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- PDB-8ec6: Cryo-EM structure of the Glutaminase C core filament (fGAC) -

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Basic information

Entry
Database: PDB / ID: 8ec6
TitleCryo-EM structure of the Glutaminase C core filament (fGAC)
ComponentsIsoform 2 of Glutaminase kidney isoform, mitochondrial
KeywordsHYDROLASE / Mitochondria / Filament
Function / homologyglutaminase / PHOSPHATE ION / Isoform 2 of Glutaminase kidney isoform, mitochondrial
Function and homology information
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsAmbrosio, A.L. / Dias, S.M. / Quesnay, J.E. / Portugal, R.V. / Cassago, A. / van Heel, M.G. / Islam, Z. / Rodrigues, C.T.
Funding support Brazil, 1items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2017/11766-5 Brazil
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.
Authors: Douglas Adamoski / Marilia Meira Dias / Jose Edwin Neciosup Quesñay / Zhengyi Yang / Ievgeniia Zagoriy / Anna M Steyer / Camila Tanimoto Rodrigues / Alliny Cristiny da Silva Bastos / Bianca ...Authors: Douglas Adamoski / Marilia Meira Dias / Jose Edwin Neciosup Quesñay / Zhengyi Yang / Ievgeniia Zagoriy / Anna M Steyer / Camila Tanimoto Rodrigues / Alliny Cristiny da Silva Bastos / Bianca Novaes da Silva / Renna Karoline Eloi Costa / Flávia Mayumi Odahara de Abreu / Zeyaul Islam / Alexandre Cassago / Marin Gerard van Heel / Sílvio Roberto Consonni / Simone Mattei / Julia Mahamid / Rodrigo Villares Portugal / Andre Luis Berteli Ambrosio / Sandra Martha Gomes Dias /
Abstract: Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, ...Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.
History
DepositionSep 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Database references / Category: citation
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.title / _citation.year
Revision 1.2Nov 1, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Dec 27, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 2 of Glutaminase kidney isoform, mitochondrial
B: Isoform 2 of Glutaminase kidney isoform, mitochondrial
C: Isoform 2 of Glutaminase kidney isoform, mitochondrial
D: Isoform 2 of Glutaminase kidney isoform, mitochondrial
E: Isoform 2 of Glutaminase kidney isoform, mitochondrial
F: Isoform 2 of Glutaminase kidney isoform, mitochondrial
G: Isoform 2 of Glutaminase kidney isoform, mitochondrial
H: Isoform 2 of Glutaminase kidney isoform, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)427,37516
Polymers426,6168
Non-polymers7608
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative staining, as published earlier
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Isoform 2 of Glutaminase kidney isoform, mitochondrial / GLS


Mass: 53326.961 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gls, Gls1, Kiaa0838 / Plasmid: pET28a / Details (production host): standard / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta-2 / References: UniProt: D3Z7P3-2, glutaminase
#2: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Glutaminase C filament, bound to inorganic phosphate / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.53 kDa/nm / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse) / Cellular location: Matrix / Organelle: Mitochondria
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta-2 / Plasmid: pET28a
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium chlorideNaClSodium chloride1
250 mMTris hydrochlorideTRIS-HClTris1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
420 mMDipotassium hydrogen phosphateK2HPO41
SpecimenConc.: 0.33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: In the presence of phosphate, GAC polymerizes and becomes polydisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 59000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 3000 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
4cryoSPARCCTF correction
7PHENIX1.20.1model fittingphenix.dock_in_map
9PHENIX1.20.1model refinementphenix.real_space_refine
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 422097
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229037 / Symmetry type: POINT
Atomic model buildingB value: 162.1 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: run = *minimization_global *rigid_body local_grid_search *adp *occupancy *nqh_flips
Atomic model buildingPDB-ID: 3SS3

3ss3


Pdb chain-ID: A / Accession code: 3SS3 / Pdb chain residue range: 128-674 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.011719596
ELECTRON MICROSCOPYf_angle_d0.851326504
ELECTRON MICROSCOPYf_chiral_restr0.04152863
ELECTRON MICROSCOPYf_plane_restr0.00663438
ELECTRON MICROSCOPYf_dihedral_angle_d3.55022615

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