Table 1 In vitro properties of purified NTnC compared to GCaMP6s.

		Proteins
		NTnC		GCaMP6s
Properties		apo	sat	apo	sat
Absorbance maximum (nm)		505		402	500
Emission maximum (nm)		518		518	515
Quantum yield ^a		0.71 ± 0.05	0.65 ± 0.04	0.11 ± 0.01	0.61
ε (mM⁻¹ cm⁻¹) ^b		108 ± 6	52 ± 1 ^c	33.3 ± 0.6	77 ± 3
Brightness (%)		163	72	7.8	100
Fluorescence contrast (fold)	0 mM Mg²⁺	2.0 ± 0.3		44 ± 6
Fluorescence contrast (fold)	1 mM Mg²⁺	2.0 ± 0.7		47 ± 24
pKa		6.09 ± 0.07	6.08 ± 0.02	9.6 ± 0.3	6.16 ± 0.08
K_d (nM)	0 mM Mg²⁺	84 ± 6 [n = 1.9 ± 0.1] ^d		144 ± 9 [n = 4.0 ± 0.6] ^d
K_d (nM)	1 mM Mg²⁺	192 ± 40		227.3 ± 0.2
K_d^kin (nM) ^e		94 ± 9 [n = 2.3 ± 0.1]		152 ± 20 [n = 3.2 ± 0.1]
k_on (s⁻¹ × M⁻ⁿ) ^e		6 × 10¹⁵		6 × 10²¹
k_off (s⁻¹) ^f		0.8 ± 0.1; 0.05 ± 0.01 ^g		0.8 ± 0.1
k^onset _limit (s⁻¹) ^h		50 ± 20		270 ± 50
Protein state		monomer		monomer
Maturation half-time (min) ⁱ		23	28	ND	ND
Photobleaching half-time (sec) ^j		40 ± 8	70 ± 5	111 ± 15	186 ± 13

^aGCaMP6s in the saturated state (QY = 0.61 ref. 14) and mTagBFP2 (QY = 0.64 ref. 28) were used as reference standards for 500- to 505- and 402-nm absorbing states, respectively.
^bExtinction coefficient was determined by alkaline denaturation.
^cExtinction coefficient was estimated relative to apo NTnC with the same concentration.
^dHill coefficient is shown in square brackets.
^eK_d^kin, Hill coefficients and k_on values were obtained via fitting the observed association rates (Supplementary Fig. 8a) at 0–350 nM Ca²⁺ concentrations to the equation k_obs = k_on × [Ca²⁺]ⁿ + k_off (Fig. 2d). K_d^kinetic = (k_off/k_on)^1/n. Hill coefficients are shown in square brackets.
^fRefined k_off values were determined from the dissociation kinetics records (Supplementary Fig. 8b).
^gUnlike GCaMP6s kinetics, NTnC kinetics do not agree with the two-state model. NTnC kinetic curves were fitted to double exponentials. k_off values were estimated from double exponential decay with individual exponent contributions of 0.48:0.52.
^hk^onset _limit values are saturation levels of the observed association rates (at >600–800 nM Ca²⁺ concentrations; Supplementary Fig. 9).
ⁱmEGFP had a maturation half-time of 14 min; we could not estimate the maturation rate for GCaMP6s because of its low expression level in bacteria.
^jmEGFP had a photobleaching half-time of 170 ± 20 sec.

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