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- EMDB-35300: The Arabidopsis CLCa transporter bound with nitrate, ATP and PIP2 -

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Basic information

Entry
Database: EMDB / ID: EMD-35300
TitleThe Arabidopsis CLCa transporter bound with nitrate, ATP and PIP2
Map data
Sample
  • Complex: The Arabidopsis CLCa transporter
    • Protein or peptide: Chloride channel protein CLC-a
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: NITRATE ION
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
KeywordsTransporter / ATP / PIP2 / MEMBRANE PROTEIN
Function / homology
Function and homology information


nitrate:proton symporter activity / nitrate transmembrane transport / nitrate transmembrane transporter activity / response to nitrate / plant-type vacuole membrane / voltage-gated chloride channel activity / chloride transport / chloride channel complex
Similarity search - Function
Chloride channel ClC-plant / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
Chloride channel protein CLC-a
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsYang Z / Zhang X / Zhang P
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
Chinese Academy of Sciences China
CitationJournal: Nat Commun / Year: 2023
Title: Molecular mechanism underlying regulation of Arabidopsis CLCa transporter by nucleotides and phospholipids.
Authors: Zhao Yang / Xue Zhang / Shiwei Ye / Jingtao Zheng / Xiaowei Huang / Fang Yu / Zhenguo Chen / Shiqing Cai / Peng Zhang /
Abstract: Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. ...Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO and to a less extent Cl from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO and Cl, respectively. Both structures are captured in ATP and PI(4,5)P bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P or PI(3,5)P binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.
History
DepositionFeb 8, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35300.png

Downloads & links

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Map

FileDownload / File: emd_35300.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.41873285 - 0.6610467
Average (Standard dev.)0.00026104404 (±0.01964114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unharden map

Fileemd_35300_additional_1.map
AnnotationUnharden map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35300_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35300_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : The Arabidopsis CLCa transporter

EntireName: The Arabidopsis CLCa transporter
Components
  • Complex: The Arabidopsis CLCa transporter
    • Protein or peptide: Chloride channel protein CLC-a
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: NITRATE ION
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Supramolecule #1: The Arabidopsis CLCa transporter

SupramoleculeName: The Arabidopsis CLCa transporter / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Chloride channel protein CLC-a

MacromoleculeName: Chloride channel protein CLC-a / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 85.488906 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MDEDGNLQIS NSNYNGEEEG EDPENNTLNQ PLLKRHRTLS STPLALVGAK VSHIESLDYE INENDLFKHD WRSRSKAQVF QYIFLKWTL ACLVGLFTGL IATLINLAVE NIAGYKLLAV GYYIAQDRFW TGLMVFTGAN LGLTLVATVL VVYFAPTAAG P GIPEIKAY ...String:
MDEDGNLQIS NSNYNGEEEG EDPENNTLNQ PLLKRHRTLS STPLALVGAK VSHIESLDYE INENDLFKHD WRSRSKAQVF QYIFLKWTL ACLVGLFTGL IATLINLAVE NIAGYKLLAV GYYIAQDRFW TGLMVFTGAN LGLTLVATVL VVYFAPTAAG P GIPEIKAY LNGIDTPNMF GFTTMMVKIV GSIGAVAAGL DLGKEGPLVH IGSCIASLLG QGGPDNHRIK WRWLRYFNND RD RRDLITC GSASGVCAAF RSPVGGVLFA LEEVATWWRS ALLWRTFFST AVVVVVLRAF IEICNSGKCG LFGSGGLIMF DVS HVEVRY HAADIIPVTL IGVFGGILGS LYNHLLHKVL RLYNLINQKG KIHKVLLSLG VSLFTSVCLF GLPFLAECKP CDPS IDEIC PTNGRSGNFK QFNCPNGYYN DLSTLLLTTN DDAVRNIFSS NTPNEFGMVS LWIFFGLYCI LGLITFGIAT PSGLF LPII LMGSAYGRML GTAMGSYTNI DQGLYAVLGA ASLMAGSMRM TVSLCVIFLE LTNNLLLLPI TMFVLLIAKT VGDSFN LSI YEIILHLKGL PFLEANPEPW MRNLTVGELN DAKPPVVTLN GVEKVANIVD VLRNTTHNAF PVLDGADQNT GTELHGL IL RAHLVKVLKK RWFLNEKRRT EEWEVREKFT PVELAEREDN FDDVAITSSE MQLYVDLHPL TNTTPYTVVQ SMSVAKAL V LFRSVGLRHL LVVPKIQASG MSPVIGILTR QDLRAYNILQ AFPHLDKHKS GKAR

UniProtKB: Chloride channel protein CLC-a

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: NITRATE ION

MacromoleculeName: NITRATE ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: NO3
Molecular weightTheoretical: 62.005 Da
Chemical component information

ChemComp-NO3:
NITRATE ION

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Macromolecule #5: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 5 / Number of copies: 2 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92708
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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