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- EMDB-28013: Cryo-EM structure of the Glutaminase C core filament (fGAC) -

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Basic information

Entry
Database: EMDB / ID: EMD-28013
TitleCryo-EM structure of the Glutaminase C core filament (fGAC)
Map dataLocal refinement, after helical refinement with D2 symmetry expansion
Sample
  • Organelle or cellular component: Glutaminase C filament, bound to inorganic phosphate
    • Protein or peptide: Isoform 2 of Glutaminase kidney isoform, mitochondrial
  • Ligand: PHOSPHATE IONPhosphate
KeywordsMitochondria / Filament / HYDROLASE
Function / homologyglutaminase / Isoform 2 of Glutaminase kidney isoform, mitochondrial
Function and homology information
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsAmbrosio AL / Dias SM / Quesnay JE / Portugal RV / Cassago A / van Heel MG / Islam Z / Rodrigues CT
Funding support Brazil, 1 items
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, 2022-
Header (metadata) releaseSep 20, 2023-
Map releaseSep 20, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28013.png

Downloads & links

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Map

FileDownload / File: emd_28013.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement, after helical refinement with D2 symmetry expansion
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-5.1829495 - 8.758822
Average (Standard dev.)-0.0005444609 (±0.15703604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map A

Fileemd_28013_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_28013_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 : Glutaminase C filament, bound to inorganic phosphate

EntireName: Glutaminase C filament, bound to inorganic phosphate
Components
  • Organelle or cellular component: Glutaminase C filament, bound to inorganic phosphate
    • Protein or peptide: Isoform 2 of Glutaminase kidney isoform, mitochondrial
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: Glutaminase C filament, bound to inorganic phosphate

SupramoleculeName: Glutaminase C filament, bound to inorganic phosphate / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse) / Organelle: Mitochondria / Location in cell: Matrix
Molecular weightTheoretical: 0.53 kDa/nm

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Macromolecule #1: Isoform 2 of Glutaminase kidney isoform, mitochondrial

MacromoleculeName: Isoform 2 of Glutaminase kidney isoform, mitochondrial
type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: glutaminase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 53.326961 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMVAAGDN KIKQGLLPSL EDLLFYTIAE GQEKIPVHKF ITALKSTGLR TSDPRLKECM DMLRLTLQTT SDGVMLDKDL FKKCVQSNI VLLTQAFRRK FVIPDFMSFT SHIDELYESA KKQSGGKVAD YIPQLAKFSP DLWGVSVCTV DGQRHSIGDT K VPFCLQSC ...String:
GSHMVAAGDN KIKQGLLPSL EDLLFYTIAE GQEKIPVHKF ITALKSTGLR TSDPRLKECM DMLRLTLQTT SDGVMLDKDL FKKCVQSNI VLLTQAFRRK FVIPDFMSFT SHIDELYESA KKQSGGKVAD YIPQLAKFSP DLWGVSVCTV DGQRHSIGDT K VPFCLQSC VKPLKYAIAV NDLGTEYVHR YVGKEPSGLR FNKLFLNEDD KPHNPMVNAG AIVVTSLIKQ GVNNAEKFDY VM QFLNKMA GNEYVGFSNA TFQSERESGD RNFAIGYYLK EKKCFPEGTD MVGILDFYFQ LCSIEVTCES ASVMAATLAN GGF CPITGE RVLSPEAVRN TLSLMHSCGM YDFSGQFAFH VGLPAKSGVA GGILLVVPNV MGMMCWSPPL DKMGNSVKGI HFCH DLVSL CNFHNYDNLR HFAKKLDPRR EGGDQRHSFG PLDYESLQQE LALKDTVWKK VSPESSDDTS TTVVYRMESL GERS

UniProtKB: Isoform 2 of Glutaminase kidney isoform, mitochondrial

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Macromolecule #2: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.33 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chlorideSodium chloride
50.0 mMTRIS-HClTrisTris hydrochloride
0.5 mMTCEPtris(2-carboxyethyl)phosphine
20.0 mMK2HPO4Dipotassium hydrogen phosphate
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsIn the presence of phosphate, GAC polymerizes and becomes polydisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 422097
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ss3


Details: One monomer was used for docking eight copies
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 229037
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3ss3


Chain - Chain ID: A / Chain - Residue range: 128-674 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsrun = *minimization_global *rigid_body local_grid_search *adp *occupancy *nqh_flips
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 162.1 / Target criteria: Correlation Coefficient
Output model

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

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