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- PDB-9upe: Glycogen phosphorylase dimer from E. coli in complex with AMP. -

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Basic information

Entry
Database: PDB / ID: 9upe
TitleGlycogen phosphorylase dimer from E. coli in complex with AMP.
ComponentsAlpha-1,4 glucan phosphorylase
KeywordsSTRUCTURAL PROTEIN / glycogen metabolism / cryo-EM / gut bacteria
Function / homologyADENOSINE MONOPHOSPHATE / :
Function and homology information
Biological speciesEscherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsTakai, M. / 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
DepositionApr 28, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 1, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 1, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-1,4 glucan phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)92,4902
Polymers92,1431
Non-polymers3471
Water00
1
A: Alpha-1,4 glucan phosphorylase
hetero molecules

A: Alpha-1,4 glucan phosphorylase
hetero molecules

A: Alpha-1,4 glucan phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)277,4706
Polymers276,4293
Non-polymers1,0423
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2

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Components

#1: Protein Alpha-1,4 glucan phosphorylase


Mass: 92142.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Gene: ECBD_0314 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140N6M9, glycogen phosphorylase
#2: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / 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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: glycogen phosphorylase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium chlorideNaCl1
220 mMTris1
35 mMAMP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 microliters droplet, 20 seconds delay before blotting, 3 seconds blot, 0 second delay before plunging.

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7656

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126140 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313328
ELECTRON MICROSCOPYf_angle_d0.63918088
ELECTRON MICROSCOPYf_dihedral_angle_d8.191804
ELECTRON MICROSCOPYf_chiral_restr0.0411966
ELECTRON MICROSCOPYf_plane_restr0.0052344

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