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- EMDB-14587: Cryo-EM structure of the human GS-GN complex in the inhibited state -

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Basic information

Entry
Database: EMDB / ID: EMD-14587
TitleCryo-EM structure of the human GS-GN complex in the inhibited state
Map dataD2
Sample
  • Complex: Glycogen synthase-Glycogenin complex
    • Protein or peptide: Glycogen [starch] synthase, muscle
    • Protein or peptide: Glycogen [starch] synthase, muscle
    • Protein or peptide: Isoform GN-1 of Glycogenin-1
Function / homology
Function and homology information


glycogen synthase activity, transferring glucose-1-phosphate / Glycogen storage disease type XV (GYG1) / Glycogen storage disease type 0 (muscle GYS1) / glycogen(starch) synthase / Glycogen storage disease type II (GAA) / glycogenin glucosyltransferase / glycogenin glucosyltransferase activity / UDP-alpha-D-glucose:glucosyl-glycogenin alpha-D-glucosyltransferase activity / glycogen (starch) synthase activity / D-glucose binding ...glycogen synthase activity, transferring glucose-1-phosphate / Glycogen storage disease type XV (GYG1) / Glycogen storage disease type 0 (muscle GYS1) / glycogen(starch) synthase / Glycogen storage disease type II (GAA) / glycogenin glucosyltransferase / glycogenin glucosyltransferase activity / UDP-alpha-D-glucose:glucosyl-glycogenin alpha-D-glucosyltransferase activity / glycogen (starch) synthase activity / D-glucose binding / glycogen biosynthetic process / Glycogen breakdown (glycogenolysis) / glycosyltransferase activity / inclusion body / Myoclonic epilepsy of Lafora / Glycogen synthesis / lysosomal lumen / manganese ion binding / heart development / secretory granule lumen / ficolin-1-rich granule lumen / Neutrophil degranulation / protein homodimerization activity / extracellular region / membrane / cytosol / cytoplasm
Similarity search - Function
Glycogen synthase / Glycogen synthase / Glycosyl transferase, family 8 / Glycosyl transferase family 8 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Glycogen [starch] synthase, muscle / Glycogenin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsMarr L / Zeqiraj E
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mechanism of glycogen synthase inactivation and interaction with glycogenin.
Authors: Laura Marr / Dipsikha Biswas / Leonard A Daly / Christopher Browning / Sarah C M Vial / Daniel P Maskell / Catherine Hudson / Jay A Bertrand / John Pollard / Neil A Ranson / Heena Khatter / ...Authors: Laura Marr / Dipsikha Biswas / Leonard A Daly / Christopher Browning / Sarah C M Vial / Daniel P Maskell / Catherine Hudson / Jay A Bertrand / John Pollard / Neil A Ranson / Heena Khatter / Claire E Eyers / Kei Sakamoto / Elton Zeqiraj /
Abstract: Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS) ...Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 Å resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite "arginine cradle". Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases.
History
DepositionMar 23, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14587.png

Downloads & links

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Map

FileDownload / File: emd_14587.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationD2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.71 Å/pix.
x 288 pix.
= 204.48 Å		0.71 Å/pix.
x 288 pix.
= 204.48 Å		0.71 Å/pix.
x 288 pix.
= 204.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.71 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.1610524 - 0.31264323
Average (Standard dev.)0.000501294 (±0.008719949)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 204.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14587_msk_1.map
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Additional map: C1

Fileemd_14587_additional_1.map
AnnotationC1
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Half map: #2

Fileemd_14587_half_map_1.map
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Half map: #1

Fileemd_14587_half_map_2.map
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Sample components

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Entire : Glycogen synthase-Glycogenin complex

EntireName: Glycogen synthase-Glycogenin complex
Components
  • Complex: Glycogen synthase-Glycogenin complex
    • Protein or peptide: Glycogen [starch] synthase, muscle
    • Protein or peptide: Glycogen [starch] synthase, muscle
    • Protein or peptide: Isoform GN-1 of Glycogenin-1

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Supramolecule #1: Glycogen synthase-Glycogenin complex

SupramoleculeName: Glycogen synthase-Glycogenin complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 485 KDa

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Macromolecule #1: Glycogen [starch] synthase, muscle

MacromoleculeName: Glycogen [starch] synthase, muscle / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: glycogen(starch) synthase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.88543 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPLNRTLSMS SLPGLEDWED EFDLENAVLF EVAWEVANKV GGIYTVLQTK AKVTGDEWGD NYFLVGPYTE QGVRTQVELL EAPTPALKR TLDSMNSKGC KVYFGRWLIE GGPLVVLLDV GASAWALERW KGELWDTCNI GVPWYDREAN DAVLFGFLTT W FLGEFLAQ ...String:
MPLNRTLSMS SLPGLEDWED EFDLENAVLF EVAWEVANKV GGIYTVLQTK AKVTGDEWGD NYFLVGPYTE QGVRTQVELL EAPTPALKR TLDSMNSKGC KVYFGRWLIE GGPLVVLLDV GASAWALERW KGELWDTCNI GVPWYDREAN DAVLFGFLTT W FLGEFLAQ SEEKPHVVAH FHEWLAGVGL CLCRARRLPV ATIFTTHATL LGRYLCAGAV DFYNNLENFN VDKEAGERQI YH RYCMERA AAHCAHVFTT VSQITAIEAQ HLLKRKPDIV TPNGLNVKKF SAMHEFQNLH AQSKARIQEF VRGHFYGHLD FNL DKTLYF FIAGRYEFSN KGADVFLEAL ARLNYLLRVN GSEQTVVAFF IMPARTNNFN VETLKGQAVR KQLWDTANTV KEKF GRKLY ESLLVGSLPD MNKMLDKEDF TMMKRAIFAT QRQSFPPVCT HNMLDDSSDP ILTTIRRIGL FNSSADRVKV IFHPE FLSS TSPLLPVDYE EFVRGCHLGV FPSYYEPWGY TPAECTVMGI PSISTNLSGF GCFMEEHIAD PSAYGIYILD RRFRSL DDS CSQLTSFLYS FCQQSRRQRI IQRNRTERLS DLLDWKYLGR YYMSARHMAL SKAFPEHFTY EPNEADAAQG YRYPRPA SV PPSPSLSRHS SPHQSEDEED PRNGPLEEDG ERYDEDEEAA KDRRNIRAPE WPRRASCTSS TSGSKRNSVD TATSSSLS T PSEPLSPTSS LGEERN

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Macromolecule #2: Glycogen [starch] synthase, muscle

MacromoleculeName: Glycogen [starch] synthase, muscle / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: glycogen(starch) synthase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.965406 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPLNRTLSMS SLPGLEDWED EFDLENAVLF EVAWEVANKV GGIYTVLQTK AKVTGDEWGD NYFLVGPYTE QGVRTQVELL EAPTPALKR TLDSMNSKGC KVYFGRWLIE GGPLVVLLDV GASAWALERW KGELWDTCNI GVPWYDREAN DAVLFGFLTT W FLGEFLAQ ...String:
MPLNRTLSMS SLPGLEDWED EFDLENAVLF EVAWEVANKV GGIYTVLQTK AKVTGDEWGD NYFLVGPYTE QGVRTQVELL EAPTPALKR TLDSMNSKGC KVYFGRWLIE GGPLVVLLDV GASAWALERW KGELWDTCNI GVPWYDREAN DAVLFGFLTT W FLGEFLAQ SEEKPHVVAH FHEWLAGVGL CLCRARRLPV ATIFTTHATL LGRYLCAGAV DFYNNLENFN VDKEAGERQI YH RYCMERA AAHCAHVFTT VSQITAIEAQ HLLKRKPDIV TPNGLNVKKF SAMHEFQNLH AQSKARIQEF VRGHFYGHLD FNL DKTLYF FIAGRYEFSN KGADVFLEAL ARLNYLLRVN GSEQTVVAFF IMPARTNNFN VETLKGQAVR KQLWDTANTV KEKF GRKLY ESLLVGSLPD MNKMLDKEDF TMMKRAIFAT QRQSFPPVCT HNMLDDSSDP ILTTIRRIGL FNSSADRVKV IFHPE FLSS TSPLLPVDYE EFVRGCHLGV FPSYYEPWGY TPAECTVMGI PSISTNLSGF GCFMEEHIAD PSAYGIYILD RRFRSL DDS CSQLTSFLYS FCQQSRRQRI IQRNRTERLS DLLDWKYLGR YYMSARHMAL SKAFPEHFTY EPNEADAAQG YRYPRPA (SEP)V PPSPSLSRHS SPHQSEDEED PRNGPLEEDG ERYDEDEEAA KDRRNIRAPE WPRRASCTSS TSGSKRNSVD TATS SSLST PSEPLSPTSS LGEERN

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Macromolecule #3: Isoform GN-1 of Glycogenin-1

MacromoleculeName: Isoform GN-1 of Glycogenin-1 / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO / EC number: glycogenin glucosyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.469348 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MADQAFVTLT TNDAYAKGAL VLGSSLKQHR TTRRLVVLAT PQVSDSMRKV LETVFDEVIM VDVLDSGDSA HLTLMKRPEL GVTLTKLHC WSLTQYSKCV FMDADTLVLA NIDDLFDREE LSAAPDPGWP DCFNSGVFVY QPSVETYNQL LHLASEQGSF D GGDQGILN ...String:
MADQAFVTLT TNDAYAKGAL VLGSSLKQHR TTRRLVVLAT PQVSDSMRKV LETVFDEVIM VDVLDSGDSA HLTLMKRPEL GVTLTKLHC WSLTQYSKCV FMDADTLVLA NIDDLFDREE LSAAPDPGWP DCFNSGVFVY QPSVETYNQL LHLASEQGSF D GGDQGILN TFFSSWATTD IRKHLPFIYN LSSISIFSYL PAFKVFGASA KVVHFLGRVK PWNYTYDPKT KSVKSEAHDP NM THPEFLI LWWNIFTTNV LPLLQQFGLV KDTCSYVNVE DVSGAISHLS LGEIPAMAQP FVSSEERKER WEQGQADYMG ADS FDNIKR KLDTYLQ

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
25.0 mMHEPES
1.0 mMTCEP
1.5 %Glycerol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 34.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Model generated in RELION using SGD from particles from 2D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 739232
FSC plot (resolution estimation)

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