Supplementary Figure 1 Sequence alignment of

C1Chrimson Chrimson CsChrimson ChR1 ChR2 C1C2 CaChR1 VChR1 ReaChR BR

..MSRRPWLLALALAVALAAG....SAGASTGSDATVPVATQDGPDYVFHRAHER..MLFQTSYTLENNGSVICIPNNGQ ..MAE...LISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGV ..MSR...LVAASWLLALLLCGITSTTTASSAPAASS..TDGTAAAAVSHYAMN...GFDELAKGAVVPEDHFVCGPADK ..MSRRPWLLALALAVALAAGS....AGASTGSDATVPVATQDGPDYVFHRAHER..MLFQTSYTLENNGSVICIPNNGQ .................MDYGG....ALSAVG.....................RE..LLFVTNPVVVN.GSVLVPED..Q ..MSRRPWLLALALAVALAAGS....AGASTGSDATVPVATQDGPDYVFHRAHER..MLFQTSYTLENNGSVICIPNNGQ .MDTLAWVARELLSTAHDATPATATPSTDHSTPSTDHGSGETFNVTITIGGGHHGGHAGPVDNSIVIGGIDGWIAIPAGD .............................MDYPVARS...............LIVRYPTDLGNGTVCMPR........GQ MVSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNN.......GQ ................................................................................

TM1 C1Chrimson Chrimson CsChrimson ChR1 ChR2 C1C2 CaChR1 VChR1 ReaChR BR

80

90

TM2

100

110

120

140

150

CFCLAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKA..TCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLS EKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKA..TCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLS CYCSAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKA..TCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLS CFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKS..TCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSS CYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKS..TCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLA CFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKS..TCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSS CYCAGWYVSHGSSFEATFAHVCQWSIFAVCILSLLWYAWQYWKA..TCGWEEVYVCCIELVFICFELYHEFDSPCSLYLS CYCEGWLRSRGTSIEKTIAITLQWVVFALSVACLGWYAYQAWRA..TCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLS CFCLAWLKSNGTNAEKLAANILQWVVFALSVACLGWYAYQAWRA..TCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLS ......QAQITGRPEWIWLALGTALMGLGTLYFLVKGMGVSDPDAKKFYAITTLVPAIAFTMYLSMLLGYGLTMVPFGGE

TM3 C1Chrimson Chrimson CsChrimson ChR1 ChR2 C1C2 CaChR1 VChR1 ReaChR BR

130

160

170

TM4 180

190

200

TM5 210

220

230

TGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLA.TDWLKWLLYIVSCIYGGYMYF TGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLA.TDWLKWLLYIVSCIYGGYMYF TGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLA.TDWLKWLLYIVSCIYGGYMYF NGNKTVWLRYAEWLLTCPVILIHLSNLTGLANDYNKRTMGLLVSDIGTIVWGTTAALS.KGYVRVIFFLMGLCYGIYTFF TGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMA.TGYVKVIFFCLGLCYGANTFF NGNKTVWLRYAEWLLTCPVILIHLSNLTGLANDYNKRTMGLLVSDIGTIVWGTTAALS.KGYVRVIFFLMGLCYGIYTFF TANIVNWLRYSEWLLCCPVILIHLSNVTGLSDDYGRRTMGLLVSDIATIVFGITAAML.VSWPKIIFYLLGFTMCCYTFY SGNGVVWMRYGEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMC.TGWTKILFFLISLSYGMYTYF SGNGVVWMRYGEWLLTCPVILIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMC.TGWTKILFFLISLSYGMYTYF QN.PIYWARYADWLFTTPLLLLDLALLVDA...DQGTILALVGADGIMIGTGLVGALTKVYSYRFVWWAISTAAMLYILY

TM6

TM7

240

250

260

270

320

330

340

350

280

290

300

310


QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK NAAKVYIEAYHTVPKGICRDLVRYLAWLYFCSWAMFPVLFLLGPEGFGHINQFNSAIAHAILDLASKNAWSMMGHFLRVK HAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVL NAAKVYIEAYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVL LAAKVLIESFHQVPKGICRHLVKAMAITYYVGWSFFPLIFLFGQSGFKKISPYADVIASSFGDLISKNMFGLLGHFLRVK HAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVK HAAKVYIEAFHTVPKGLCRQLVRAMAWLFFVSWGMFPVLFLLGPEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVK VLFFGFTSKAES.MRPEVASTFKVLRNVTVVLWSAYPVVWLIGSEGAGIVPLNIETLLFMVLDVSAKVGFGLILLRSRAI


IHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTV.... IHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTV.... IHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTV.... IHEHILLYGDIRKKQKVNVAGQEMEVETMVHEEDDETQKV.. IHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAV... IHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAV... IHEHILKHGDIRKTTHLRIAGEEKEVETFVEEEDED....TV IHEHILLYGDIRKKQKITIAGQEMEVETLVAEEEDDTVKQST IHEHILLYGDIRKKQKITIAGQEMEVETLVAEEEDK..YESS FGEAEAPEPSAGDGAAATSD......................

Supplementary Figure 1│Sequence alignment of C1Chrimson and ChR variants. Amino acid sequences of C1Chrimson, Chrimson (CnChR1), CsChrimson, CrChR1, CrChR2, C1C2, CaChR1, VChR1, ReaChR, and BR are aligned. Secondary structures of C1Chrimson are indicated above the sequences. The N-terminal disulfide bond-forming cysteine residues are indicated by yellow arrowheads. The completely conserved sequences are highlighted in cyan, and the conserved lysine residues forming a linkage to the chromophore retinal are indicated in the blue panels. Residues important for the red-shift absorption are indicated by arrow heads, according to their contribution manners: counterion protonation (orange) and polarity of the retinal binding pocket (pink). The position of the red-shift-causing point mutation (S169A) is indicated (red).

a

c mol A 90°

a

a

mol B b

b

b

c D322(A)

K325(A) N63(B)

N63(A)

C69(B)

C78(B)

C69(A)

C76(A)

C78(A)

C76(B)

Supplementary Figure 2│Crystal packing of C1Chrimson. a, The Chrimson proteins in the crystals are shown in ribbons, viewed from within the LCP crystal layer (left) and from the top (right). The a-, b- and c-axes are indicated by yellow arrows, and the two protomers (mol A and mol B) are shown. b, The N-terminal CrChR1-derived residues are involved in the crystal packing interactions between the layers. Disulfide bond-forming cysteine and hydrogen bonding residues are shown as sticks, with the hydrogen bonding interactions indicated by yellow dotted lines. The letters in brackets indicate the respective molecules (A and B for mol A and mol B, respectively). c, Two protomers are superimposed, and colored according to the distance of the Cα atoms between the protomers.

C224

C224 P269

P269

M201

M201 Y268

ATR

C170

C198 S169

Y268

ATR

C170

C198 D295

K299

S169

D295

K299

Supplementary Figure 3│Electron density of retinal. A stereo view of the 2Fo-Fc electron density map for the retinal binding pocket is shown as a mesh representation, contoured at 1.1 σ. The all-trans retinal (ATR) and the surrounding residues are indicated by sticks.

b

600 nm 250 pA

250

Normalized photocurrent

ns

***

100

C

n im

so

hr

im hr

so

n

n so

im

hr

C

C

C

im

hr

sC

so

im

im

hr

1C

so

sC

700

n

n

n

so

C

500 600 Wavelength (nm)

-1.0

0

0 400

30 Holding potential (mV)

-0.5

200

pHe 9.0 +30 mV

hr

0.0

pHe 7.2 +30 mV

500 100 ms

0.5

-30

e

pHe 7.2 -60 mV 750

0.5

-60

1C

650

250 pA

1.0

Normalized photocurent

d

400 C1Chrimson Chrimson CsChrimson

110 mM NaCl pHe9.0

30 mV 15 mV 0 mV -15 mV -30 mV -45 mV -60 mV

Photocurrent density (pA/pF)

c

1 mM NaCl pHe 7.2

τapparent, off (ms)

C1Chrimson

500 ms

110 mM NaCl pHe 7.2

1.0

110 mM NaCl pHe 7.2 1 mM NaCl pHe 7.2 110 mM NaCl pHe 9.0

C

a

Supplementary Figure 4│Photocurrents of Chrimson variants. a, Representative photocurrents of C1Chrimson at different voltages with different extracellular solutions of 110 mM extracellular NaCl and pHe 7.2 (left), 1 mM NaCl and pHe 7.2 (middle) and 110 mM NaCl and pHe 9.0 (right) with intracellular 110 mM NaCl pHi 7.2. b, The current-voltage dependence of the normalized peak photocurrents C170

of Chrimson under the ionic conditions described in (a) (Mean ± SD, n=5-16). c, Normalized peak photocurrents after 10 ms excitation at different wavelengths with equal photon count (Mean ± SD; n=6-10; Inlet: Representative photocurrents). d, Apparent off-kinetics (τapparent, off) at positive and negative voltages and pHe 7.2 and pHe 9.0 (Mean ± SD; n=7-16). (e) Head-to-head comparison of photocurrent amplitudes of the Chrimson variants at -60 mV in symmetric 110 mM NaCl and pHe,i 7.2 (Mean ± SD, n=5-7). Significance was assessed by t-tests using Welch's correction in Origin 9.1®, with significance thresholds of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). p=0.5 for C1Chrimson and CsChrimson and p=0.0003 for C1Chrimson and Chrimson.

Chrimson

C1C2

CrChR2

IC2

intracellular

IC2 IC2

inner gate

IC1

central gate

EC2 outer gate EC1

inner gate

inner gate

IC1 IC1

central gate

central gate

EC2

EC EG (extracellular gate) EC1

extracellular

Supplementary Figure 5│Comparison of the putative ion translocating pathway. Protein internal cavities are shown as blue surface representations, for Chrimson (left), C1C2 (center) and CrChR2 (right). By observing discontinuities and locations, we named these cavities according to the combination of extracellular cavity (EC) or intracellular cavity (IC) and the sequential numbering from the extracellular to the intracellular side. We observed three constrictions (inner, central and outer gates) and four cavities (EC1, EC2, IC1 and IC2) for Chrimson, two constrictions (inner and central gates) and three cavities (EC, IC1, and IC2) for C1C2, and three constrictions (inner, central and extracellular gates) and four cavities (EC1, EC2, IC1 and IC2) for CrChR2. Putative ion translocating pathways are indicated by navy arrows in each structure, and constriction sites are highlighted by circles.

a

b

pH5 pH6 pH7 pH8 pH9

1.0

relative absorbance

0.8

c

d

0.6 0.4

pH5

0.2 0 400

500 600 wavelength (nm)

pH6

pH7

f N H+ + OH O

N H+

or

O-

F135 Chrimson (low and neutral pH)

N H+ O-

O

N H+

O - D295 O

O-

O

E165 F135

K296 (K257)

K299

O - D295 O H+

E165

E165

g

K299

O - D295 O

pH9

700

e K299

pH8

F135

Chrimson (high pH)

E162 (E123)

O O O

D292 (D253)

+ NH3 K132 (K93)

C1C2, CrChR2

Supplementary Figure 6│pH-dependent peak shift and Schiff base environment. a-b, Absorption spectra of the purified wild type Chrimson protein measured at the respective pH conditions (a). The color of the retinal chromophore is highly dependent on the solution pH. The Chrimson protein is blue at a lower pH, and red at higher pH. c-d, Crystals of Chrimson were obtained under low pH conditions, and accordingly the color of the crystal was blue. e-g, Proposed hydrogen bonding networks around the protonated Schiff base for Chrimson at low to neutral pH (e) and at high pH (f) and in C1C2 and CrChR2 (g).

a

b pH5 pH6 pH7 pH8 pH9

Relative absorbance

0.6

0.8

500

d 1.0


650

450

500

e 1.0


650

0.8

1.0

500

g


650

650

0.2

450

500


650

700

500


650

700

0 400

450

500


650

M201T

Relative absorbance

0.6

0.6

0.4

0.4

0.2

0.2

450

500


650

700

0 400

700

pH5 pH6 pH7 pH8 pH9

E300Q

0.8

Relative absorbance

0.8

1.0


0.4

h

pH5 pH6 pH7 pH8 pH9

E143Q (E5)

0 400

700

500

0.8

0.2

450

450

f

0.6

0.4

0.2

0 400

700

pH5 pH6 pH7 pH8 pH9

E139Q (E4)

0.6

0.4

0 400

0.2

Relative absorbance

Relative absorbance

0.6

1.0

0 400

700

pH5 pH6 pH7 pH8 pH9

E132Q (E3)

0.8

0 400

0.8

0.2

450

pH5 pH6 pH7 pH8 pH9

D295N (Ci2)

0.4

0.4

0.2

1.0

0.6

0.6

0.4

0 400

c pH5 pH6 pH7 pH8 pH9

E165Q (Ci1)

Relative absorbance

0.8

1.0

Relative absorbance

E165A (Ci1)

Relative absorbance

1.0

450

Supplementary Figure 7│Absorption spectra of Chrimson mutants. a-h, Absorption spectra of Chrimson mutants measured at different pH conditions are shown. Gray dotted lines indicate the absorption spectrum of wild type Chrimson at pH 7. The mutations of E165, E132, E139 and E300 all caused large blue-shifted absorption, while the mutation of E143 did not affect either the absorption spectrum or pH-dependency. The M201T mutant showed the largest blue-shift among the tested mutants.

700

a 540 nm

E132Q

E165T

E165D

C198A

Y220F

S223G

Y268F

D295N

M230T

W272F

D295E

Y231F

N287A

250 pA

580 nm

1s

30 mV E165Q

b

M201N

A298S

-60 mV

d

300

E132Q R162H 200

R162K E165D E165Q

C198A M201T

A298S

D295E

N287E

D295N

E277A

N287A

Y268F

W272F

Y231F

M230T

Y220F

S223G

M201T

M201N

C198A

C198D

C170A

E165T

S169A

E165Q

C198D R162K

104

C170A

E165D

c

S169A

R162H

WT

0

R162A non functional

E165T

100

E132Q

Photocurrent density (pA/pF)

WT

M201N Y220F S223G Y231F

103

Y268F W272F E277A

10

N287A

2

N287E D295N D295E

10 D295E

D295N

N287E

N287A

E277A

W272F

M230T

Y220F

E165T

E165Q

E165D

R162K

R162H

E132Q

A298S WT

τapparent,off, -60 mV (ms)

M230T

500

550

600

Peak activity wavelength (nm)

Supplementary Figure 8│Photocurrent traces and channel kinetics of mutant Chrimson. a, Representative photocurrent traces of Chrimson mutants in symmetric 110 mM NaCl pHi,e 7.2 at different voltages, illuminated with either 530 nm light (green line) or 580 nm light (orange line), depending on the peak absorption. b–d, Characterization of Chrimson mutants. Photocurrent density (b), channel closing kinetics at -60 mV (c) and peak activity wavelength (Mean ± SD) (d) are shown.

0.8 0.6

wt(pH7)

b

0.4

1.0

WT (n=4) S169A (n=4)

0.8 0.6 0.4 0.2

0.2

450

500 550 600 Wavelength (nm)

635 nm, 10 ms

20 mV

WT S169A

200 ms

0.0 300

650

700

150

current light

400


d 150

100

700

current light

100

dV/dt (V/s)

0 400

c

pH5 pH6 pH7 pH8 pH9

S169A

Relative response

1.0

dV/dt (V/s)

Relative absorbance

a

50 WT

0 -40

0 Voltage (mV)

50 S169A

0 40

-40

0 Voltage (mV)

40

Supplementary Figure 9│pH-dependency and neuronal characterization of ChrimsonSA. a, Absorption spectra of ChrimsonSA, measured under different pH conditions. Gray dotted lines indicate the absorption spectrum of wild type Chrimson at pH 7. b, Peak-normalized photocurrents in neurons expressing either WT Chrimson (black) or Chrimson-S169A (red, Mean ± SEM). c, Single action potentials in neurons expressing either WT Chrimson (black) or ChrimsonSA (red). d, Phase plots of action potentials evoked with 300 pA current injections (dashed lines) or with 8-ms red light pulses (solid lines) in neurons expressing either WT Chrimson (black) or ChrimsonSA.

Supplementary Table 1 | Data collection and refinement statistics

Chrimson BL32XU P212121 13

Data collection Space group Number of crystals Cell dimensions a, b, c (Å) α, β, γ (°)

60.99,81.44, 170.14 90.0, 90.0, 90.0

Wavelength Resolution (Å) Rmeas CC1/2 I / σI Completeness (%) Redundancy

1.0000 50-2.60 (2.69-2.60) 0.144 (0.591) 0.980 (0.595) 13.8 (1.7) 100.0 (100.0) 9.3 (9.2)

Refinement Resolution (Å) 50-2.60 (2.69-2.60) No. reflections 26744 (2618) Rwork / Rfree 0.229/0.277 (0.313/0.358) No. atoms Protein 4410 Water/Ion/Lipid 411 B-factors Protein 85.6 Water/Ion/Lipid 80.8 R.m.s. deviations Bond lengths (Å) 0.005 0.96 Bond angles (°) Ramachandran plot Favored (%) 95.6 Allowed (%) 4.4 Disallowed (%) 0 *Values in parentheses are for highest-resolution shell.