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- EMDB-35904: AtSLAC1 8D mutant in closed state -

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Basic information

Entry
Database: EMDB / ID: EMD-35904
TitleAtSLAC1 8D mutant in closed state
Map data
Sample
  • Complex: Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN and CHS micelle
    • Protein or peptide: Guard cell S-type anion channel SLAC1,Green fluorescent protein
  • Ligand: CHLORIDE IONChloride
  • Ligand: CHOLESTEROL HEMISUCCINATE
KeywordsStomatal closure / anion channel / phosphorylation-dependent activation / MEMBRANE PROTEIN
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
Green fluorescent protein / Guard cell S-type anion channel SLAC1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsLee Y / Lee S
Funding support Korea, Republic Of, 4 items
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
DepositionApr 11, 2023-
Header (metadata) releaseNov 22, 2023-
Map releaseNov 22, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35904.png

Downloads & links

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Map

FileDownload / File: emd_35904.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.858 Å
Density
Contour LevelBy AUTHOR: 0.352
Minimum - Maximum-2.0443795 - 3.1347518
Average (Standard dev.)0.0027837956 (±0.059491523)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 257.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35904_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35904_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35904_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN an...

EntireName: Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN and CHS micelle
Components
  • Complex: Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN and CHS micelle
    • Protein or peptide: Guard cell S-type anion channel SLAC1,Green fluorescent protein
  • Ligand: CHLORIDE IONChloride
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN an...

SupramoleculeName: Trimer of closed state AtSLAC1 8D mutant with sfGFP tag in GDN and CHS micelle
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 280 KDa

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Macromolecule #1: Guard cell S-type anion channel SLAC1,Green fluorescent protein

MacromoleculeName: Guard cell S-type anion channel SLAC1,Green fluorescent protein
type: protein_or_peptide / ID: 1 / Details: sfGFP tag / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 94.073008 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MERKQSNAHS TFADINEVED EAEQELQQQE NNNNKRFSGN RGPNRGKQRP FRGFSRQVDL EDGFDVLNRE SRERDDKKSL PRSGRDFGG FESGGIINGG DGRKTDFDMF RTKSTLSKQK SLLPDIIRER DIENSLRTED GETKDDDINE NVDAGRYFAA L RGPELDEV ...String:
MERKQSNAHS TFADINEVED EAEQELQQQE NNNNKRFSGN RGPNRGKQRP FRGFSRQVDL EDGFDVLNRE SRERDDKKSL PRSGRDFGG FESGGIINGG DGRKTDFDMF RTKSTLSKQK SLLPDIIRER DIENSLRTED GETKDDDINE NVDAGRYFAA L RGPELDEV KDNEDILLPK EEQWPFLLRF PIGCFGICLG LSSQAVLWLA LAKSPATNFL HITPLINLVV WLFSLVVLVS VS FTYILKC IFYFEAVKRE YFHPVRVNFF FAPWVVCMFL AISVPPMFSP NRKYLHPAIW CVFMGPYFFL ELKIYGQWLS GGK RRLCKV ANPSSHLSVV GNFVGAILAS KVGWDEVAKF LWAVGFAHYL VVFVTLYQRL PTSEALPKEL HPVYSMFIAA PSAA SIAWN TIYGQFDGCS RTCFFIALFL YISLVARINF FTGFKFSVAW WSYTFPMTTA SVATIKYAEA VPGYPSRALA LTLSF ISTA MVCVLFVSTL LHAFVWQTLF PNDLAIAITK RKLTREKKPF KRAYDLKRWT KQALAKKISA EKDFEAEEES HHGSEN LYF QSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYP DH MKRHDFFKSA MPEGYVQERT ISFKDDGTYK TRAEVKFEGD TLVNRIELKG IDFKEDGNIL GHKLEYNFNS HNVYITAD K QKNGIKANFK IRHNVEDGSV QLADHYQQNT PIGDGPVLLP DNHYLSTQSV LSKDPNEKRD HMVLLEFVTA AGITHGMDE LYKYPYDVPD YAGGGSHHHH HHHHHH

UniProtKB: Guard cell S-type anion channel SLAC1, Green fluorescent protein

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Macromolecule #2: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration2.3 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMNaClSodium chlorideSodium chloride
1.0 mMTCEPTCEP
0.01 % (w/v)GDNGDN
0.002 % (w/v)CHSCHS
GridModel: C-flat / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: Au-Flat 1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 18 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8gw6

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1) / Details: NU-refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1) / Details: NU-refinement
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 181365
FSC plot (resolution estimation)

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