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- EMDB-77146: Focused refinement of turnover filament interface of glutamine sy... -

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Basic information

Entry
Database: EMDB / ID: EMD-77146
TitleFocused refinement of turnover filament interface of glutamine synthetase
Map dataFocused refinement of glutamine synthetase turnover filament interface sharpened
Sample
  • Complex: Glutamine synthetase filament under turnover conditions
    • Protein or peptide: Glutamine synthetase
KeywordsGlutamine synthetase / glutamate-ammonia ligase / filament / Type II / LIGASE
Function / homology
Function and homology information


intracellular ammonium homeostasis / protein palmitoylation / protein S-acyltransferase / Astrocytic Glutamate-Glutamine Uptake And Metabolism / protein-cysteine S-palmitoyltransferase activity / regulation of protein localization to nucleolus / regulation of sprouting angiogenesis / regulation of endothelial cell migration / glutamine synthetase / : ...intracellular ammonium homeostasis / protein palmitoylation / protein S-acyltransferase / Astrocytic Glutamate-Glutamine Uptake And Metabolism / protein-cysteine S-palmitoyltransferase activity / regulation of protein localization to nucleolus / regulation of sprouting angiogenesis / regulation of endothelial cell migration / glutamine synthetase / : / glutamine synthetase activity / Glutamate and glutamine metabolism / L-glutamate catabolic process / glial cell projection / response to glucose / positive regulation of erythrocyte differentiation / cellular response to starvation / ribosome biogenesis / cell body / angiogenesis / cell population proliferation / mitochondrion / extracellular exosome / ATP binding / metal ion binding / identical protein binding / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
: / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain ...: / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain / Glutamine synthetase, catalytic domain / Glutamine synthetase (GS) catalytic domain profile. / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
Glutamine synthetase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsGreene ER / Muniz RS / Kollman JM / Fraser JS
Funding support United States, France, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F32GM144981-01 United States
Human Frontier Science Program (HFSP)RGP0054 France
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM123159 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM145238 United States
CitationJournal: bioRxiv / Year: 2025
Title: Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity.
Authors: Eric Greene / Richard Muniz / Hiroki Yamamura / Samuel E Hoff / Priyanka Bajaj / D John Lee / Erin M Thompson / Angelika Arada / Gyun Min Lee / Massimiliano Bonomi / Justin M Kollman / James S Fraser /
Abstract: To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can ...To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 Å conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.
History
DepositionMay 13, 2026-
Header (metadata) releaseJun 17, 2026-
Map releaseJun 17, 2026-
UpdateJun 17, 2026-
Current statusJun 17, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_77146.png

Downloads & links

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Map

FileDownload / File: emd_77146.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused refinement of glutamine synthetase turnover filament interface sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 512 pix.
= 427.52 Å		0.84 Å/pix.
x 512 pix.
= 427.52 Å		0.84 Å/pix.
x 512 pix.
= 427.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0
Minimum - Maximum-52.892969999999998 - 81.943473999999995
Average (Standard dev.)-0.000000000003995 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 427.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Focused refinement of glutamine synthetase turnover filament interface...

Fileemd_77146_half_map_1.map
AnnotationFocused refinement of glutamine synthetase turnover filament interface half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Focused refinement of glutamine synthetase turnover filament interface...

Fileemd_77146_half_map_2.map
AnnotationFocused refinement of glutamine synthetase turnover filament interface half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Glutamine synthetase filament under turnover conditions

EntireName: Glutamine synthetase filament under turnover conditions
Components
  • Complex: Glutamine synthetase filament under turnover conditions
    • Protein or peptide: Glutamine synthetase

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Supramolecule #1: Glutamine synthetase filament under turnover conditions

SupramoleculeName: Glutamine synthetase filament under turnover conditions
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Glutamine synthetase

MacromoleculeName: Glutamine synthetase / type: protein_or_peptide / ID: 1
Details: Tagless human glutamine synthetase recombinantly expressed in E. coli as a His6-SUMO fusion where the fused proteins were cleaved by Ulp1 protease. Wild-type sequence with omitted Met1.
Enantiomer: LEVO / EC number: glutamine synthetase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TTSASSHLNK GIKQVYMSLP QGEKVQAMYI WIDGTGEGLR CKTRTLDSEP KCVEELPEWN FDGSSTLQSE GSNSDMYLVP AAMFRDPFRK DPNKLVLCEV FKYNRRPAET NLRHTCKRIM DMVSNQHPWF GMEQEYTLMG TDGHPFGWPS NGFPGPQGPY YCGVGADRAY ...String:
TTSASSHLNK GIKQVYMSLP QGEKVQAMYI WIDGTGEGLR CKTRTLDSEP KCVEELPEWN FDGSSTLQSE GSNSDMYLVP AAMFRDPFRK DPNKLVLCEV FKYNRRPAET NLRHTCKRIM DMVSNQHPWF GMEQEYTLMG TDGHPFGWPS NGFPGPQGPY YCGVGADRAY GRDIVEAHYR ACLYAGVKIA GTNAEVMPAQ WEFQIGPCEG ISMGDHLWVA RFILHRVCED FGVIATFDPK PIPGNWNGAG CHTNFSTKAM REENGLKYIE EAIEKLSKRH QYHIRAYDPK GGLDNARRLT GFHETSNIND FSAGVANRSA SIRIPRTVGQ EKKGYFEDRR PSANCDPFSV TEALIRTCLL NETGDEPFQY KN

UniProtKB: Glutamine synthetase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
60.0 mMC8H18N2O4SHEPES
50.0 mMNaClSodium Chloride
50.0 mMKClPotassium Chloride
10.0 mMMgCl2Magnesium Chloride
0.5 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 106000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: Initial particle set of / Number images used: 5626596
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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