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- EMDB-64393: Glycogen phosphorylase dimer from E. coli in complex with AMP. -

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Basic information

Entry
Database: EMDB / ID: EMD-64393
TitleGlycogen phosphorylase dimer from E. coli in complex with AMP.
Map data
Sample
  • Complex: glycogen phosphorylase
    • Protein or peptide: Alpha-1,4 glucan phosphorylase
  • Ligand: ADENOSINE MONOPHOSPHATE
Keywordsglycogen metabolism / cryo-EM / gut bacteria / STRUCTURAL PROTEIN
Function / homology:
Function and homology information
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsTakai M / Fukuda Y / Inoue T
Funding support Japan, 1 items
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, 2025-
Header (metadata) releaseApr 1, 2026-
Map releaseApr 1, 2026-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64393.png

Downloads & links

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Map

FileDownload / File: emd_64393.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0537
Minimum - Maximum-0.28380966 - 0.37164328
Average (Standard dev.)0.00003165044 (±0.008202462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64393_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_64393_half_map_2.map
Projections & Slices
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Sample components

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Entire : glycogen phosphorylase

EntireName: glycogen phosphorylase
Components
  • Complex: glycogen phosphorylase
    • Protein or peptide: Alpha-1,4 glucan phosphorylase
  • Ligand: ADENOSINE MONOPHOSPHATE

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Supramolecule #1: glycogen phosphorylase

SupramoleculeName: glycogen phosphorylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Alpha-1,4 glucan phosphorylase

MacromoleculeName: Alpha-1,4 glucan phosphorylase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: glycogen phosphorylase
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 92.142867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TLSVEALKHS IAYKLMFTIG KDPVVANKHE WLNATLFAVR DRLVERWLRS NRAQLSQETR QVYYLSMEFL IGRTLSNAML SLGIYEDVQ GALEAMGLNL EELIDEENDP GLGNGGLGRL AACFLDSLAT LGLPGRGYGI RYDYGMFKQN IVNGSQKESP D YWLEYGNP ...String:
TLSVEALKHS IAYKLMFTIG KDPVVANKHE WLNATLFAVR DRLVERWLRS NRAQLSQETR QVYYLSMEFL IGRTLSNAML SLGIYEDVQ GALEAMGLNL EELIDEENDP GLGNGGLGRL AACFLDSLAT LGLPGRGYGI RYDYGMFKQN IVNGSQKESP D YWLEYGNP WEFKRHNTRY KVRFGGRIQQ EGKKTRWIET EEILGVAYDQ IIPGYDTDAT NTLRLWSAQA SSEINLGKFN QG DYFAAVE DKNHSENVSR VLYPDDSTYS GRELRLRQEY FLVSSTIQDI LSRHYQLHKT YDNLADKIAI HLNDTHPVLS IPE MMRLLI DEHQFSWDDA FEVCCQVFSY TNHTLMSEAL ETWPVDMLGK ILPRHLQIIF EINDYFLKTL QEQYPNDTDL LGRA SIIDE SNGRRVRMAW LAVVVSHKVN GVSELHSNLM VQSLFADFAK IFPGRFTNVT NGVTPRRWLA VANPSLSAVL DEHLG RNWR TDLSLLNELQ QHCDFPMVNH AVHQAKLENK KRLAEYIAQQ LNVVVNPKAL FDVQIKRIHE YKRQLMNVLH VITRYN RIK ADPDAKWVPR VNIFGGKAAS AYYMAKHIIH LINDVAKVIN NDPQIGDKLK VVFIPNYSVS LAQLIIPAAD LSEQISL AG TEASGTSNM(LLP) FALNGALTIG TLDGANVEML DHVGADNIFI FGNTAEEVEE LRRQGYKPRE YYEKDEELHQ VLTQ IGSGV FSPEDPGRYR DLVDSLINFG DHYQVLADYR SYVDCQDKVD ELYELQEEWT AKAMLNIANM GYFSSDRTIK EYADH IWHI DPV

UniProtKB: UNIPROTKB: A0A140N6M9

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Macromolecule #2: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.00 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloride
20.0 mMTris
5.0 mMAMP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters droplet, 20 seconds delay before blotting, 3 seconds blot, 0 second delay before plunging..

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Electron microscopy

MicroscopeJEOL CRYO ARM 200
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7656 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 126140
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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