C

CA S51

CB S51

Hn Q52

CA S5/S43

CB S5/S43

Hn E44 13C (ppm)

1H (ppm)

13C (ppm)

1H (ppm)

B

Figure S1

A

-0.4

-0.2

0

0.2

0.4

38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68

ERM AA#

A.U

SS

P s

core

Helix

Strand

15N ERM38-68

+ MED25 (1:0.1)

Figure S2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68ERM AA#

1H + 0.215N (ppm)A

i3Q5415N ERM38-68

+ MED25 (1:0.2)

i3Q54 15N ERM38-68

+ MED25 (1:2)

1H

1H

NHW57

1H

15N

B

% Bound

0

100

50

Figure S3

B

Bound Free

Delta kex (~ Kd)

A

Delta kex (~ Kd)

Bound Free

Figure S3

ERM/MED25

0.150.300.500.600.701.00

0.00ERM/MED25

1.201.502.002.503.00

1.00

Gly524

Gly532

Gly436

Gly524

Gly532

Gly436

C D

1H

15N

1H

15N

Gly532

Gly436

E F

1H

15N

1H

15N

ERM-bound MED25

Free MED25

ERM-bound MED25

0

20

40

60

80

100

120

0 2 4 6 8 10

Kd (µM)

Relative CSD (%) n=10r2=-0.97

0

20

40

60

80

100

120

0 2 4 6 8 10

Kd (µM)

Relative CSD (%) S525S525

15N

1H

A

B

Figure S4

15N MED25+ ERM38-68

+ ERM38-68 F47L+ ERM38-68 W57L+ ERM38-68 F47L/W57L

0

0.2

0.4

0.6

0.8

390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550

Figure S5

Intensity ratio Bound/free I/I0

1 2 3 4 5 6 71 2 3

ERM38-68 F47L – MED25 WT

1

4 7 6

2

5

3

31

180°

A B

180°

C D

Figure S6

Intensity ratio Bound/free I/I0

1 2 3 4 5 6 71 2 3

ERM38-68 W57L – MED25 WT

4 7 65

12

3

31

180°

A B

180°

C D

0

0.2

0.4

0.6

0.8

390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550

1H (ppm)

15N(ppm)

Cter-NO°B

1H (ppm)

15N(ppm)

Nter-NO°A

Figure S7

ERM38-68 F47L/W57L - MED25 Q451E

2

5

3

31

1

4

7 6

A B

180°

C D

Figure S8

Intensity ratio Bound/free MED25

1 2 3 4 5 6 71 2 3

0

0.2

0.4

0.6

0.8

1

390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550

Q451E MED25 AA#

Figure S9

Q451

H499

W57

BA