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- EMDB-65042: Glycogen phosphorylase dimer from E. coli in complex with glycogen -

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Basic information

Entry
Database: EMDB / ID: EMD-65042
TitleGlycogen phosphorylase dimer from E. coli in complex with glycogen
Map data
Sample
  • Complex: glycogen phosphorylase
    • Protein or peptide: Alpha-1,4 glucan phosphorylase
Keywordsglycogen metabolism / cryo-EM / gut bacteria / STRUCTURAL PROTEIN
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 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
DepositionJun 12, 2025-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_65042.png

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Map

FileDownload / File: emd_65042.map.gz / Format: CCP4 / Size: 282.6 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 420 pix.
= 348.6 Å		0.83 Å/pix.
x 420 pix.
= 348.6 Å		0.83 Å/pix.
x 420 pix.
= 348.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.10685085 - 0.20246616
Average (Standard dev.)0.00030030447 (±0.011681619)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 348.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_65042_half_map_1.map
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Half map: #2

Fileemd_65042_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

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Supramolecule #1: glycogen phosphorylase

SupramoleculeName: glycogen phosphorylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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 / Enantiomer: LEVO / EC number: glycogen phosphorylase
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH ENLYFQGMNA PFTYSSPTLS VEALKHSIAY KLMFTIGKDP VVANKHEWLN ATLFAVRDRL VERWLRSNRA QLSQETRQVY YLSMEFLIGR TLSNAMLSLG IYEDVQGALE AMGLNLEELI DEENDPGLGN GGLGRLAACF LDSLATLGLP GRGYGIRYDY ...String:
MGSSHHHHHH ENLYFQGMNA PFTYSSPTLS VEALKHSIAY KLMFTIGKDP VVANKHEWLN ATLFAVRDRL VERWLRSNRA QLSQETRQVY YLSMEFLIGR TLSNAMLSLG IYEDVQGALE AMGLNLEELI DEENDPGLGN GGLGRLAACF LDSLATLGLP GRGYGIRYDY GMFKQNIVNG SQKESPDYWL EYGNPWEFKR HNTRYKVRFG GRIQQEGKKT RWIETEEILG VAYDQIIPGY DTDATNTLRL WSAQASSEIN LGKFNQGDYF AAVEDKNHSE NVSRVLYPDD STYSGRELRL RQEYFLVSST IQDILSRHYQ LHKTYDNLAD KIAIHLNDTH PVLSIPEMMR LLIDEHQFSW DDAFEVCCQV FSYTNHTLMS EALETWPVDM LGKILPRHLQ IIFEINDYFL KTLQEQYPND TDLLGRASII DESNGRRVRM AWLAVVVSHK VNGVSELHSN LMVQSLFADF AKIFPGRFTN VTNGVTPRRW LAVANPSLSA VLDEHLGRNW RTDLSLLNEL QQHCDFPMVN HAVHQAKLEN KKRLAEYIAQ QLNVVVNPKA LFDVQIKRIH EYKRQLMNVL HVITRYNRIK ADPDAKWVPR VNIFGGKAAS AYYMAKHIIH LINDVAKVIN NDPQIGDKLK VVFIPNYSVS LAQLIIPAAD LSEQISLAGT EASGTSNMKF ALNGALTIGT LDGANVEMLD HVGADNIFIF GNTAEEVEEL RRQGYKPREY YEKDEELHQV LTQIGSGVFS PEDPGRYRDL VDSLINFGDH YQVLADYRSY VDCQDKVDEL YELQEEWTAK AMLNIANMGY FSSDRTIKEY ADHIWHIDPV RL

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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
5.0 mg/mLGlycogen
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: PDB ENTRY
PDB model - PDB ID:

9m9p

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 156019
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD

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