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- PDB-9otp: Human glutamine synthetase R298A decamer under turnover conditions -

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Basic information

Entry
Database: PDB / ID: 9otp
TitleHuman glutamine synthetase R298A decamer under turnover conditions
ComponentsGlutamine synthetase
KeywordsLIGASE / Glutamine synthetase / glutamate-ammonia ligase / Type II
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 / glutamine biosynthetic process ...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 biosynthetic process / 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 / endoplasmic reticulum / 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
ADENOSINE-5'-DIPHOSPHATE / Glutamine synthetase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.95 Å
AuthorsGreene, E.R. / Muniz, R.S. / Kollman, J.M. / Fraser, J.S.
Funding support United States, France, 4items
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 27, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 2, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 2, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamine synthetase
B: Glutamine synthetase
C: Glutamine synthetase
D: Glutamine synthetase
E: Glutamine synthetase
F: Glutamine synthetase
G: Glutamine synthetase
H: Glutamine synthetase
I: Glutamine synthetase
J: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)425,08140
Polymers420,32310
Non-polymers4,75830
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Glutamine synthetase / GS / Glutamate--ammonia ligase / Palmitoyltransferase GLUL


Mass: 42032.285 Da / Num. of mol.: 10 / Mutation: R298A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GLUL, GLNS / Variant: R298A / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P15104, glutamine synthetase, protein S-acyltransferase
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Glutamine synthetase R298A decamer under turnover conditions
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
160 mMHEPESC8H18N2O4S1
250 mMSodium ChlorideNaCl1
350 mMPotassium ChlorideKCl1
410 mMMagnesium ChlorideMgCl21
50.5 mMTCEPC9H15O6P1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 106000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 1.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 619435 / Symmetry type: POINT

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