C1Chrimson. Chrimson. CsChrimson. ChR1. ChR2. C1C2. CaChR1. VChR1. ReaChR. BR. 80. 90. 100. 110. 120. 130. 140. 150. C1Chrimson. L. V. Chrimson.
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
C1Chrimson Chrimson CsChrimson ChR1 ChR2 C1C2 CaChR1 VChR1 ReaChR BR
QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK QAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIK NAAKVYIEAYHTVPKGICRDLVRYLAWLYFCSWAMFPVLFLLGPEGFGHINQFNSAIAHAILDLASKNAWSMMGHFLRVK HAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVL NAAKVYIEAYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVL LAAKVLIESFHQVPKGICRHLVKAMAITYYVGWSFFPLIFLFGQSGFKKISPYADVIASSFGDLISKNMFGLLGHFLRVK HAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVK HAAKVYIEAFHTVPKGLCRQLVRAMAWLFFVSWGMFPVLFLLGPEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVK VLFFGFTSKAES.MRPEVASTFKVLRNVTVVLWSAYPVVWLIGSEGAGIVPLNIETLLFMVLDVSAKVGFGLILLRSRAI
C1Chrimson Chrimson CsChrimson ChR1 ChR2 C1C2 CaChR1 VChR1 ReaChR BR
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
550 600 Wavelength (nm)
650
450
500
e 1.0
550 600 Wavelength (nm)
650
0.8
1.0
500
g
550 600 Wavelength (nm)
650
650
0.2
450
500
550 600 Wavelength (nm)
650
700
500
550 600 Wavelength (nm)
650
700
0 400
450
500
550 600 Wavelength (nm)
650
M201T
Relative absorbance
0.6
0.6
0.4
0.4
0.2
0.2
450
500
550 600 Wavelength (nm)
650
700
0 400
700
pH5 pH6 pH7 pH8 pH9
E300Q
0.8
Relative absorbance
0.8
1.0
550 600 Wavelength (nm)
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
500 600 Wavelength (nm)
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.