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N501K E514D V16E L463F T467S S33N E43D E43D E16V S33N S90T M113L F463L S467T R509K D514E S33N H35Q N33S D43E E16V D514E N104T V16E L270F L463F T467S N196D L463F I224M F463L S467T T104S T104S N196D N196D S467T S104T N196D N196D N334S S104T D196N D196N N334S S104T S467T D196N S334N S334N M224I T104S Q78R D196N M224I D196N F463L D196N T467S T104S T467S Q206R S252N K352R R273H P75Q S252N S252N R273H N254I R273H S104T Q78R S104T R206Q R206Q R273H R78Q R78Q T104S N196D I224M N252S R352K P75Q R352K R273H R352K K202Q H273R Q206R H273R P75Q S252N S252N N254I K343E S252N D385E N254I G333T N254I D196W N254I S252N N254I Q34R R273H Q75P N252S Q75P K352R R273H Q75P R273H K222R R273H R273H K319R V182S R273H D196W R273H Q34R Q75P N252S H273R Q75P N153S D385E K222R N254I K343E Q75P D385E K319R Q75P N254I V182S D196W Q34R P75Q N252S H273R H273R K352R N252S P75Q H273R P75Q E277Q E381V P75Q K222R K202Q D385E D385E P75Q K319R N252S N254I N254I V23D N254I N254I G373H S33G N254I Q34G N254I Q34R N254I Y82V N254I N254I L463W P75Q V182S P75Q D196W Q277E V381E S153N E385D Q75P S153N S33G Q34G P75Q P75Q G373H I254N E343K P75Q S252N P75Q P248E S252N G333T E343K E343K D196W E343K S252N E343K V182S Q202K Q34R R222K R222K H273R R222K Q75P R222K R222K K319R V182S R222K D196W R222K Q34G N252S N252S G373H I254N E343K Q75P N252S N252S Q75P P248E Q75P E343K Q202K E385D Q202K N252S E385D P75Q E385D E385D P75Q N153S K202Q N254I E385D Q34R R319K H273R R319K R319K Q75P R319K K222R R319K V182S R319K D196W R319K S33G Q75P Q34G G373H Q75P S252N Q75P E343K Q75P V182S E248P N252S P75Q E248P E248P E343K V182S I254N T333G T333G K343E T333G M224I T333Q D196W T333G S252N T333G V23K V23A V23D S33G S33G Q34R Q34R Q34G S33G Q34G V23D V311F Y82V G373H L463W V23K K193L V23K F245G V23K F28G V23K H273G V23K V311F V23K S33G V23K L463F V23K Q34R V23A V311F V182S V23D V182S V23R Q34G V182S S33G K343E I254N H273R I254N S252N I254N K343E P75Q K343E G333T M224I G333Q D196W S252N V23K G333T V23A G333T V23D G333T V23D S252N S33G G333T Q34R G333T Q34G G333T S252N G373H S33G S252N Q34G S252N V182S K343E Y82V G333T G333T G373H G333T L463W Q34R S252N Y82V S252N S252N L463W V182S G333T V23R G333T I224M Q333T I224M Q333G V23K V23R V23D V23D V311F S33G Q34G S33G Q34G Q34R Y82V G373H L463W S33G Q34R V182S V311F V182S W196D I254N W196D K343E W196D G333T W196D W196D S252N N252S I254N P75Q I254N I254N N252S K343E P75Q K343E I254N D385E P248E K343E N252S G333T N252S D196W N252S V23D S33G Q34R G373H S33G S33G Q34G Q34G Q34R Y82V L463W V23D V311F H273R G373H S33G G373H V23D G373H N334I G373H G373H L463F Q34R G373H V23D V182S K23V K23V T333G M224I T333Q K23A K23D K23V S33G K23V Q34R K23V Q34G K23D V311F K23V Y82V K23V G373H K23V L463W K193L F245G F28G H273G V311F S33G L463F Q34R K23A V311F K23V V182S K23D V182S K23R K23V I224M Q333T K23R K23D K23D V311F K23V S33G K23V Q34G K23V Q34R K23V Y82V K23V G373H K23V L463W K23V V182S A23V A23V T333G A23K A23D A23V S33G A23V Q34R A23V Q34G A23D V311F A23V Y82V A23V G373H A23V L463W A23K K193L A23K F245G A23K F28G A23K H273G A23K V311F A23K S33G A23K L463F A23K Q34R V311F A23V V182S A23D V182S A23R R23V R23K R23D R23D V311F R23V S33G R23V Q34G R23V Q34R R23V Y82V R23V G373H R23V L463W R23V V182S I224M Q333T D23V D23V T333G D23K D23A S252N T333G M224I T333Q D23V S33G D23V Q34R D23V Q34G V311F D23V Y82V D23V G373H D23V L463W D23K K193L D23K F245G D23K F28G D23K H273G D23K V311F D23K S33G D23K L463F D23K Q34R V311F T333Q D23A V311F D23V V182S V182S D23R D23V I254N D23V N252S D23V N252S G333T D23V G373H D23V S33G D23V Q34G D23V Q34R D23V Y82V D23V L463W V311F G373H V182S D23V D23K D23R I224M Q333T V311F D23V S33G D23V Q34G D23V Q34R D23V Y82V D23V G373H D23V L463W I224M V311F D23V V182S T333G K343E G33S G33S T333G V23K G33S V23A G33S V23D G33S Q34R G33S Q34R G33S Q34G S252N T333G Q34G G33S Y82V G33S G373H G33S L463W M224I T333Q V23K G33S V182S V23R G33S Q34R V182S G33S R34Q R34Q G33S G33S R34G S252N T333G R34G M224I T333Q V23K R34Q V23K G33S V182S R34Q R34Q T333G V23K R34Q V23A R34Q V23D R34Q S33G R34Q S33G R34G S33G R34G R34Q Y82V R34Q G373H R34Q L463W M224I T333Q S252N T333G V23K R34Q V182S V23R R34Q R34G V182S S33G V182S G34Q G34Q T333G V23K G34Q V23A G34Q V23D G34Q S33G G34Q S33G G34R G34R S252N T333G S33G G34Q Y82V G34Q G373H G34Q L463W M224I T333Q V23K G34R G34Q V182S V23R G34Q V182S G33S R34Q N252S G333T R34Q R34G G33S R34G G33S R34Q G373H G33S G373H I254N H373G N252S H373G H373G V23D H373G S33G H373G Q34G H373G N252S G333T Q34R H373G Y82V H373G H373G L463W H273R S33G V23D N334I L463F Q34R N252S K343E G33S I254N G33S N252S G33S V23D G33S N252S G333T Q34R G33S G373H Q34G G33S Q34G G33S Q34R G33S Y82V G33S L463W G373H G33S G34Q G34R G34Q G33S N252S G333T G33S G34R G34Q G373H G34Q I254N G34Q N252S G34Q V23D G34Q N252S G333T S33G G34R G34Q G373H S33G G34Q S33G G34R G34Q Y82V G34Q L463W G34R G373H G33S G34Q G34Q G34R G33S G34R G33S S252N T333G M224I T333Q V23K G34Q G33S V182S G34R V182S S33G S182V S182V P75Q S182V S182V E343K P248E S252N G333T E343K S182V E248P N252S D23V F311V D23K F311V D23A F311V F311V M224I T333Q S252N T333G D23K T333Q D23A V182S F311V D23R F311V D23V F311V D23K F311V D23R F311V F311V I224M Q333T D23V V182S I224M V82Y V82Y T333G V23K V82Y V23A V82Y V23D V82Y S33G V82Y Q34R V82Y Q34G V82Y V82Y G373H V82Y L463W M224I T333Q S252N T333G V82Y V182S V23R V82Y H373G T333G H373G V23K H373G V23A H373G V23D H373G S33G H373G Q34R H373G Q34G H373G S252N T333G Y82V H373G H373G L463W M224I T333Q V182S H373G V23R H373G W463L T333G W463L V23K W463L V23A W463L V23D W463L S33G W463L Q34R W463L Q34G W463L Y82V W463L G373H W463L M224I T333Q S252N T333G V23K W463F V182S W463L V23R W463L G33S V23K G33S V23R G33S V23D G33S I224M Q333T G33S Q34G Q34G G33S Q34R G33S Y82V G33S G373H G33S L463W Q34R G33S V182S G34Q V23K G34Q V23R G34Q V23D G34Q I224M Q333T S33G G34Q S33G G34R G34Q Y82V G34Q G373H G34Q L463W S33G G34R G34Q V182S G33S G34Q I224M Q333T G34Q G33S G33S G34R G34R R34Q V23K R34Q V23R R34Q V23D R34Q I224M Q333T S33G R34Q R34G S33G R34G R34Q Y82V R34Q G373H R34Q L463W S33G R34Q V182S V82Y V23K V82Y V23R V82Y V23D V82Y I224M Q333T S33G V82Y Q34G V82Y Q34R V82Y V82Y G373H V82Y L463W V82Y V182S H373G V23K H373G V23R H373G V23D H373G I224M Q333T S33G H373G Q34G H373G Q34R H373G Y82V H373G H373G L463W V182S H373G W463L V23K W463L V23R W463L V23D W463L I224M Q333T S33G W463L Q34G W463L Q34R W463L Y82V W463L G373H W463L V182S W463L G33S R34Q I224M Q333T R34Q G33S R34G R34G G33S P75Q I254N R34Q I254N R34Q N252S R34Q V23D R34Q N252S G333T S33G R34Q G373H S33G R34Q S33G R34G R34G R34Q Y82V R34Q L463W G373H V82Y I254N V82Y N252S V82Y V23D V82Y V82Y G373H S33G V82Y Q34G V82Y N252S G333T Q34R V82Y V82Y L463W I254N W463L N252S W463L W463L V23D W463L G373H W463L S33G W463L Q34G W463L N252S G333T Q34R W463L Y82V W463L G373H W463F Q34R S182V S182V H273R S182V Q75P S182V S182V K222R S182V K319R S182V D196W Q34R W196D W196D H273R W196D Q75P W196D W196D K222R W196D K319R V182S W196D S182V F311V V23D S182V F311V D23V F311V F311V N252S G333T F311V G373H N252S G333Q V182S F311V I224M M224I K23V L193K L193K K23A L193K K23D L193K L193K F245G F28G L193K L193K H273G L193K V311F S33G L193K L193K L463F Q34R L193K K23R L193K K23V G245F G245F K23A G245F K23D G245F K193L G245F F28G G245F G245F H273G G245F V311F S33G G245F G245F L463F Q34R G245F K23R G245F K23V G28F G28F K23A G28F K23D G28F G28F K193L G28F F245G G28F H273G G28F V311F G28F S33G G28F L463F G28F Q34R K23R G28F K23V G273H G273H K23A G273H K23D G273H K193L G273H F245G G273H F28G G273H G273H V311F S33G G273H G273H L463F Q34R G273H K23R G273H K23V F311V F311V K23A F311V K23D F311V K23D K193L F311V F245G F311V F28G F311V H273G F311V S33G F311V F311V L463F Q34R F311V K23D T333Q K23A K23R F311V K23V G33S G33S K23A G33S K23D G33S K23V K23V Q34R K23V Q34G G33S K193L G33S F245G F28G G33S G33S H273G G33S V311F G33S L463F G33S Q34R K23R G33S K23V F463L F463L K23A F463L K23D F463L K23V F463W K193L F463L F245G F463L F28G F463L H273G F463L V311F F463L S33G F463L Q34R F463L K23R F463L K23V R34Q R34Q K23A R34Q K23D R34Q K23V S33G K23V K23V R34G R34Q K193L R34Q F245G F28G R34Q R34Q H273G R34Q V311F S33G R34Q R34Q L463F K23R R34Q R273H H373G R273H S33G R273H V23D R273H R273H N334I R273H L463F Q34R R273H G33S H373G Q34R H373G G33S H373G Q34G H373G G33S H273R V23D G33S G33S N334I G33S L463F G33S Q34R D23V H373G H373G D23V V311F H373G D23V H273R D23V S33G D23V N334I D23V L463F D23V Q34R V182S H373G I334N H373G I334N H273R I334N S33G I334N V23D I334N I334N L463F Q34R I334N H373G F463L F463L H373G F463W H273R F463L S33G F463L V23D F463L N334I F463L Q34R F463L R34Q H373G S33G H373G R34Q R34G H373G H373G R34Q H273R S33G R34Q V23D R34Q R34Q N334I R34Q L463F F311V Q333T M224I F311V Q333T M224I S252N Q333G D23K Q333T D23A Q333T A23V F311V A23K F311V F311V A23D F311V A23D A23K A23D T333Q A23R F311V S182V V23K S182V V23R S182V V23D S182V S33G S182V Q34G S182V Q34R S182V Y82V S182V S182V G373H S182V L463W V311F I224M Q333T D23V S182V S182V S182V V311F S182V G373H N252S G333T S182V S182V T333G V23K S182V V23A S182V V23D S182V S33G S182V Q34R S182V Q34G S182V T333G K343E Y82V S182V S182V G373H S182V L463W M224I T333Q V23D V23R S182V Q34G S33G Q34R D23V S182V D23K S182V D23A S182V S182V S182V V311F S252N T333G D23V D23R S182V D23V Q34G R23V R23V T333G R23K R23A M224I T333Q R23D R23V S33G R23V Q34R R23V Q34G R23D V311F R23V Y82V R23V G373H R23V L463W R23K K193L R23K F245G R23K F28G R23K H273G R23K V311F R23K S33G R23K L463F R23K Q34R R23A V311F R23V V182S R23D V182S G34Q S182V G34R S182V S182V S33G S182V G34Q V23D G34Q S33G G34R R34Q S182V S182V G33S S182V R34G S182V G33S R34Q G33S R34G Wildtype [0] (80.0 um) 1E3 [1] (0.0 um) 1A2 [2] (0.0 um) 1A4 [3] (0.0 um) 1E8 [4] (0.0 um) 2G6 [5] (0.0 um) 2H6 [6] (0.0 um) 3A1 [7] (0.0 um) 3A2 [8] (0.0 um) 3B1 [9] (0.0 um) 3C1 [10] (0.0 um) 3D8 [11] (0.0 um) 3A10 [12] (0.0 um) 3F3 [13] (0.0 um) 4A1 [14] (0.0 um) 4B5 [15] (0.0 um) 4C3 [16] (0.0 um) 4C5 [17] (0.0 um) 4D4 [18] (0.0 um) 4D10 [19] (0.0 um) 4G4 [21] (0.0 um) 8F11 [23] (0.0 um) 8F9 [24] (0.0 um) 8F1 [25] (0.0 um) 8E10 [26] (0.0 um) 9D3 [27] (0.0 um) 9D11 [28] (0.0 um) 9D7 [29] (0.0 um) 9B6 [30] (0.0 um) 11F12 [31] (0.0 um) 11H1 [32] (0.0 um) 11G1 [33] (0.0 um) 11G8 [34] (0.0 um) 12H4 [35] (0.0 um) 12H11 [36] (0.0 um) 13C6 [37] (0.0 um) 13G3 [38] (0.0 um) 13H12 [39] (0.0 um) 13C2 [40] (0.0 um) 15E11 [43] (0.0 um) 15D6 [44] (0.0 um) 16B5 [46] (0.0 um) 16G11 [47] (0.0 um) 17G11 [48] (0.0 um) 18B5 [49] (0.0 um) 19C1 [51] (0.0 um) 19C12 [52] (0.0 um) 20F3 [54] (0.0 um) 20C7 [55] (0.0 um) 21G11 [56] (0.0 um) 21F10 [57] (0.0 um) 21G6 [58] (0.0 um) 21C5 [59] (0.0 um) 21D8 [60] (0.0 um) 22C8 [61] (0.0 um) 22A7 [62] (0.0 um) 22H10 [63] (0.0 um) 23C1 [64] (0.0 um) 23D12 [65] (0.0 um) 23F10 [66] (0.0 um) 23F11 [67] (0.0 um) 23B5 [68] (0.0 um) 24B8 [69] (0.0 um) 24F5 [71] (0.0 um) 24H9 [72] (0.0 um) 24C9 [73] (0.0 um) 24D11 [75] (0.0 um) 24D12 [76] (0.0 um) 25B5 [77] (0.0 um) 25C10 [78] (0.0 um) 25B4 [79] (0.0 um) 25G3 [80] (0.0 um) 26E7 [82] (0.0 um) 27B8 [83] (0.0 um) 27B12 [84] (0.0 um) 28G9 [85] (0.0 um) 29E8 [86] (0.0 um) 29A8 [87] (0.0 um) 34H9 [88] (0.0 um) 36G8 [89] (0.0 um) 37D5 [91] (0.0 um) 37B1 [92] (0.0 um) 37G9 [93] (0.0 um) 37H4 [94] (0.0 um) 37E3 [95] (0.0 um) 33D12 [96] (0.0 um) 38B6 [97] (0.0 um) 38F10 [98] (0.0 um) 38A12 [99] (0.0 um) 38B5 [100] (0.0 um) 39F1 [101] (0.0 um) 39H6 [102] (0.0 um) 39F4 [104] (0.0 um) 40A10 [105] (0.0 um) 40B8 [107] (0.0 um) 40F11 [108] (0.0 um) 40D8 [110] (0.0 um) 41C8 [113] (0.0 um) 41H5 [115] (0.0 um) 41A12 [116] (0.0 um) 42A11 [117] (0.0 um) 42F10 [118] (0.0 um) 42D1 [119] (0.0 um) 43E7 [120] (0.0 um) 43B7 [121] (0.0 um) 43C12 [122] (0.0 um) 43H4 [123] (0.0 um) 43A4 [124] (0.0 um) 43D10 [125] (0.0 um) 43A3 [126] (0.0 um) 40F1 [127] (0.0 um) 41A8 [128] (0.0 um) 44A12 [130] (0.0 um) 44C3 [131] (0.0 um) 45B8 [132] (0.0 um) 46E6 [133] (0.0 um) 47E12 [134] (0.0 um) 44A1 [135] (0.0 um) 48A8 [136] (0.0 um) 48D10 [137] (0.0 um) 49H8 [138] (0.0 um) 49H10 [139] (0.0 um) 50E11 [140] (0.0 um) 50A2 [141] (0.0 um) 50B11 [142] (0.0 um) 50F7 [143] (0.0 um) 50A11 [144] (0.0 um) 51A8 [145] (0.0 um) 52E11 [146] (0.0 um) 52G12 [147] (0.0 um) 53G10 [149] (0.0 um) 54C6 [150] (0.0 um) 54F6 [152] (0.0 um) 54D1 [154] (0.0 um) 57D2 [162] (0.0 um) 57D4 [166] (0.0 um) 58G11 [167] (0.0 um) 58H9 [168] (0.0 um) 58D3 [169] (0.0 um) 60B3 [170] (0.0 um) 61A4 [171] (0.0 um) 61B4 [172] (0.0 um) 62D9 [173] (0.0 um) 62F8 [174] (215.0 um) 63A10 [175] (0.0 um) 66H7 (Sample 1) [180] (0.0 um) 66D11 (Sample 6) [184] (0.0 um) 68H5 (Sample 26) [187] (0.0 um) 68H6 (Sample 27) [188] (0.0 um) 71B10 (Sample 49) [191] (0.0 um) 67C1 (Sample 12) [194] (0.0 um) 70H5 (Sample 38) [204] (0.0 um) 84D11 (Hybrid) [205] (0.0 um) Combo 1A1 [206] (209.1 um) Combo 2G1 [207] (0.0 um) Combo 2F3 [208] (32.45 um) Combo 2F1 [210] (53.88 um) Combo 4A7 [212] (130.4 um) Combo 1F1_E23K [213] (176.4 um) Combo 2F3_E23K [214] (25.87 um) Combo 1F1_E23A [215] (132.9 um) Combo 2F3_E23R [216] (33.86 um) 1F1_E23D [217] (870.0 um) 4A7_E23D [218] (1700.0 um) 2F3_E23D [219] (33.3 um) 1F1_S33G [220] (918.2 um) 1F1_S33G_Q34R [221] (599.9 um) 1F1_Q34R [222] (0.0 um) 1F1_Q34G [223] (1801.0 um) 4A7_S33G_Q34R [224] (403.9 um) 4A7_G373H [225] (106.0 um) 4A7_S33G [226] (319.1 um) 4A7_S33G_Q34G [227] (335.9 um) 4A7_Q34G [228] (243.0 um) 1F1_S33G_Q34G [229] (462.1 um) Sample 52_A184S [230] (32.95 um) Hybrid_A184S [231] (73.33 um) 1F1_E23D_V311F [232] (1044.0 um) 2F3_E23D_V311F [233] (105.0 um) 1F1_Y82V [234] (0.0 um) 1F1_G373H [235] (0.0 um) 1F1_L463W [236] (0.0 um) 2F3_S33G [237] (0.0 um) 2F3_Q34G [238] (0.0 um) 2F3_S33G_Q34G [239] (0.0 um) 2F3_Q34R [240] (0.0 um) 2F3_Y82V [241] (0.0 um) 2F3_G373H [242] (0.0 um) 2F3_L463W [243] (0.0 um) 2F3_S33G_Q34R [244] (0.0 um) 4A7_Q34R [245] (0.0 um) 4A7_Y82V [246] (0.0 um) 4A7_L463W [247] (0.0 um) Sample 23_A184S [248] (0.0 um) Sample 23_N196W [249] (0.0 um) 2F3_A184S_V311F [250] (1.244 um) 4A7_E23D_V311F [251] (481.5 um) 2F3_trunc_332_335 [252] (1.11 um) 1F1_trunc_332_335 [253] (7.457 um) 1F1_E23K_K193L (103E4) [254] (0.0 um) 1F1_E23K_F245G (103E5) [255] (0.0 um) 1F1_E23K_F28G (103E6) [257] (0.0 um) 1F1_E23K_R273G (103E7) [258] (0.0 um) 1F1_E23K_V311F (103D7) [260] (0.0 um) 1F1_E23K_S33G (103D8) [261] (0.0 um) 1F1_E23K_F463F (103E2) [263] (0.0 um) 1F1_E23K_Q34R (103E3) [264] (0.0 um) 4A7_G373H_R273R (103D1) [266] (0.0 um) 4A7_G373H_N33G (103D2) [267] (0.0 um) 4A7_G373H_E23D (103D3) [268] (0.0 um) 4A7_G373H_I334I (103D4) [269] (0.0 um) 4A7_G373H_F463F (103D5) [270] (0.0 um) 4A7_G373H_Q34R (103D6) [271] (0.0 um) 1F1_E23D_V311F_G333Q (103E8) [272] (0.0 um) 1F1_V311F_E23A (103F1) [273] (0.0 um) 2F3_A184S [274] (0.0 um) 4A7_E23D_A184S [275] (0.0 um) 1F1_A184S [276] (0.0 um) 1F1_E23D_A184S [277] (0.0 um) 1F1_E23R [278] (0.0 um) 1F1_Q34G_A184S [279] (0.0 um) 1F1_S33G_Q34R_A184S [280] (0.0 um) 0 50 100 150 200 250 Var# Variant number 0 50 100 150 200 250 Var# Variant number 0 10 20 30 40 50 60 dF/F fluorescence response 0 10 20 30 40 50 60 dF/F fluorescence response 0 200 400 600 800 1000 1200 1400 1600 1800 Kd binding affinity 0 200 400 600 800 1000 1200 1400 1600 1800 Kd binding affinity 0 100 200 300 400 500 600 Kon response rise time, 1/sec 0 100 200 300 400 500 600 Kon response rise time, 1/sec 0 1 2 3 4 dF1 change in F, 1 action potential 0 1 2 3 4 dF1 change in F, 1 action potential 0 100 200 300 400 500 600 Dk1 decay in F after 1 action potential 0 100 200 300 400 500 600 Dk1 decay in F after 1 action potential 0 2 4 6 8 10 12 14 dF20 change in F after 20 action potentials 0 2 4 6 8 10 12 14 dF20 change in F after 20 action potentials 0 200 400 600 800 1000 Dk20 decay in F after 20 action potentials 0 200 400 600 800 1000 Dk20 decay in F after 20 action potentials 0 2 4 6 8 Fitn estimate of fitness based on dF/F and Kon 0 2 4 6 8 Fitn estimate of fitness based on dF/F and Kon Color -> Kd Click on mutations (edges) to toggle identical ones w/ other bg If things get confusing, reload (F5) Data / protein engineering / etc by Abhi Aggarwal and Kaspar Podgorski. Programming & analysis by Tim Hanson.