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- PDB-8tfk: Cryo-EM structure of the Methanosarcina mazei glutamine synthetas... -

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Basic information

Entry
Database: PDB / ID: 8tfk
TitleCryo-EM structure of the Methanosarcina mazei glutamine synthetase (GS) with Met-Sox-P and ADP
ComponentsGlutamine synthetase
KeywordsLIGASE / glutamine synthetase / GS / transition state / MSO / Met-Sox-P / ADP
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
ADENOSINE-5'-DIPHOSPHATE / L-METHIONINE-S-SULFOXIMINE PHOSPHATE / Glutamine synthetase
Similarity search - Component
Biological speciesMethanosarcina mazei Go1 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsSchumacher, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM130290 United States
CitationJournal: Nat Commun / Year: 2023
Title: M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.
Authors: Maria A Schumacher / Raul Salinas / Brady A Travis / Rajiv Ranjan Singh / Nicholas Lent /
Abstract: Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi- ...Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.
History
DepositionJul 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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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
K: Glutamine synthetase
L: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)643,05972
Polymers633,93512
Non-polymers9,12460
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


Mass: 52827.926 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-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-P3S / L-METHIONINE-S-SULFOXIMINE PHOSPHATE


Mass: 260.205 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C5H13N2O6PS / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: M. mazei Glutamine Synthetase (GS) transition state complex
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Methanosarcina mazei Go1 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2700 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164400 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00643037
ELECTRON MICROSCOPYf_angle_d0.75758480
ELECTRON MICROSCOPYf_dihedral_angle_d8.0476012
ELECTRON MICROSCOPYf_chiral_restr0.1016276
ELECTRON MICROSCOPYf_plane_restr0.0047571

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