complex / 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 ...ribonucleoside monophosphate biosynthetic process / ribose phosphate diphosphokinase complex / 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 / cytosol / cytoplasm
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
Ribose-phosphate pyrophosphokinase
Similarity search - Component
Biological speciesEscherichia coli str. K-12 substr. MG1655 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsHu HH / Lu GM / Chang CC / Liu JL
Funding support China, 2 items
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, 2022-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33306.png

Downloads & links

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Map

FileDownload / File: emd_33306.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.1983182 - 0.3076565
Average (Standard dev.)0.000098876866 (±0.010989766)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33306_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33306_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33306_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : E.coli PRPP syhthetase filament structure complex with ADP,AMP, M...

EntireName: E.coli PRPP syhthetase filament structure complex with ADP,AMP, Mg2+ and R5P
Components
  • Organelle or cellular component: E.coli PRPP syhthetase filament structure complex with ADP,AMP, Mg2+ and R5P
    • Protein or peptide: Ribose-phosphate pyrophosphokinase
  • Ligand: 5-O-phosphono-alpha-D-ribofuranose
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: water

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Supramolecule #1: E.coli PRPP syhthetase filament structure complex with ADP,AMP, M...

SupramoleculeName: E.coli PRPP syhthetase filament structure complex with ADP,AMP, Mg2+ and R5P
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: a new allosteric for AMP binding
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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Macromolecule #1: Ribose-phosphate pyrophosphokinase

MacromoleculeName: Ribose-phosphate pyrophosphokinase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: ribose-phosphate diphosphokinase
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Molecular weightTheoretical: 35.08409 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MPDMKLFAGN ATPELAQRIA NRLYTSLGDA AVGRFSDGEV SVQINENVRG GDIFIIQSTC APTNDNLMEL VVMVDALRRA SAGRITAVI PYFGYARQDR RVRSARVPIT AKVVADFLSS VGVDRVLTVD LHAEQIQGFF DVPVDNVFGS PILLEDMLQL N LDNPIVVS ...String:
MPDMKLFAGN ATPELAQRIA NRLYTSLGDA AVGRFSDGEV SVQINENVRG GDIFIIQSTC APTNDNLMEL VVMVDALRRA SAGRITAVI PYFGYARQDR RVRSARVPIT AKVVADFLSS VGVDRVLTVD LHAEQIQGFF DVPVDNVFGS PILLEDMLQL N LDNPIVVS PDIGGVVRAR AIAKLLNDTD MAIIDKRRPR ANVSQVMHII GDVAGRDCVL VDDMIDTGGT LCKAAEALKE RG AKRVFAY ATHPIFSGNA ANNLRNSVID EVVVCDTIPL SDEIKSLPNV RTLTLSGMLA EAIRRISNEE SISAMFEHHH HHH H

UniProtKB: Ribose-phosphate pyrophosphokinase

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Macromolecule #2: 5-O-phosphono-alpha-D-ribofuranose

MacromoleculeName: 5-O-phosphono-alpha-D-ribofuranose / type: ligand / ID: 2 / Number of copies: 6 / Formula: HSX
Molecular weightTheoretical: 230.11 Da
Chemical component information

ChemComp-HSX:
5-O-phosphono-alpha-D-ribofuranose

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 810 / 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 statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: blot for 3.5 seconds with blot force of -1 before plunge-freezing.
DetailsThe purifide monomer or oligomer protein was incubated with ADP,AMP and MgCl2 to form this type A(AMP/ADP) filament.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2566 / Average exposure time: 4.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1066797
Startup modelType of model: OTHER
Details: The featureless cylinder was reconstructed using the relion_helix_toolbox command and applied as a reference model for 3D classification.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1-beta) / Number images used: 53045
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 4 / Avg.num./class: 40411 / Software - Name: RELION (ver. 3.1-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4s2u


Chain - Chain ID: A / Chain - Residue range: 2-309 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7xmv:
E.coli phosphoribosylpyrophosphate (PRPP) synthetase type A(AMP/ADP) filament bound with ADP, AMP and R5P

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