0.75 1.00 0.25 n = 2.1 ± 0.2 K d = 2.1 ± 1.1 μM 0.50 log 10 [P](log 10 μM) -1 0 1 2 log 10 θ/(1-θ) 0.0 1.0 2.0 2.5 3.0 3.5 4.0 5.0 6.0 9.0 pri-miR-16-1 DGCR8M- pri-miR-16-1 a b DGCR8M (µM) Supplementary Figure 1. The binding analysis between DGCR8M and pri-miR-16-1 (a) The binding between wild type DGCR8M (0 to 9 uM) and pri-miR-16-1 (0.25nM) was analyzed by EMSA. (b) The Hill plot of integration data from (a) is shown. The Hill constant (n) and Kd are mean values of three independent experiments. Crystal structure of human DGCR8 core Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho
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Crystal structure of human DGCR8 core Sun Young Sohn, Won ... · Crystal structure of human DGCR8 core Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and
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0.75 1.000.25
n = 2.1 ± 0.2Kd = 2.1 ± 1.1 μM
0.50
log10[P](log10μM)-1
0
1
2
log 10
θ/(1
-θ)
0.0 1.0 2.0 2.5 3.0 3.5 4.0 5.0 6.0 9.0
pri-miR-16-1
DGCR8M- pri-miR-16-1
a bDGCR8M (µM)
Supplementary Figure 1. The binding analysis between DGCR8M and pri-miR-16-1
(a) The binding between wild type DGCR8M (0 to 9 uM) and pri-miR-16-1 (0.25nM) was analyzed by EMSA.
(b) The Hill plot of integration data from (a) is shown. The Hill constant (n) and Kd are mean values of three independent experiments.
Crystal structure of human DGCR8 core
Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho
y = -1.6294x + 7.7709R2 = 0.995
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
Ve/Vo
log(
Mw
)
a b
pri-miR-16-1& DGCR8M
100 150 200 250 3000
35
70
280 nm260 nm
DGCR8M
100 150 200 250 3000
5
10
Abs
orba
nce
(mA
U)
pri-miR-16-1
100 150 200 250 3000
50
100
280 nm260 nm
280 nm260 nm
669 440 150 66 29 12.4 kDa
Elution volume (ml)
200 ml (101.8 kDa)
230ml (38.4 kDa)
204 ml (87.1 kDa)
Supplementary Figure 2. SEC analysis of the DGCR8M-pri-miR-16-1 complex (a) The standard curves for SEC experiments. The formula (indicated in figure) was derived from the linear regression curve and used to estimate the molecular weight of the DGCR8M-pri-miR-16-1 complex. The standard curves for SEC experiments were obtained from the following proteins: cytochrome C, 12.4 kDa; carbinic anhydrorase, 29 kDa; Bovin serum albumin, 66 kDa; Alcohol dehydroganase, 150 kDa; Ferritin, 440 kDa; thyroglobulin, 480 kDa. The formula was derived from the linear regression curve and used to estimate the molecular weight of the hDGCR8M-pri-miR-16-1 complex.
(b) SEC results of the DGCR8M-pri-miR-16-1 complex (top), DGCR8M (middle), and pri-miR-16-1 (bottom).
Crystal structure of human DGCR8 core
Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho
Molecular weight of DGCR8M−pri-miR-16-1 complex from equilibrium sedimentation
Concentration of
DGCR8M (mM)
Concentration of
pri-miR-16-1 (mM)
Estimated MW
(kDa)
Monomer MW of
DGCR8M (kDa)
Monomer MW of
pri-miR-16-1(kDa)
MW estimated /
MW monomer of
DGCR8M and
pri-miR-16-1
1 0.5 62.4 27 35 1.01
Model: Single ideal speciesVariance: 5.94153E-5Speed:12000 rpmTemp: 15 ℃ V-bar: 0Rho : 1MW: 62417 Da
Radius (cm)
Abo
sorb
ance
Res
idua
lsCrystal structure of human DGCR8 core
Sun Young Sohn, Won Jin Bae, Jeong Joo Kim, Kyu-Hyeon Yeom, V. Narry Kim and Yunje Cho
The equilibrium fit results of analytical ultracentrifuge for the DGCR8M-pri-miR-16-1 complex. Fitted overlay (red line) to experimental data (blue circles) is shown in the bottom panel; residuals are shown in upper panel. The fitted parameter for the weight-average molecular weight (Mwapp) was estimated at 62,417 gm-1.
5′ ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT-TMR 5′
b
RNA−TMR
5′ FAM ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT 5′
RNA−FAM a
5′ FAM ag c - A C U GAUUC 3′ gucagc ugc uUAGCAGCAC GU AAUAUUGG G UAA caguug aug AGUCGUCGUG CA UUAUGACC C AUU 3′ cauac ga A U A U U AAAAT-TMR 5′