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- PDB-9v16: Crystal structure of E. coli glycogen phosphorylase N185A/R267E m... -

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Basic information

Entry
Database: PDB / ID: 9v16
TitleCrystal structure of E. coli glycogen phosphorylase N185A/R267E mutant in complex with AMP
ComponentsGlycogen phosphorylase
KeywordsTRANSFERASE / glycogen phosphorylase
Function / homology
Function and homology information


glycogen phosphorylase / glycogen phosphorylase activity / glycogen catabolic process / pyridoxal phosphate binding / protein homodimerization activity / cytoplasm
Similarity search - Function
Glycogen/starch/alpha-glucan phosphorylase / Phosphorylase pyridoxal-phosphate attachment site / Phosphorylase pyridoxal-phosphate attachment site. / Glycosyl transferase, family 35 / Carbohydrate phosphorylase
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / Glycogen phosphorylase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.45 Å
AuthorsTakai, M. / Shobu, K. / Fukuda, Y. / Inoue, T.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Structural and mechanistic diversity of glycogen phosphorylases from gut bacteria.
Authors: Keigo Shobu / Mayu Takai / Hiroki Tanino / Yohta Fukuda / Tsuyoshi Inoue /
Abstract: Glycogen phosphorylase (GP) plays a central role in glycogen metabolism. While the structure and regulation of mammalian GPs have been extensively studied, the corresponding mechanisms in gut ...Glycogen phosphorylase (GP) plays a central role in glycogen metabolism. While the structure and regulation of mammalian GPs have been extensively studied, the corresponding mechanisms in gut bacterial GPs remain poorly understood. Here, we investigate GPs from (GP), (GP), and (GP), which represent three phylogenetic clades of GPs, using enzymatic assays, cryo-electron microscopy (cryo-EM), and X-ray crystallography. We find that GP forms a unique pentamer that undergoes adenosine monophosphate (AMP)-dependent assembly into a dimer-of-pentamer, which inhibits activity by restricting substrate access to the catalytic site. GP exists in equilibrium among monomers, dimers, and tetramers, with AMP promoting tetramer dissociation and enhancing catalytic efficiency. In contrast, GP remains predominantly monomeric and is unresponsive to AMP. These findings uncover structural and regulatory diversity among gut bacterial GPs. Notably, the oligomeric states of GPs modulate substrate accessibility and enzyme activation, suggesting a distinct mode of allosteric regulation beyond the canonical T-to-R transition model. Because bacterial GPs contribute to the generation of glucose, their regulation may influence the composition of gut-derived metabolites that affect host glucose homeostasis and insulin sensitivity. Our study provides mechanistic insight into the structural and functional diversity of gut bacterial GPs and lays a foundation for future exploration of microbiome-mediated metabolic interactions.
History
DepositionMay 19, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2026Group: Database references / Category: citation / citation_author
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Revision 1.2Mar 25, 2026Group: Database references / Category: citation / citation_author
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Revision 1.3Apr 1, 2026Group: Database references / Category: citation / citation_author
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glycogen phosphorylase
B: Glycogen phosphorylase
C: Glycogen phosphorylase
D: Glycogen phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)375,8958
Polymers374,5064
Non-polymers1,3894
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11510 Å2
ΔGint-24 kcal/mol
Surface area113290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)185.604, 185.604, 451.242
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein
Glycogen phosphorylase


Mass: 93626.562 Da / Num. of mol.: 4 / Mutation: N185A/R267E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: glgP, glgY, b3428, JW3391 / Plasmid: pET28-a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AC86, glycogen phosphorylase
#2: Chemical
ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 5.19 Å3/Da / Density % sol: 76.29 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 2% v/v 1,4-dioxane, 0.1M Tris pH 8.0, 15% w/v polyethylene glycol 3350 (The crystal was soaked in a solution supplemented with 40 mM AMP before data collection)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Apr 23, 2025
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 3.45→49.5 Å / Num. obs: 98722 / % possible obs: 95.3 % / Redundancy: 9.6 % / Biso Wilson estimate: 111.35 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.164 / Rpim(I) all: 0.054 / Rrim(I) all: 0.173 / Net I/σ(I): 12
Reflection shellResolution: 3.45→3.51 Å / Redundancy: 10 % / Rmerge(I) obs: 2 / Mean I/σ(I) obs: 1.5 / Num. unique obs: 4913 / CC1/2: 0.81 / Rpim(I) all: 0.652 / Rrim(I) all: 2.11 / % possible all: 96.7

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Processing

Software
NameVersionClassification
PHENIX(1.19.2_4158: ???)refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.45→49.45 Å / SU ML: 0.49 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.12 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2351 4971 5.04 %
Rwork0.1921 --
obs0.1942 98610 94.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.45→49.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms25677 0 92 0 25769
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01126349
X-RAY DIFFRACTIONf_angle_d1.40835758
X-RAY DIFFRACTIONf_dihedral_angle_d17.669708
X-RAY DIFFRACTIONf_chiral_restr0.0713901
X-RAY DIFFRACTIONf_plane_restr0.0144623
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.45-3.490.42831710.35613106X-RAY DIFFRACTION96
3.49-3.530.4621620.33953123X-RAY DIFFRACTION96
3.53-3.570.31481630.3173131X-RAY DIFFRACTION96
3.57-3.620.39811690.3063103X-RAY DIFFRACTION96
3.62-3.670.31871650.28973109X-RAY DIFFRACTION96
3.67-3.720.33681610.28943149X-RAY DIFFRACTION96
3.72-3.770.35041580.26893098X-RAY DIFFRACTION96
3.77-3.830.29081530.25913125X-RAY DIFFRACTION96
3.83-3.890.26761590.2473142X-RAY DIFFRACTION96
3.89-3.950.23971700.23043128X-RAY DIFFRACTION96
3.95-4.020.2721880.2293079X-RAY DIFFRACTION96
4.02-4.090.29781700.2333105X-RAY DIFFRACTION95
4.09-4.170.30011770.23013134X-RAY DIFFRACTION95
4.17-4.250.24651760.22013087X-RAY DIFFRACTION95
4.25-4.350.25141570.20223119X-RAY DIFFRACTION95
4.35-4.450.26481850.19513108X-RAY DIFFRACTION95
4.45-4.560.24291530.19133118X-RAY DIFFRACTION95
4.56-4.680.19381590.1872972X-RAY DIFFRACTION91
4.68-4.820.19591460.17053070X-RAY DIFFRACTION93
4.82-4.970.24791840.17663119X-RAY DIFFRACTION95
4.98-5.150.19931610.16063145X-RAY DIFFRACTION95
5.15-5.360.20391740.17013089X-RAY DIFFRACTION94
5.36-5.60.20931610.17483155X-RAY DIFFRACTION95
5.6-5.90.20461750.17083090X-RAY DIFFRACTION94
5.9-6.270.20661740.18293147X-RAY DIFFRACTION94
6.27-6.750.21851630.16793135X-RAY DIFFRACTION94
6.75-7.420.20481490.17343181X-RAY DIFFRACTION94
7.43-8.490.18521560.13553167X-RAY DIFFRACTION93
8.5-10.680.15081560.12343134X-RAY DIFFRACTION91
10.69-49.450.22631760.17693271X-RAY DIFFRACTION90

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