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- EMDB-34304: AtSLAC1 6D mutant in open state -

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Basic information

Entry
Database: EMDB / ID: EMD-34304
TitleAtSLAC1 6D mutant in open state
Map data
Sample
  • Complex: Trimer of open state AtSLAC1 6D mutant with sfGFP tag in GDN and CHS micelle
    • Protein or peptide: Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
  • Ligand: CHLORIDE ION
  • Ligand: CHOLESTEROL HEMISUCCINATE
KeywordsStomatal closure / anion channel / phosphorylation-dependent activation / MEMBRANE PROTEIN
Function / homology
Function and homology information


inorganic anion transport / response to humidity / stomatal closure / regulation of stomatal opening / voltage-gated monoatomic anion channel activity / regulation of stomatal closure / stomatal movement / response to ozone / intracellular monoatomic ion homeostasis / response to carbon dioxide ...inorganic anion transport / response to humidity / stomatal closure / regulation of stomatal opening / voltage-gated monoatomic anion channel activity / regulation of stomatal closure / stomatal movement / response to ozone / intracellular monoatomic ion homeostasis / response to carbon dioxide / monoatomic anion transmembrane transporter activity / response to abscisic acid / multicellular organismal-level water homeostasis / organic anion transport / abscisic acid-activated signaling pathway / monoatomic anion transport / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 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
Citation
Journal: 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.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography
Authors: Pavel VA
History
DepositionSep 16, 2022-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_34304.png

Downloads & links

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Map

FileDownload / File: emd_34304.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 240 pix.
= 201.6 Å		0.84 Å/pix.
x 240 pix.
= 201.6 Å		0.84 Å/pix.
x 240 pix.
= 201.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.139
Minimum - Maximum-0.5810045 - 0.8453114
Average (Standard dev.)0.0016088666 (±0.026731262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34304_msk_1.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: #2

Fileemd_34304_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34304_half_map_2.map
Projections & Slices
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Sample components

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Entire : Trimer of open state AtSLAC1 6D mutant with sfGFP tag in GDN and ...

EntireName: Trimer of open state AtSLAC1 6D mutant with sfGFP tag in GDN and CHS micelle
Components
  • Complex: Trimer of open state AtSLAC1 6D mutant with sfGFP tag in GDN and CHS micelle
    • Protein or peptide: Guard cell S-type anion channel SLAC1,Green fluorescent protein (Fragment)
  • Ligand: CHLORIDE ION
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Trimer of open state AtSLAC1 6D mutant with sfGFP tag in GDN and ...

SupramoleculeName: Trimer of open state AtSLAC1 6D 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 (Fragment)
type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 94.016984 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MERKQSNAHS TFADINEVED EAEQELQQQE NNNNKRFSGN RGPNRGKQRP FRGFSRQVSL EDGFDVLNRE SRERDDKKSL PRSGRSFGG FESGGIINGG DGRKTDFDMF RTKSTLSKQK SLLPDIIRER DIENSLRTED GETKDDDINE NVDAGRYFAA L RGPELDEV ...String:
MERKQSNAHS TFADINEVED EAEQELQQQE NNNNKRFSGN RGPNRGKQRP FRGFSRQVSL EDGFDVLNRE SRERDDKKSL PRSGRSFGG 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: 6 / 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

Concentration3.3 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMNaClSodium chloride
1.0 mMTCEPTCEP
0.01 % (w/v)GDNGDN
0.002 % (w/v)CHSCHS
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS TALOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio model
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 55882
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3) / Details: NU-refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3) / Details: Local-refinement
FSC plot (resolution estimation)

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