Table 1. List of parameters used in the model.

Estimated parameters for the N lobe (site: a,b)		Estimated parameters for the C lobe (site: c,d)
Allosteric parameters:
cN	0.01	cC	5 ⋅ 10−5
LN	299	LC	195865
Dissociation constants of calcium from calcium lobes:
KNT	9.38 ⋅ 10−5 M	KCT	9.38 ⋅ 10−5 M
KNR	KNT × cN = 9.38 ⋅ 10−7 M	KCR	KCT × cC = 4.69 ⋅ 10−9 M
Calcium binding rates:
kon_N	1.22 ⋅ 1010 M−1 ⋅ s−1	kon_C	1.22 ⋅ 108 M−1 ⋅ s−1
Calcium dissociation rates:
koff_NT	1.14 ⋅ 106 s−1	koff_CT	1.14 ⋅ 104 s−1
koff_NR	1.14 ⋅ 104 s−1	koff_CR	0.6 s−1
CaM state transition rates (0,1,2: number of calcium ions bound to that lobe):
k_TtoR_N0	316227 s−1	k_TtoR_C0	316227 s−1
k_RtoT_N0	k_TtoR_N0 ⋅ LN ≈ 9.46 ⋅ 107 s−1	k_RtoT_C0	k_TtoR_C0 ⋅ LC ≈ 6.2 ⋅ 1010 s−1
k_TtoR_N1	$k\_TtoR\_N0/\sqrt{cN}\approx 3.16\cdot {10}^6{s}^{-1}$	k_TtoR_C1	$k\_TtoR\_C0/\sqrt{cC}\approx 4.47\cdot {10}^7{s}^{-1}$
k_RtoT_N1	$k\_RtoT\_N0\cdot \sqrt{cN}\approx 9.46\cdot {10}^6{s}^{-1}$	k_RtoT_C1	$k\_RtoT\_C0\cdot \sqrt{cC}\approx 4.38\cdot {10}^8{s}^{-1}$
k_TtoR_N2	k_TtoR_N0/cN ≈ 3.16 ⋅ 107 s−1 s−1	k_TtoR_C2	k_TtoR_C0/cC ≈ 6.32 ⋅ 109 s−1
k_RtoT_N2	k_RtoT_N0 ⋅ cN ≈ 9.46 ⋅ 105 s−1	k_R2T_C2	k_R2T_C0 ⋅ cC ≈ 3.1 ⋅ 106 s−1
Parameters relating to CaM-binding substrates:
k_CaMKII_autop	adjusted_coefficient × 6.3 s−1	This is adjusted at each time step during simulation. Base rate is from [62].
koff_CaMKII_RR	1.1 s−1	CaMKII’s dissociation rate from active CaM [58].
kon_CaMKII_RR	2.87 ⋅ 106 M−1 ⋅ s−1	Estimated CaMKII’s binding rate to active CaM
koff_CaMKIIp_RR	8.7 ⋅ 10−5 s−1	Thr286-phospho-CaMKII’s dissociation rate from active CaM [58]
kon_CaMKIIp_RR	3.85 ⋅ 107 M−1 ⋅ s−1	Estimated Thr286-phospho-CaMKII’s binding rate to active CaM
kon_CaN_Ca1, 2, 3, 4	2 ⋅ 107 M−1 ⋅ s−1	CaN B’s binding rate to Ca [10]
koff_CaN_Ca	0.0092 s−1	[10]
koff_CaN_Ca_Ca	0.0312 s−1	[10]
koff_CaN_Ca2_Ca	0.352 s−1	[10]
koff_CaN_Ca3_Ca	0.9 s−1	[10]
kon_CaN_RR	2.3 ⋅ 107 M−1 ⋅ s−1	Estimated CaN’s binding rate to activated CaM
Kd_CaN_RR	3.26 ⋅ 10−9 M	Estimated CaN’s dissociation constant from activated CaM
kon_Ng_RT kon_Ng_TT	1 ⋅ 1014 M−1 ⋅ s−1	Estimated Ng’s binding rate to CaM when C lobe is closed
Kd_Ng_RT Kd_Ng_TT	1.2 ⋅ 10−6 M	Estimated Ng’s dissociation constant from CaM, when C lobe is closed
kcat_CaN_CaMKIIp	0.8 s−1	CaN’s catalytic efficiency on dephosphorylating CaMKII Used in this paper
Km_CaN_CaMKIIp	2 ⋅ 10−5 M	CaN’s Michaelis-Menten constant on dephosphorylating CaMKII Used in this paper
Concentrations used in the model (unless specified):
basal Ca 2+	8 ⋅ 10−8 M [63]	CaM	4 ⋅ 10−5 M [64]
CaN	8 ⋅ 10−6 M [64]	CaMKII	8 ⋅ 10−5 M [64]
Ng	4 ⋅ 10−5 M [64]	compartment volume	1 ⋅ 10−15 L
Other Parameters):
Ca 2+ leak	→ Ca 2+	3.85 ⋅ 10−6 M ⋅ s−1
Ca 2+ pump	Ca 2+ →	Km = 2 ⋅ 10−6 M	Vmax = 1 ⋅ 10−4 M ⋅ s−1

For parameter estimations, please see the method and supplementary sections. R: open; T: closed. When combined, the first letter indicates the state of CaM’s N lobe while the second one indicates the state of the C lobe. Since kon, koff, and Kd are linked (Kd = koff/kon), only two of these three are displayed, depending on which ones were actually estimated. For parameters concerning sequential dissociation of Ca²⁺ ions from CaN, the first “Ca” indicates the number of Ca²⁺ ions bound to CaN, the second “Ca” shows the one dissociating from CaN.