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- PDB-7xa9: Structure of Arabidopsis thaliana CLCa -

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Basic information

Entry
Database: PDB / ID: 7xa9
TitleStructure of Arabidopsis thaliana CLCa
ComponentsChloride channel protein CLC-a
KeywordsMEMBRANE PROTEIN / nitrate / antiporter / transport protein
Function / homology
Function and homology information


nitrate transmembrane transport / nitrate transmembrane transporter activity / response to nitrate / 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
ADENOSINE-5'-TRIPHOSPHATE / NITRATE ION / Chloride channel protein CLC-a
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsJi, S. / Jin, H. / Kaiming, Z. / Mingxing, W. / Shanshan, L. / Long, C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071201 China
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM structure of the plant nitrate transporter AtCLCa reveals characteristics of the anion-binding site and the ATP-binding pocket.
Authors: Jin He / Mingxing Wang / Shanshan Li / Long Chen / Kaiming Zhang / Ji She /
Abstract: Nitrate is one of the major nitrogen sources for most plants. Chloride channel (CLC) proteins mediate the transport and vacuole storage of nitrate in plants, but the structural basis of nitrate ...Nitrate is one of the major nitrogen sources for most plants. Chloride channel (CLC) proteins mediate the transport and vacuole storage of nitrate in plants, but the structural basis of nitrate transport by plant CLC proteins remains unknown. Here, we solved the cryo-EM structure of ATP-bound Arabidopsis thaliana CLCa (AtCLCa) at 2.8 Å resolution. Structural comparison between nitrate-selective AtCLCa and chloride-selective CLC-7 reveals key differences in the central anion-binding site. We observed that the central nitrate is shifted by ∼1.4 Å from chloride, which is likely caused by a weaker interaction between the anion and Pro160; the side chains of aromatic residues around the central binding site are rearranged to accommodate the larger nitrate. Additionally, we identified the ATP-binding pocket of AtCLCa to be located between the cytosolic cystathionine β-synthase domains and the N-terminus. The N-terminus may mediate the ATP inhibition of AtCLCa by interacting with both ATP and the pore-forming transmembrane helix. Together, our studies provide insights into the nitrate selectivity and ATP regulation of plant CLCs.
History
DepositionMar 17, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chloride channel protein CLC-a
B: Chloride channel protein CLC-a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,78912
Polymers176,3542
Non-polymers1,43510
Water0
1


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

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Components

#1: Protein Chloride channel protein CLC-a / / AtCLC-a / CBS domain-containing protein CBSCLC5


Mass: 88176.820 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: CLC-A, CBSCLC5, At5g40890, MHK7.12 / Production host: Homo sapiens (human) / References: UniProt: P92941
#2: Chemical
ChemComp-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: NO3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: AtCLCa / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2913673 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00911276
ELECTRON MICROSCOPYf_angle_d1.24715348
ELECTRON MICROSCOPYf_dihedral_angle_d28.8461528
ELECTRON MICROSCOPYf_chiral_restr0.0651794
ELECTRON MICROSCOPYf_plane_restr0.011914

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