1

Supplemental Information

Structure and Activity of SLAC1 Channels for Stomatal Signaling in Leaves

Ya-nan Denga,b,c, Hamdy Kashtoha,b,1, Quan Wangd,1, Guang-xiao Zhena,b,c, Qi-

yu Lia,b,c, Ling-hui Tanga,b,c, Hai-long Gaod, Chun-rui Zhanga,b,c, Li Qina,b,c, Min

Sua,b, Fei Lie, Xia-he Huanga,b, Ying-chun Wanga,b,c, Qi Xiea,b,c, Oliver B.

Clarkeef,g,2, Wayne A. Hendricksonf,h,2 & Yu-hang Chena,b,c,i,2

*Correspondence to:

Yu-hang Chen, E-mail: yuhang.chen@genetics.ac.cn; or

Wayne A. Hendrickson, E-mail: wah2@cumc.columbia.edu; or

Oliver B. Clarke, E-mail: oc2188@cumc.columbia.edu.

This PDF file includes: Figs. S1 to S7

Tables S1 to S2

Supplementary text(includes SI reference citations)

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B

C D

E

F G

A

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Figure S1. Purification and cryo-EM analyses of BdSLAC1 in SMALP (A) Size-exclusion elution profile and SDS-PAGE analysis of the purified BdSLAC1 in SMALP. (B) A representative negative-stain EM micrograph of purified BdSLAC1 in SMALP. (C) Example micrograph of BdSLAC1. (D) Representative 2D class averages of BdSLAC1. (E) cryo-EM map of BdSLAC1, top view (left), and side view (right). (F) FSC curve of BdSLAC1 final non-uniform refinement. (G) Orientation distribution plot of particles used for final reconstruction. (H) The workflow for cryo-EM structure determination of BdSLAC1.

H

4

BdSLAC1 MAADPSS----------STTTTGHGLHATEEARQAAMSGPISARRPPPPASQRAFSRQVSLGSGV 55 AtSLAC1 MERKQSNAHSTFADINEVEDEAEQELQQQENNNNKRFSG-NRGPNRGKQRPFRGFSRQVSLETGF 64 HiTehA ----------------------------------------------------------------- 0 BdSLAC1 TVLGMDRVGGGGRNGGRRSALPRSGKSLGVLNNNINHSGALGGGGGERRGGGDFSMFRTKSTLNKQNSML 125 AtSLAC1 SVLNRE-----SRERDDKKSLPRSGRSFGGFES----GGIINGGDG---RKTDFSMFRTKSTLSKQKSLL 122 HiTehA ---------------------------------------------------------------------- 0 BdSLAC1 PSRIREELDDVDLGRVEGGGQSAGRPDEDPLNKSVPAGRYFAALRGPELDEVRDYEDILLPKD 188 AtSLAC1 PSIIRERDIENSLRT------EDGETKDDSINENVSAGRYFAALRGPELDEVKDNEDILLPKE 179 HiTehA --------------------------------------------------------------- 0 H01 TM1 TM2 BdSLAC1 EVWPFLLRFPIGCFGVCLGLGSQAILWGALAASPAMRFLRVTPMINLAVWLLAAAVLAATSVTYALKCVFY 259 AtSLAC1 EQWPFLLRFPIGCFGICLGLSSQAVLWLALAKSPATNFLHITPLINLVVWLFSLVVLVSVSFTYILKCIFY 250 HiTehA MNITKPFPLPTGYFGIPLGLAALSLAWFHLENLFPA-----ARMVSDVLGIVASAVWILFILMYAYKLRYY 66 : :* * **: ***.: :: * * *.: : :: . : :: * . * * :* H23 TM3 TM4 BdSLAC1 FEAIRREFFHPVRVNFFFTPSIAAMFLAIGLPRALAPADGRAMHPAVWCASVAPLFALELKIYGQWLSG 328 AtSLAC1 FEAVKREYFHPVRVNFFFAPWVVCMFLAISVPPMFSPN-RKYLHPAIWCVFMGPYFFLELKIYGQWLSG 318 HiTehA FEEVRAEYHSPVRFSFIALIPITTMLVGDIL----YRWNPLIAEVLIWIGTIGQLLFSTLRVSELWQGG 131 ** :: *:. ***..*: :. *::. : . :* :. : *:: * .* TM5 TM6 BdSLAC1 GKRRLCKVANPSSHLS-VVGNFVGAILAARVGWVEAGKFLWAIGVAHYIVVFVTLY 383 AtSLAC1 GKRRLCKVANPSSHLS-VVGNFVGAILASKVGWDEVAKFLWAVGFAHYLVVFVTLY 373 HiTehA VFEQK-S-THPSFYLPAVAANFTSASSLALLGYHDLGYLFFGAGMIAWIIFEPVLL 185 .: :: ** :* :*..**..* : :*: : . :::. *. :::. .* TM7 TM8 BdSLAC1 QRLPTNEALPMELHPVYSMFIATPSAASLAWAA-IYGSFD---AVARTFFFMALFLYMSLVVRIN 444 AtSLAC1 QRLPTSEALPKELHPVYSMFIAAPSAASIAWNT-IYGQFD---GCSRTCFFIALFLYISLVARIN 434 HiTehA QHLRISSLEPQFR-ATMGIVLAPAFVCVSAYLSINHGEVDTLAKILWGYGFLQLFFLLRLFPWI- 248 *:* .. * . .:.:* .. *: :*..* :: *: **: : * : TM9 TM10 BdSLAC1 FFRGFRFSIAWWSYTFPMTTASLATVKYAEA-VPCFLSRALALSLSLMSTTMVSLLLVSTLLHAFVWRS 512 AtSLAC1 FFTGFKFSVAWWSYTFPMTTASVATIKYAEA-VPGYPSRALALTLSFISTAMVCVLFVSTLLHAFVWQT 502 HiTehA --VEKGLNIGLWAFSFGLASMANSATAFYHGNV-LQGVSIFAFVFSNVMIGLLVLMTIYKLTKGQFFLK 314 :: :.:. *:::* ::: : :: ::.: * .: ..*: :*.: : : : : :* : BdSLAC1 LFPNDLAIAITKDRQNGGARPHGKGRKAGKRVYDIKRWAKQAPLSLVSSITKTNSADKEEEEKTD 577 AtSLAC1 LFPNDLAIAITKRKLTRE-------KKPFKRAYDLKRWTKQALAKKISAEKDFE---AEEESHH- 556 HiTehA ----------------------------------------------------------------- 314

Figure S2. Sequence Alignment for BdSLAC1, AtSLAC1 and HiTehA

Structure-based sequence alignment for BdSLAC1 (Barachypodium distachyon), AtSLAC1 (Arabidopsis thaliana) and HiTehA (Haemophilus influenzae). The structures for both BdSLAC1 and HiTehA have been used to restrict sequence gaps to inter-helical segments. Superior coils define extents of the BdSLAC1 helical segments， and the helical regions for HiTehA are indicated by underscore. Boxes are drawn for pore lining residues. Red letters mark conserved positive-charged residues in the TMs connecting loops on the cytoplasmic side. The residues that are identified as phosphorylation sites by mass spectrometry are highlighted in green. The kink-mediated residues in the middle of TM helices are highlight in yellow. A residue insertion (A146) in HiTehA, resulting in a wider p-helical turn in the middle of TM5, is highlighted in magenta. The disordered region in BdSLAC1 is highlight in cyan.

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Figure S3. Electrophysiological Measurements for Arabidopsis SLAC1::OST1

(A) Current-voltage (I-V) relations from oocytes expressing wild type or mutant channels. Wild-type SLAC::OST1 (black), SLAC::OST1 (D160A) (blue), ABI alone (magenta) and co-expression of SLAC::OST1/ABI1 (red). (B) Mean chloride currents, measured at -90 mV, are shown comparing SLAC1::OST1, SLAC1::OST1 (D160A), and SLAC1::OST1/ABI1 co-expression. The OST1 kinase stimulates macroscopic currents in SLAC1, while the functionally impaired OST1 (D160A) abolished the channel activity. This observation validates SLAC1 activation by OST1. Co-expression of SLAC1::OST1 with ABI1 shows no conductance confirming ABI1 inhibition to OST1 kinase activity.

Curr

ent a

t –90

mV

(μA)

A

B

A

V (mV)

I (μA)

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A

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E F

a B

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Figure S4. Mass Spectrometry Analyses of Phosphorylated Peptides

(A) Purification of Arabidopsis SLAC1::OST1 fusion protein from yeast S. pombe. To prepare SLAC1::OST1 fusion protein, Arabidopsis SLAC1 was fused directly to OST1, and this construct was introduced into a modified pREP1 vector with a C-terminal Flag-10XHis tag. The resulting construct was transformed into S. pombe for expression.The fusion protein was purified by Ni2+-affinity chromatography and analysed on 12% SDS-PAGE. (B-F) The band corresponding to the SLAC1::OST1 fusion protein, as shown in (A), was recovered, subjected to tryptic digestion, and further analysed with a nano LC-MS/MS system. At least 14 sites were found being phosphorylated, including 13 in the N-terminal domain and 1 at the C-terminus. Six of these residues were shown to be critical for SLAC1 activation in our electrophysiological studies in Xenopus oocytes. MS/MS spectra of the five phospho-peptides were shown, with m/z 764.87, 853.36, 422.21, 590.27 and 915.47, representing peptides that contain phosphorylated S59 (B), S86 (C), S113 (D), T114/S116 (E) and S116/S120 (F), respectively.

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C

T111D T513D

10 μ

A

1s

D E

S59/S120-2D S86/S113-2D S59/S86/S113/S120-4D

S59/S86/S113/T114/S120-5D S59/S86/S113/S116/S120-5D S59/S86/S113/T114/S116/S120-6D

5 μA

1s

10 μ

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1s

F

B A

V (mV)

I (μA)

V (mV)

I (μA)

V (mV)

I (μA)

V (mV)

I (μA)

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Figure S5. Electrophysiological analysis of SLAC1 phosphorylation mutants (A) Current-voltage (I-V) relations of Arabidopsis SLAC1::OST1, wild-type and mutants. Mutations in SLAC1::OST1 are shown and coloured as indicated. (B) Current-voltage (I-V) relations for Arabidopsis SLAC1-13A::OST1 mutant channels. SLAC1-13A::OST1 contains 13 mutations at the N-terminus of SLAC1, including: S6A, S38A, S59A, S86A, S113A, T114A, S116A, S120A, S134A, T137A, T142A, S146A and S152A, whereas F450A SLAC1-13A::OST1 contains the additional F450A mutation. F450A SLAC1::OST1 served as a positive control for proper surface expression of the SLAC1-13A::OST1 construct. This observation shows that the lack of current in SLAC1-13A::OST1 is due to loss of phosphorylation, rather than failure of surface expression in oocytes. (C, D) Representative current traces and current-voltage (I-V) relations of Arabidopsis SLAC1 phosphomimetic mutants. Mutations are shown and coloured as indicated. (E, F) Representative current traces and current-voltage (I-V) relations of other Arabidopsis SLAC1 phosphomimetic mutants.

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Figure S6. Electrophysiological analysis of the Conserved Pro in TM9 of SLAC1 (A) Comparison of the water-mediated kink in HiTehA (right) and the proline-kinked TM9 in BdSLAC1(left); (B) Current-voltage (I-V) relations for wild-type Arabidopsis SLAC1::OST1 (black), P451A SLAC1::OST1 (blue) and P451G SLAC1::OST1 (red); (C) Mean chloride currents, measured at -90 mV, are shown comparing wild-type SLAC1::OST1 with its mutant variants P451G and P451A. P451 substitutions to glycine (as conserved in bacterial TehA) or to alanine impair channel activity in SLAC1::OST.

A B

C

HiTehA BdSLAC1

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F450A SLAC1

SLAC1::OST1

F276A SLAC1::OST1

SLAC1

F276A SLAC1

5 μA

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1s

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F276A/F450A SLAC1

A

F450A SLAC1::OST1

F276A/F450A SLAC1::OST1

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I (μA)

V (mV)

I (μA)

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Figure S7. Electrophysiological Analysis of SLAC1 Gating Residues (A) Representative current traces and current-voltage (I-V) relations for wild-type SLAC1and F276A SLAC1; (B) Representative current traces and current-voltage (I-V) relations for F450A SLAC1 and F276A/F450A SLAC1; (C) Representative current traces and current-voltage (I-V) relations for wild-type SLAC1::OST1 and F276A SLAC1::OST1; (D) Representative current traces and current-voltage (I-V) relations for F450A SLAC1::OST1 and F276A/F450A SLAC1::OST1. Note: different Y-axis scales.

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Table S1

Cryo-EM data collection, refinement and validation statistics

Data collection and processing Microscope Titan Krios (Krios 3, SEMC) Detector Gatan K2 Magnification 130,000x Voltage (kV) 300 Electron exposure (e-/Å2) 71.3 Nominal defocus range (µm) 0.7-2 Movies (total) 7265 Movies (final) 4792 Frames per movie 50 Pixel size (Å) 1.0475 Symmetry imposed C3 Initial particle images (no.) 2938351 Final particle images (no.) 95704 Map resolution (Å) 2.97 FSC threshold 0.143 Map sharpening B factor (Å2) -131.7 Refinement and model validation EMringer score 2.49 Initial model used (PDB code) 3M71 Model resolution (Å, FSC=0.5) 3.24 CC(mask) 0.78 Ramachandran plot Favored (%) 95.47 Allowed (%) 4.53 Disallowed (%) 0.0 Molprobity score 2.41 Clash score 7.21 Model composition Non hydrogen atoms 7017 Protein residues 939 Ligands 0 B factors (Å2) Protein 149.04 R.M.S deviations Bond lengths (Å) 0.006 Bond angles (degrees) 0.675

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Table S2

Mass Spectrum analyses of phosphorylation sites in Arabidopsis SLAC1::OST1

When performed mass spectrometry analyses, we found 14 potential phosphorylation sites, 13 at N terminus (S6, S38, S59, S86, S113, T114, S116, S120, S134, T137, T142, S146 and S152) and 1 at C terminus (S543) in SLAC1 channel. In addition, three sites (S29, S71 and S171) were also identified in OST1 kinase.

*Potential phosphorylation site is indicated in lower case and colored in red.

Site Protein Sequence* m/z S6 KQsNAHSTFADINEVEDEAEQELQQQENN

NNK 1260.55

S6 KQsNAHSTFADINEVEDEAEQELQQQENNNNKR

1312.57

S38 RFsGNR 408.18 S38 RFsGNRGPNR 620.29

S59 QVsLETGFSVLNR 764.87 S86 sFGGFESGGIINGGDGR 853.36

S86 sFGGFESGGIINGGDGRK 917.41 S86 SGRsFGGFESGGIINGGDGRK 711.66

S113 TKsTLSK 422.21 T114, S116 TKStLsKQK 590.27

S116, 120 STLsKQKsLLPSIIR 915.47 S134, T137, T142

ERDIENsLRtEDGEtK 1025.92 S134 ERDIENsLR 605.77

S134 DIENsLR 463.20 S134, T137 DIENsLRtEDGETK 883.35

T137 ERDIENSLRtEDGETK 985.93 T142 DIENSLRtEDGETK 843.36

T137, S146 DIENSLRtEDGETKDDsINENVSAGR 1034.74 S146 TEDGETKDDsINENVSAGR 1058.43

S146, S152 DDsINENVsAGR 718.26 S146, S152 TEDGETKDDsINENVsAGR 732.28

S152 TEDGETKDDSINENVsAGRYFAALR 946.08 S152 DIENSLRTEDGETKDDSINENVsAGR 736.07

S152 DDSINENVsAGR 678.27 S543 KIsAEKDFEAEEESHH 655.27 S543 QALAKKIsAEK 633.34

S543 KIsAEK 377.68 OST1 S29 DIGsGNFGVAR 586.25

OST1 S71 sLRHPNIVR 390.54 OST1 S171 SSVLHsQPK

531.25

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Materials and Methods

Cloning and Expression in Yeast S. pombe

SLAC1 proteins from 15 plant species were selected, corresponding cDNAs

were synthesized and cloned into a modified pREP1 vector, which contains a

TEV protease cleavage site, a FLAG tag and a deca-histidine tag at the C-

terminus (34). The resulting constructs were transformed into a leucine

auxotrophic S. pombe strain by the lithium acetate method, and standard media

for fission yeast were used. The transformants were negatively selected by

plating on EMM plates without leucine, but 25mM thiamine inhibits the promoter.

Among those, BdSLAC1 had the best expression level, and passed for further

biochemical and structural studies.

Scaled-up Production and Purification

In brief, for preparation of the seed, the transformed yeast S. pombe cells were

inoculated into ~ 150 ml EMM culture medium supplemented with 25 mM

thiamine, and shaken at 200 rpm, 30 °C for ~ 24 hours. For protein expression

and scale up, the cells were spun down, and washed with sterile ddH2O twice

before further inoculation to large culture of ~ 0.5-1.0 liter. Cells were first grown

for ~12 hours while at 30° C and 200 rpm, then supplemented with an equal

volume of fresh media and continued in growth for another 12 hours, and finally

harvested by centrifugation and stored at -80°C for future use.

Cells were suspended in lysis buffer (50 mM Tris-HCl pH 8.0 and 200 mM

NaCl) at 1:10 (w/v) ratio and then lysed by passing through a high-pressure cell

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disrupter (JNBIO, Guangzhou) at ~15-20,000 psi 4 times. Cell debris was

removed by centrifugation at 10,000 rpm for 20 min, and the supernatant was

subjected to ultra-centrifugation at 150,000g for 1 hour for membrane isolation.

The membrane fraction was homogenized in a solubilization buffer containing

40 mM Tris-HCl (pH 8.0) and 150 mM NaCl at 1:10 (w/v) ratio. To prepare

SMALP nanoparticles of BdSLAC1, SMA was added to the cell lysate at 2%

SMA (w/v) final concentration, and the mixture was stirred gently at room

temperature for 2 hours. The insoluble matter was removed by

ultracentrifugation at 150,000g for 30 min, and the supernatant was loaded to a

1ml HiTrap Ni2+-NTA affinity column (GE Healthcare), pre-equilibrated with

solubilization buffer. After a 10 column-volume buffer wash, the protein was

eluted with 10 column volumes of 500 mM imidazole in solubilization buffer. The

protein sample was concentrated to around 5 mg/ml for further purification on a

Superose-6 10/300 GL (GE Healthcare) column without addition of detergents

or SMAs in solubilization buffer. The elution was analyzed by SDS-PAGE, and

peak fractions for the resulting BdSLAC1 SMALP were concentrated to ~ 2

mg/ml for protein for making cryo-EM grids.

Cryo-EM Structure Determination

3µL of purified BdSLAC1 (1.8mg/ml) was applied to a plasma-cleaned Quantifoil

UltrAuFoil holey gold grid (0.6µm holes with 1 µm spacing) in a TFS Vitrobot,

with a blot time of 7s and a wait time of 30s. Data were collected on a Titan Krios

equipped with a Gatan K2 direct electron detector, operated in electron counting

17

mode. 7265 50-frame stacks, each with a total electron exposure of 71.3 e-/Å2,

were collected at a nominal magnification of 130kx and a calibrated pixel size of

1.0475 Å. Initial motion correction was carried out using the Relion 3 CPU-based

implementation of Motioncor2 (46), and whole frame CTF estimation was

performed using CTFFIND4 (47). Approximately 1000 particles were manually

picked in Relion and 2D classes derived from these particles were used as

templates for picking the entire dataset using relion_autopick. 3M initial particles

(4x binned) were subjected to extensive 2D classification in cryoSPARC (48),

performing separate classification runs for top views and side views, giving a

final set of 110k high quality particles. After re-extraction at full scale, a single-

class ab initio reconstruction run without imposed symmetry produced a volume

with the features of a trimeric channel. Using the non-uniform refinement module

of cryoSPARC and this set of particles, the ab initio model refined to a

reconstruction at 3.29 Å. Further improvement was achieved to 3.09 Å by

Bayesian polishing in Relion(49), 3.02 Å after refinement of per particle defocus,

and 2.97 Å after cleaning up the particle set further by a multi class ab initio

reconstruction followed by heterogeneous refinement. An initial model was

generated from the structure of TehA using Phyre (50). The model was initially

fit to the map as a rigid body in Chimera (51), and flexible fitting and model

completion was carried out in Coot (52). The final map was sharpened with

phenix.auto_sharpen, and the model was refined with phenix.real_space_refine

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(53). Validation statistics of the final model were calculated with phenix.validate

and EMringer (54).

Electrophysiology

All constructs were cloned into plasmid pGHME2, which contains inserts of 5’

UTR (60nt) and 3’ UTR (300nt) from the Xenopus beta-globin gene in the

expression cassette, linearized and transcribed into cRNA using T7 polymerase

(33). Oocytes were injected with indicated cRNA solution as followings：when

injected with cRNA of SLAC1 or SLAC1::OST1, their concentrations were

diluted to 1.0 mg ml-1, and injected 36 nl of cRNA for each oocyte； when co-

injected with cRNAs of SLAC1 (Wild-type or mutant) with OST1, or

SLAC1::OST1 with ABI1, their concentrations were diluted to 2.0 mg ml-1, then

mixed at the ratio of 1:1, and injected 36 nl of cRNA for each oocyte.

Two-microelectrode voltage-clamp recordings were performed 2 days after

cRNA injection to measure SLAC1 currents as described (33). The

microelectrode solutions contained 3 M KCl. For voltage-clamp current

recordings, the bath solution contained 1 mM MgCl2, 1 mM CaCl2, 10 mM

Tris/MES (pH5.6) and 30 mM CsCl; Osmolarity was adjusted with D-mannitol to

220 mOsmol*kg-1. The bath electrode was a 3 M KCl agar bridge. Voltage-clamp

currents were measured in response to 7.5-s-long voltage steps to test

potentials that ranged from -110 mV to 70 mV in 20 mV increments. Prior to

each voltage step the membrane was held at 0 mV for 1.60 s, and following

each voltage step the membrane was returned to 0 mV for 2.0 s. I–V relations

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for AtSLAC1 channels were generated from currents measured 0.5 s after the

start of each test voltage step.

Current responses from voltage-clamped oocyte experiments are directly

proportional to the amount of cRNA injection for a particular genetic construct,

and proportional to the moles of individual genetic constructs when the masses

per construct differ. With a fixed mass of injection, the mass, the number of

transcribed channels per oocytes will be inversely proportional to the construct

length. As there are 3126 nt per SLAC1::OST1 construct vs. 2031 nt per

SLAC1 construct, the ratio of SLAC1::OST1 to SLAC1 channels is 2031/3126

from the fixed amount of SLAC1 cRNA injected into each oocyte. We normalize

our results to the currents from SLAC1 injections; i.e. measured currents from

SLAC1::OST1 constructs were increased by the factor of 1.539.

Mass Spectrometry Analysis

Protein bands were excised from SDS-PAGE gels and digested with trypsin

according to a modified procedure(54). In brief, the protein-containing gel was

cut into pieces, and de-stained in 25mM ammonium bicarbonate / 50%

acetonitrile buffer. The proteins were reduced with 10 mM DTT at 56° C for 1

hour and alkylated with 55 mM iodo-acetamide at room temperature for 1 hour

in the dark. Trypsin (Sigma T1426; enzyme-to-substrate ratio 1:50) in 25 mM

ammonium bicarbonate was applied to digest the peptides at 37°C overnight.

Digested peptides were extracted from the gel with stripping buffer (5%

trifluoroacetic acid and 50% acetonitrile) by sonication. The liquid was dried by

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SpeedVac, and peptides were re-solubilized in 0.1% formic acid and filtered by

0.45 μm centrifugal filter.

The peptides were analyzed by LC-MS/MS using LTQ-Orbitrap elite mass

spectrometer (Thermo Fisher Scientific) coupled to an online Easy-nLC 1000

(Thermo Fisher Scientific) with enabled multistage activation. Phosphopeptides

were identified by searching the Arabidopsis thaliana proteome sequences

(downloadRed from TAIR) using the Thermo Scientific Proteome DiscovererTM

software (version 1.4).

References

46. J. Zivanov, et al., New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, 1–22 (2018).

47. A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol 192, 216–221 (2015).

48. A. Punjani, J. L. Rubinstein, D. J. Fleet, M. A. Brubaker, CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290–296 (2017).

49. J. Zivanov, T. Nakane, S. H. W. Scheres, Estimation of High-Order Aberrations and Anisotropic Magnification from Cryo-EM Datasets in RELION-3.1. bioRxiv, 798066 (2019).

50. L. A. Kelley, S. Mezulis, C. M. Yates, M. N. Wass, M. J. E. Sternberg, The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10, 845–858 (2015).

51. E. F. Pettersen, et al., UCSF Chimera - A visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612 (2004).

52. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr 66, 486–501 (2010).

53. P. V. Afonine, et al., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr Sect D Struct Biol 74, 531–544 (2018).

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54. B. A. Barad, et al., EMRinger: Side chain-directed model and map validation for 3D cryo-electron microscopy. Nat Methods 12, 943–946 (2015).

55. A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850–858 (1996).