0

1020

3040

5060

70

0 5 10 15 20 25

Abs

orba

nce

at 2

80 n

m, m

AU

Retention volume, ml

0

10

20

30

40

50

0 5 10 15 20 25

Abs

orba

nce

at 2

80 n

m, m

AU

Retention volume, ml

1

2

3

4

3

1

2 4

A

B

C

Supplementary Figure S1: Calibration of Superdex 200 10/300 GL column with known standards. (A) Low molecular weight standards; (1) Aldolase 158 kDa, 9.62 nm (2) Ovalbumin 43 kDa, 6.1 nm (3) Ribonuclease A 13.7 kDa, 3.28 nm (4) Aprotinin 6.5 kDa. (B) High molecular weight standards; (1) Ferritin 440 kDa, 12.2 nm (2) Conalbumin 75 kDa, 7.28 nm (3) Carbonic Anhydrase 29 kDa, 4 nm (4) Ribonuclease A 13.7 kDa, 3.28 nm. (C) Plot of partition coefficient versus stokes diameter. The partition coefficient is calculated as: Kav=Ve-Vo/Vc-Vo

, where Ve, Vc, and Vo, are the retention volume, geometric bead volume, and void volume, respectively. The void volume was determined to be 7.5 ml using Blue Dextran 2000. Only the four middle protein standards were used for fitting the standard curve to obtain the best fit in the expected range of the nanodiscs.

y = 42.4x -17.987R² = 0.9979

0

2

4

6

8

10

12

14

0 0.2 0.4 0.6 0.8 1

Stok

es d

iam

eter

, nm

√(-log(Kav))

Ribonuclease ACarbonic Anhydrase

Ovalbumin

Conalbumin

Aldolase

Ferritin

y = 83.683x

y = 143.86x

0

40

80

120

160

0 0.2 0.4 0.6 0.8 1.0 1.2

Tota

l Den

sity

, OD

Protein, µg

aha2∆73MSP1D1(-)

A B

C

Supplementary Figure S2: Calculation of MSP:aha2∆73 stoichiometry from densitometry analysis of Coomassie stained SDS-PAGE gels. (A) SDS-PAGE with standards of known values of MSP1D1(-) and aha2∆73 loaded in lanes 1 to 10. Lanes A and B are loaded with 0.5 µL and 1 µL of reconstituted and purified aha2∆73-containing nanodiscs respectively. Lane M contains a protein marker*. Regions 1 to 24 are indicated by red squares. (B) Table containing measured density for each region and calculated protein amounts in µg and moles from the standard curve. The MSP:aha2∆73 stoichiometry is calculated from the division of region 23 by 11, and 24 by 12. (C) Plots and standard curves of measured density against protein amount in µg.

Region DensityOD

12345678

Loaded Protein µg

Calculated valuesµg mole

9101112131415161718192021222324

76.1467.7976.1765.3552.9640.8533.3131.6912.398.4058.2026.48137.92127.97112.7082.3184.6074.1070.9990.3938.5019.4970.2122.26

1.00.90.80.70.60.50.40.30.20.1--

1.00.90.80.70.60.50.40.30.20.1--

0.910.810.910.780.630.490.40.380.150.101.020.320.960.890.780.570.590.520.490.630.270.140.490.15

9.37×10-12

8.35×10-12

9.38×10-12

8.05×10-12

6.52×10-12

5.03×10-12

4.10×10-12

3.90×10-12

1.53×10-12

1.03×10-12

1.05×10-11

3.26×10-12

4.35×10-11

4.04×10-11

3.55×10-11

2.60×10-11

2.67×10-11

2.34×10-11

2.24×10-11

2.85×10-11

1.21×10-11

6.14×10-12

2.21×10-11

7.02×10-12

1 2 3 4 5 6 7 8 9 10 A BM

aha2∆73

MSP1

Standards ND