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- PDB-8gw6: AtSLAC1 6D mutant in closed state -

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Basic information

Entry
Database: PDB / ID: 8gw6
TitleAtSLAC1 6D mutant in closed state
ComponentsGuard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
KeywordsMEMBRANE PROTEIN / Stomatal closure / anion channel / phosphorylation-dependent activation
Function / homology
Function and homology information


response to humidity / stomatal closure / regulation of stomatal opening / inorganic anion transport / voltage-gated monoatomic anion channel activity / regulation of stomatal closure / stomatal movement / response to ozone / intracellular monoatomic ion homeostasis / organic anion transport ...response to humidity / stomatal closure / regulation of stomatal opening / inorganic anion transport / voltage-gated monoatomic anion channel activity / regulation of stomatal closure / stomatal movement / response to ozone / intracellular monoatomic ion homeostasis / organic anion transport / monoatomic anion transmembrane transporter activity / response to abscisic acid / response to carbon dioxide / multicellular organismal-level water homeostasis / monoatomic anion transport / abscisic acid-activated signaling pathway / response to light stimulus / bioluminescence / generation of precursor metabolites and energy / protein phosphatase binding / protein kinase binding / membrane / plasma membrane
Similarity search - Function
S-type anion channel / Transporter protein SLAC1/Mae1/ Ssu1/TehA / Voltage-dependent anion channel superfamily / Voltage-dependent anion channel / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
CHOLESTEROL HEMISUCCINATE / Green fluorescent protein / Guard cell S-type anion channel SLAC1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLee, Y. / Lee, S.
Funding support Korea, Republic Of, 4items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2022R1A2C100988211 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4022936 Korea, Republic Of
National Research Foundation (NRF, Korea)2020M3A9E4039217 Korea, Republic Of
National Research Foundation (NRF, Korea)2017R1A5A1014560 Korea, Republic Of
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structures of the plant anion channel SLAC1 from Arabidopsis thaliana suggest a combined activation model.
Authors: Yeongmok Lee / Hyeon Seong Jeong / Seoyeon Jung / Junmo Hwang / Chi Truc Han Le / Sung-Hoon Jun / Eun Jo Du / KyeongJin Kang / Beom-Gi Kim / Hyun-Ho Lim / Sangho Lee /
Abstract: The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation ...The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation model and an inhibition-release model for activation have been proposed based on only the closed structures of SLAC1, rendering the structure-based activation mechanism controversial. Here we report cryo-EM structures of Arabidopsis SLAC1 WT and its phosphomimetic mutants in open and closed states. Comparison of the open structure with the closed ones reveals the structural basis for opening of the conductance pore. Multiple phosphorylation of an intracellular domain (ICD) causes dissociation of ICD from the transmembrane domain. A conserved, positively-charged sequence motif in the intracellular loop 2 (ICL2) seems to be capable of sensing of the negatively charged phosphorylated ICD. Interactions between ICL2 and ICD drive drastic conformational changes, thereby widening the pore. From our results we propose that SLAC1 operates by a mechanism combining the binding-activation and inhibition-release models.
History
DepositionSep 16, 2022Deposition site: PDBJ / Processing site: PDBE
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
B: Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
C: Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)283,6179
Polymers282,0513
Non-polymers1,5676
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 150 through 320 or resid 322 through 603))
d_2ens_1(chain "A" and (resid 150 through 320 or resid 322 through 603))
d_3ens_1(chain "C" and (resid 150 through 320 or resid 322 through 603))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASNLYSD1 - 171
d_12ens_1ARGLYSD173 - 367
d_13ens_1Y01Y01E
d_21ens_1ASNLYSA1 - 171
d_22ens_1ARGLYSA173 - 367
d_23ens_1Y01Y01B
d_31ens_1ASNLYSG1 - 171
d_32ens_1ARGLYSG173 - 367
d_33ens_1Y01Y01H

NCS oper:
IDCodeMatrixVector
1given(-0.502757841753, 0.864423155148, -0.00267607905269), (-0.864422892955, -0.502762753424, -0.00163581915608), (-0.00275947282906, 0.0014908430881, 0.999995081336)65.4093112701, 241.694446384, 0.113720556872
2given(-0.496589688779, -0.86798540187, -0.000152116265097), (0.867985398852, -0.496589637789, -0.000281104554218), (0.000168455288474, -0.000271628320126, 0.99999994892)241.286566667, 64.0769046225, 0.0246167466043

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Components

#1: Protein Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment) / Protein CARBON DIOXIDE INSENSITIVE 3 / Protein OZONE-SENSITIVE 1 / Protein RADICAL-INDUCED CELL ...Protein CARBON DIOXIDE INSENSITIVE 3 / Protein OZONE-SENSITIVE 1 / Protein RADICAL-INDUCED CELL DEATH 3 / Protein SLOW ANION CHANNEL-ASSOCIATED 1


Mass: 94016.984 Da / Num. of mol.: 3 / Mutation: T62D,S65D,S107D,S124D,S146D,T152D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: SLAC1, CDI3, OZS1, RCD3, At1g12480, F5O11.23, T12C24.3, gfp
Plasmid: pACEBac2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9LD83, UniProt: A0A059PIQ0
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
Has ligand of interestN

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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: Trimer of closed state AtSLAC1 6D mutant with sfGFP tag in GDN and CHS micelle
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.28 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMSodium chlorideNaClSodium chloride1
31 mMTCEPTCEP1
40.01 % (w/v)GDNGDN1
50.002 % (w/v)CHSCHS1
SpecimenConc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Nominal defocus max: 1900 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 14 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
4cryoSPARC3.3CTF correction
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
CTF correctionType: NONE
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69390 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00229201
ELECTRON MICROSCOPYf_angle_d0.479512600
ELECTRON MICROSCOPYf_chiral_restr0.0361425
ELECTRON MICROSCOPYf_plane_restr0.00481518
ELECTRON MICROSCOPYf_dihedral_angle_d6.1031224
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000702731486711
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000709049560173

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