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- EMDB-19730: Cryo-EM structure of the active dodecameric Methanosarcina mazei ... -

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Basic information

Entry
Database: EMDB / ID: EMD-19730
TitleCryo-EM structure of the active dodecameric Methanosarcina mazei glutamine synthetase.
Map dataCryo-EM map of the active dodecameric Methanosarcina mazei glutamine synthetase.
Sample
  • Complex: Glutamine synthetases from Methanosarcina mazei
    • Protein or peptide: Glutamine synthetase
  • Ligand: 2-OXOGLUTARIC ACID
  • Ligand: water
KeywordsGlutamine synthetases / nitrogen metabolism / ammonium assimilation / cryo-EM / CYTOSOLIC PROTEIN
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
Glutamine synthetase
Similarity search - Component
Biological speciesMethanosarcina mazei Go1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsKumar A / Schuller JM / Schmitz RA
Funding support Germany, 1 items
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 JM / Schmitz RA
History
DepositionFeb 23, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19730.png

Downloads & links

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Map

FileDownload / File: emd_19730.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the active dodecameric Methanosarcina mazei glutamine synthetase.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.72 Å/pix.
x 480 pix.
= 345.6 Å		0.72 Å/pix.
x 480 pix.
= 345.6 Å		0.72 Å/pix.
x 480 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.04
Minimum - Maximum-8.464207 - 10.8467
Average (Standard dev.)0.0024537528 (±0.30055884)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_19730_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_19730_half_map_2.map
AnnotationHalf 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 synthetases from Methanosarcina mazei

EntireName: Glutamine synthetases from Methanosarcina mazei
Components
  • Complex: Glutamine synthetases from Methanosarcina mazei
    • Protein or peptide: Glutamine synthetase
  • Ligand: 2-OXOGLUTARIC ACID
  • Ligand: water

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Supramolecule #1: Glutamine synthetases from Methanosarcina mazei

SupramoleculeName: Glutamine synthetases from Methanosarcina mazei / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Molecular weightTheoretical: 55 kDa/nm

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Macromolecule #1: Glutamine synthetase

MacromoleculeName: Glutamine synthetase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: glutamine synthetase
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Molecular weightTheoretical: 50.525371 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VQMKKCTTKE DVLEAVKERD VKFIRTQFTD TLGIIKSWAI PAEQLEEAFE NGVMFDGSSI QGFTRIEESD MKLALDPSTF RILPWRPAT GAVARILGDV YLPDGNPFKG DPRYVLKTAI KEAEKMGFSM NVGPELEFFL FKLDANGNPT TELTDQGGYF D FAPLDRAQ ...String:
VQMKKCTTKE DVLEAVKERD VKFIRTQFTD TLGIIKSWAI PAEQLEEAFE NGVMFDGSSI QGFTRIEESD MKLALDPSTF RILPWRPAT GAVARILGDV YLPDGNPFKG DPRYVLKTAI KEAEKMGFSM NVGPELEFFL FKLDANGNPT TELTDQGGYF D FAPLDRAQ DVRRDIDYAL EHMGFQIEAS HHEVAPSQHE IDFRFGDVLC TADNVVTFKY VVKSIAYHKG YYASFMPKPL FG VNGSGMH SNQSLFKDGK NVFYDPDTPT KLSQDAMYYI GGLLKHIREF TAVTNPVVNS YKRLVPGYEA PVYISWSAQN RSS LIRIPA TRGNGTRIEL RCPDPACNPY LAFALMLRAG LEGIKNKIDP GEPTNVNIFH LSDKEREERG IRSLPADLKE AIDE MKGSK FVKEALGEHV FSHYLCAKEM EWDEYKAVVH PWELSRYLSM L

UniProtKB: Glutamine synthetase

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Macromolecule #2: 2-OXOGLUTARIC ACID

MacromoleculeName: 2-OXOGLUTARIC ACID / type: ligand / ID: 2 / Number of copies: 12 / Formula: AKG
Molecular weightTheoretical: 146.098 Da
Chemical component information

ChemComp-AKG:
2-OXOGLUTARIC ACID

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 4164 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 55.0 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: 0.3 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 800000
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING

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