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- EMDB-28025: cryoEM structure of bovine bestrophin-2 and glutamine synthetase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28025
TitlecryoEM structure of bovine bestrophin-2 and glutamine synthetase complex
Map datalocal_filtered_map_locres
Sample
  • Complex: Best2-GS
    • Protein or peptide: Bestrophin
    • Protein or peptide: Glutamine synthetase
  • Ligand: CALCIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: MANGANESE (II) ION
  • Ligand: water
Keywordsion channel / transport / anion channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


Astrocytic Glutamate-Glutamine Uptake And Metabolism / Glutamate and glutamine metabolism / protein S-acyltransferase / intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / Stimuli-sensing channels / protein palmitoylation / protein-cysteine S-palmitoyltransferase activity / bicarbonate channel activity / regulation of sprouting angiogenesis ...Astrocytic Glutamate-Glutamine Uptake And Metabolism / Glutamate and glutamine metabolism / protein S-acyltransferase / intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / Stimuli-sensing channels / protein palmitoylation / protein-cysteine S-palmitoyltransferase activity / bicarbonate channel activity / regulation of sprouting angiogenesis / ligand-gated monoatomic cation channel activity / regulation of endothelial cell migration / glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / bicarbonate transport / chloride channel activity / chloride channel complex / basolateral plasma membrane / angiogenesis / endoplasmic reticulum / mitochondrion / ATP binding / metal ion binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase (GS) catalytic domain profile. / 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. ...Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase (GS) catalytic domain profile. / 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, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
Bestrophin-2 / Glutamine synthetase
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.0 Å
AuthorsOwji AP / Kittredge AK / Yang T
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28025.png

Downloads & links

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Map

FileDownload / File: emd_28025.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal_filtered_map_locres
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.9081268 - 4.38657
Average (Standard dev.)0.0015683518 (±0.05739714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 498.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28025_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_28025_msk_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: cryosparc half map1

Fileemd_28025_half_map_1.map
Annotationcryosparc_half_map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryosparc half map2

Fileemd_28025_half_map_2.map
Annotationcryosparc_half_map2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Best2-GS

EntireName: Best2-GS
Components
  • Complex: Best2-GS
    • Protein or peptide: Bestrophin
    • Protein or peptide: Glutamine synthetase
  • Ligand: CALCIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: MANGANESE (II) ION
  • Ligand: water

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Supramolecule #1: Best2-GS

SupramoleculeName: Best2-GS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Bestrophin

MacromoleculeName: Bestrophin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 47.424754 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTVTYTARVA KARFGGFSKL LLLWRGSIYK LLWRELLCFL GLFMALSAAY RFVLTEEQKR YFEKLVLYCD RYASLIPVSF VLGFYVTLV VHRWWNQYLS MPLTDALMCV VVGTVHGHDE RGRLYRRTLM RYAGLSGVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER ...String:
MTVTYTARVA KARFGGFSKL LLLWRGSIYK LLWRELLCFL GLFMALSAAY RFVLTEEQKR YFEKLVLYCD RYASLIPVSF VLGFYVTLV VHRWWNQYLS MPLTDALMCV VVGTVHGHDE RGRLYRRTLM RYAGLSGVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER KKFENLNSSY NKYWVPCVWF CNLAAQARRE GRIRDNGAFK LLLEELNVFR SKCGMLFHYD WISVPLVYTQ VV TIAVYSY FLACLIGRQF LDPAQGYKDH DLDLCVPIFT LLQFFFYAGW LKVAEQLINP FGEDDDDFET NFLIDRCFQV SML AVDEMY DDLAMLEKDL YWDAAEARAP YTAATAFLMQ QPSFQGSTFD ITLAKEDMQF QRQDGLEAPL NEAHGDFLQR LLPV GTGMG TGGLL

UniProtKB: Bestrophin-2

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Macromolecule #2: Glutamine synthetase

MacromoleculeName: Glutamine synthetase / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO / EC number: glutamine synthetase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 42.085414 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MATSASSHLN KGIKQVYMAL PQGDKVQAMY IWIDGTGEGL RCKTRTLDSE PKCIEELPEW NFDGSSTFQS EGSNSDMYLV PAAMFRDPF RKDPNKLVFC EVFKYNRKPA ETNLRHTCKR IMDMVSNQRP WFGMEQEYTL MGTDGHPFGW PSNGFPGPQG P YYCGVGAD ...String:
MATSASSHLN KGIKQVYMAL PQGDKVQAMY IWIDGTGEGL RCKTRTLDSE PKCIEELPEW NFDGSSTFQS EGSNSDMYLV PAAMFRDPF RKDPNKLVFC EVFKYNRKPA ETNLRHTCKR IMDMVSNQRP WFGMEQEYTL MGTDGHPFGW PSNGFPGPQG P YYCGVGAD KAYGRDIVEA HYRACLYAGI KIGGTNAEVM PAQWEFQIGP CEGIDMGDHL WVARFILHRV CEDFGVIATF DP KPIPGNW NGAGCHTNFS TKAMREENGL KYIEEAIEKL SKRHQYHIRA YDPKGGLDNA RRLTGFHETS NINDFSAGVA NRG ASIRIP RTVGQEKKGY FEDRRPSANC DPFAVTEALI RTCLLNETGD EPFQYKN

UniProtKB: Glutamine synthetase

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 4 / Number of copies: 11 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #5: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 5 / Number of copies: 20 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 2024 / 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: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.16 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 78623
Startup modelType of model: OTHER / Details: ab initio
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 25618
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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