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- PDB-8s59: Cryo-EM structure of the active dodecameric Methanosarcina mazei ... -

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Basic information

Entry
Database: PDB / ID: 8s59
TitleCryo-EM structure of the active dodecameric Methanosarcina mazei glutamine synthetase.
ComponentsGlutamine synthetase
KeywordsCYTOSOLIC PROTEIN / Glutamine synthetases / nitrogen metabolism / ammonium assimilation / cryo-EM
Function / homology
Function and homology information


glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Glutamine synthetase type I / 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 type I / 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
2-OXOGLUTARIC ACID / Glutamine synthetase
Similarity search - Component
Biological speciesMethanosarcina mazei Go1 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsKumar, A. / Schuller, J.M. / Schmitz, R.A.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)Schm1052/20-2 Germany
Citation
Journal: Elife / Year: 2025
Title: 2-oxoglutarate triggers assembly of active dodecameric glutamine synthetase.
Authors: Eva Herdering / Tristan Reif-Trauttmansdorff / Anuj Kumar / Tim Habenicht / Georg Hochberg / Stefan Bohn / Jan Schuller / Ruth Anne Schmitz /
Abstract: Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based ...Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon , it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK and sP26) has been reported. Here, we show that the strong activation of GS (GlnA) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of GS into a catalytically active dodecamer is not affected by GlnK and crucially depends on the presence of 2-OG.
#1: Journal: Elife / Year: 2024
Title: Cryo-EM structure of the active dodecameric Methanosarcina mazei glutamine synthetase.
Authors: Kumar, A. / Schuller, J.M. / Schmitz, R.A.
History
DepositionFeb 23, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / em_imaging
Item: _em_admin.last_update / _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
O: Glutamine synthetase
D: Glutamine synthetase
A: Glutamine synthetase
B: Glutamine synthetase
C: Glutamine synthetase
L: Glutamine synthetase
M: Glutamine synthetase
P: Glutamine synthetase
Q: Glutamine synthetase
V: Glutamine synthetase
X: Glutamine synthetase
Y: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)608,05824
Polymers606,30412
Non-polymers1,75312
Water75,0144164
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area65350 Å2
ΔGint-236 kcal/mol
Surface area177910 Å2

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Components

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


Mass: 50525.371 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina mazei Go1 (archaea) / Gene: glnA1, MM_0964 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8PY99, glutamine synthetase
#2: Chemical
ChemComp-AKG / 2-OXOGLUTARIC ACID


Mass: 146.098 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C5H6O5 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4164 / Source method: isolated from a natural source / Formula: H2O
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 synthetases from Methanosarcina mazei / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 55 kDa/nm / Experimental value: YES
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 800000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00343872
ELECTRON MICROSCOPYf_angle_d0.58959352
ELECTRON MICROSCOPYf_dihedral_angle_d5.3635894
ELECTRON MICROSCOPYf_chiral_restr0.0446288
ELECTRON MICROSCOPYf_plane_restr0.0067788

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