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- PDB-8ecy: cryoEM structure of bovine bestrophin-2 and glutamine synthetase ... -

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Basic information

Entry
Database: PDB / ID: 8ecy
TitlecryoEM structure of bovine bestrophin-2 and glutamine synthetase complex
Components
  • Bestrophin
  • Glutamine synthetase
KeywordsMEMBRANE PROTEIN / ion channel / transport / anion channel
Function / homology
Function and homology information


Astrocytic Glutamate-Glutamine Uptake And Metabolism / Glutamate and glutamine metabolism / intracellular ammonium homeostasis / Stimuli-sensing channels / intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / protein palmitoylation / protein S-acyltransferase / protein-cysteine S-palmitoyltransferase activity / regulation of sprouting angiogenesis ...Astrocytic Glutamate-Glutamine Uptake And Metabolism / Glutamate and glutamine metabolism / intracellular ammonium homeostasis / Stimuli-sensing channels / intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / protein palmitoylation / protein S-acyltransferase / protein-cysteine S-palmitoyltransferase activity / regulation of sprouting angiogenesis / bicarbonate channel activity / regulation of endothelial cell migration / glutamine synthetase / : / glutamine synthetase activity / bicarbonate transport / ligand-gated monoatomic cation channel activity / chloride channel activity / chloride channel complex / positive regulation of erythrocyte differentiation / angiogenesis / basolateral plasma membrane / endoplasmic reticulum / mitochondrion / ATP binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / 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. ...: / Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / 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
: / Bestrophin-2 / Glutamine synthetase
Similarity search - Component
Biological speciesBos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 Å
AuthorsOwji, A.P. / Kittredge, A.K. / Yang, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)5F31EY030763 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM127652 United States
CitationJournal: Nature / Year: 2022
Title: Bestrophin-2 and glutamine synthetase form a complex for glutamate release.
Authors: Aaron P Owji / Kuai Yu / Alec Kittredge / Jiali Wang / Yu Zhang / Tingting Yang /
Abstract: Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology. Here we uncover a directional permeability of BEST2 to ...Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology. Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS.
History
DepositionSep 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Bestrophin
C: Bestrophin
G: Bestrophin
K: Bestrophin
N: Bestrophin
A: Glutamine synthetase
J: Glutamine synthetase
L: Glutamine synthetase
Q: Glutamine synthetase
H: Glutamine synthetase
D: Glutamine synthetase
B: Glutamine synthetase
F: Glutamine synthetase
I: Glutamine synthetase
M: Glutamine synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)659,66751
Polymers657,97815
Non-polymers1,68936
Water36,4622024
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 15 molecules ECGKNAJLQHDBFIM

#1: Protein
Bestrophin


Mass: 47424.754 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: BEST2 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: E1BF86
#2: Protein
Glutamine synthetase / GS / Glutamate--ammonia ligase / Palmitoyltransferase GLUL


Mass: 42085.414 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: GLUL / Production host: Escherichia coli (E. coli)
References: UniProt: P15103, glutamine synthetase, protein S-acyltransferase

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Non-polymers , 4 types, 2060 molecules

#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Mn
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2024 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: Best2-GS / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
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: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 1.16 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 78623
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25618 / Symmetry type: POINT

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