Nature - USA (2020-10-15)

a

k Apo

100 nm

3',3'-cGAMP

100 nm

c-di-GMP

100 nm

2' ,3'-cGAMP

100 nm

c-di-AMP

100 nm

Apo

100 Å

3',3'-cGAMP

100 Å

c-di-GMP

100 Å

2',3'-cGAMP

100 Å

c-di-AMP

100 Å

j

37.1 kDa 35.1 kDa

d

b

39.9 kDa

20.9 kDa

77.4 kDa

36.1 kDa

14 18 22

0.0

0.2

0.4

0.6

0

50

100

150

200

Time(min)

[Relative

protein] (dRI

) Mola

rm

ass

(kDa)

ReSTING ΔTM + 3',3'-cGAMP

ReSTING ΔTM

20.1 kDa

34.1 kDa

14 18 22

0.0

0.5

1.0

0

20

40

60

80

Time(min)

[Relative

protein] (dRI

) Mola

rm

ass(kDa)

FsSTING ΔTFsSTING ΔTM + M 3',3'-cGAMP

Void

77.0 kDa

115.4 kDa

297.0 kDa

835.4 kDa

10 14 18 22

0.0

0.2

0.4

0.6

0

100

200

300

Time(min)

[Relative

protein] (dRI

) Mola

rm

ass

(kDa)

SfSfSTING STING +2+3',3 '-cGAMP',3 '-cGAMP

Calc. MW: 35.9 kDa

66.7 kDa 41.7 kDa

79.9 kDa 49.4 kDa

14 18 22

0.0

0.1

0.2

0.3

0

100

200

300

Time(min)

[Relative

protein] (dRI

) Molar

ma

ss(kDa)

SfSfSTINSTINGΔGΔTIRTIR +c-di-GMP

Calc. MW: 19.9 kDa

g

Time(min)

14 18 22

0.0

0.5

1.0

0

50

100

150

200

[Relative

protein] (dRI

) Mola

rm

ass

(kDa)

SfSTINGΔCDN-bindingdomain

32.0 kDa

Calc. MW: 16.0 kDa

14 18 22

0.0

0.5

1.0

0

50

100

150

200

Time(min)

[Relative

protein] (dRI

) Mola

rm

ass

(kDa)

CgCgSTINSTINGΔGΔTIR + TIR 3',3 '-cGAMP

Calc. MW: 18.9 kDa

Calc. MW: 18.5 kDa Calc. MW: 18.7 kDa

e f

h i

Time(min)

Mola

rm

ass

(kDa

)

Time(min)

94.4 kDa

94.7 kDa

14 18 22

0.0

0.5

1.0

0

100

200

300

[Relative

protein] (dRI

)

Oyster STING (TIROyster STING (TIR) + )2',3 '-cGAMP

Calc. MW: 47.0 kDa

44.2 kDa

45.4 kDa

14 18 22

0.0

0.5

1.0

0

20

40

60

80

[Relative

protein] (dRI

) Mola

rm

ass

(kDa)

Human STING ΔTM + 2',3 '-cGAMP

Human STING ΔTM

Calc. MW: 21.7 kDa

l

0.0

0.2

0.4^5

15

25

35

45

55

Average filament # per micrographAp

o

c-di-AMPc-di-GMP3',3'-cGAMP2',3'-cGA

MP

10 14 18 22

0.0

0.1

0.2

0.3

0.4

0.5

0

100

200

300

400

500

Time(min)

[Relative

protein] (dRI

) Molarm

ass

(kDa)

SfSTING + c-di-GMP

SfSTING

Filaments(void)

77.0kDa

167.6 kDa

115.4 kDa

Calc. MW: 35.9 kDa

c

Extended Data Fig. 7 | See next page for caption.