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- EMDB-64377: Glycogen phosphorylase tetramer from E. coli -

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Basic information

Entry
Database: EMDB / ID: EMD-64377
TitleGlycogen phosphorylase tetramer from E. coli
Map data
Sample
  • Complex: Glycogen phosphorylase
    • Protein or peptide: Alpha-1,4 glucan phosphorylase
  • Ligand: water
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: 2.63 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 25, 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_64377.png

Downloads & links

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Map

FileDownload / File: emd_64377.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å		0.83 Å/pix.
x 480 pix.
= 398.4 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033
Minimum - Maximum-0.5850934 - 0.88586235
Average (Standard dev.)-0.000040950374 (±0.020846026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 398.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64377_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_64377_half_map_2.map
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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: water

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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: 2 / Enantiomer: LEVO / EC number: glycogen phosphorylase
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 91.742398 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: EALKHSIAYK LMFTIGKDPV VANKHEWLNA TLFAVRDRLV ERWLRSNRAQ LSQETRQVYY LSMEFLIGRT LSNAMLSLGI YEDVQGALE AMGLNLEELI DEENDPGLGN GGLGRLAACF LDSLATLGLP GRGYGIRYDY GMFKQNIVNG SQKESPDYWL E YGNPWEFK ...String:
EALKHSIAYK LMFTIGKDPV VANKHEWLNA TLFAVRDRLV ERWLRSNRAQ LSQETRQVYY LSMEFLIGRT LSNAMLSLGI YEDVQGALE AMGLNLEELI DEENDPGLGN GGLGRLAACF LDSLATLGLP GRGYGIRYDY GMFKQNIVNG SQKESPDYWL E YGNPWEFK RHNTRYKVRF GGRIQQEGKK TRWIETEEIL GVAYDQIIPG YDTDATNTLR LWSAQASSEI NLGKFNQGDY FA AVEDKNH SENVSRVLYP DDSTYSGREL RLRQEYFLVS STIQDILSRH YQLHKTYDNL ADKIAIHLND THPVLSIPEM MRL LIDEHQ FSWDDAFEVC CQVFSYTNHT LMSEALETWP VDMLGKILPR HLQIIFEIND YFLKTLQEQY PNDTDLLGRA SIID ESNGR RVRMAWLAVV VSHKVNGVSE LHSNLMVQSL FADFAKIFPG RFTNVTNGVT PRRWLAVANP SLSAVLDEHL GRNWR TDLS LLNELQQHCD FPMVNHAVHQ AKLENKKRLA EYIAQQLNVV VNPKALFDVQ IKRIHEYKRQ LMNVLHVITR YNRIKA DPD AKWVPRVNIF GGKAASAYYM AKHIIHLIND VAKVINNDPQ IGDKLKVVFI PNYSVSLAQL IIPAADLSEQ ISLAGTE AS GTSNM(LLP)FALN GALTIGTLDG ANVEMLDHVG ADNIFIFGNT AEEVEELRRQ GYKPREYYEK DEELHQVLTQ IGSG VFSPE DPGRYRDLVD SLINFGDHYQ VLADYRSYVD CQDKVDELYE LQEEWTAKAM LNIANMGYFS SDRTIKEYAD HIWHI DPV

UniProtKB: UNIPROTKB: A0A140N6M9

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 10 / 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 stateparticle

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Sample preparation

Concentration1.00 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
100.0 mMsodium chlorideNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.007 kPa / Details: 10 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5 microliters droplet, 20 seconds delay before blotting, 3 seconds blot, 0 seconds 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: PDB ENTRY
PDB model - PDB ID:

9m9p

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 454086
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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