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Fig. S1. Biophysical characterization of the
Sgt2
-
C
cons
domain.
(
A
) CD spectra as in Fig. 1
C
for the conserved C
-
terminal domains of
y
Sgt2 (blue) and
hSgt2
(orange). NMR spectra as in Fig.
1
D
&
E
for
y
Sgt2
-
C
cons
(
B
,
blue) and
h
S
gt2
-
C
cons
(
C
,
o
range).
B
C
0.95 ppm
110
115
120
125
15
N (ppm)
9.0
8.5
8.0
7.5
7.0
6.5
130
105
1
H (ppm)
h
Sgt2-C
cons
8.5
9.0
8.0
7.5
7.0
6.5
110
115
120
125
15
N (ppm)
1
H (ppm)
0.91 ppm
y
Sgt2-C
cons
200
250
Wavelength (nm)
ï
20
ï
16
ï
12
ï
8
ï
4
0
Ellipticity (m deg)
210
220
230
240
h
Sgt2-C
cons
y
Sgt2-C
cons
A
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Fig. S2. Identification of minimal binding region of Sgt2.
(
A
)
The
full
image of the gel in Fig
2C.
(
B
)
An anti
-
MBP western blot of the lysate from the which the complexes in (A) were purified
from.
The load concentrations were normalized based on the total optical density of the cells when
harvested.
75
50
37
(kDa)
TA: MBP-Sbh1
His-ySgt2
His-hSgt2
MBP-
Sbh1
TPR-C
C
cons
C
TPR-C
CC
cons
TPR-C
CC
cons
у+
у+
14.4
21.5
(kDa)
6.5
His-ySgt2
His-hSgt2
TPR-C
C
C
cons
C
у+
у+
MBP-
Sbh1
TPR-C
C
cons
TA: MBP-Sbh1
A
B
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Fig. S
3
.
Structural models across prediction methods.
(
A
)
Predictions from
Quark, I
-
TASSER, Pcons,
Phyre2, RaptorX, and
Ro
b
etta.
Methods
produce between 5 and 10 model
s
.
(
B
)
Ro
b
etta
provides a residue
-
wise
estimated error in
A
ngstroms
; this is shown below the
corresponding models with a g
rey
bar
indicating the
C
cons
region
.
Quark
Robetta
I-TASSER
Pcons
A
1
2
3
6
7
8
4
5
1
2
3
4
5
6
7
8
RaptorX
Phyre2
9
10
1
2
3
4
5
1
2
3
4
5
B
Estimated
Error (Å)
5
10
220 260 300 340
Residue
220 260 300 340
Residue
220 260 300 340
Residue
220 260 300 340
Residue
220 260 300 340
Residue
5
10
5
10
5
10
5
10
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Fig. S
4
. Cysteine mutants are capable of binding to clients.
(
A
)
Schematic showing how
h
is
-
tagged
y
Sgt2
-
TPR
-
C and double cysteine mutant constructs were coexpressed with the client
11[L8]
,
and complexes were purified by nickel affinity chromatography.
(
B
)
A
coomassie stained
SDS
-
PAGE gel of the elution fractions demonst
rates that
11[L8]
was present in the elution
suggesting
double cysteine
mutations d
o
not affect
client
binding.
(
C
) An anti
-
cMyc western blot
of the fractions represented in the SDS
-
PAGE gel
also demonstrates that 11[L8]
was present in
all elu
ate
s.
Sgt2 TPR-C
(kDa)
A272C
L327C
I286C
M323C
M289C
A319C
M289C
N322C
N285C
G329C
WT
11[L8]
Co-expression
NTA-Ni
2+
cMyc-T11[L8]
His-tagged
Sgt2-TPR-C &
mutants
His-tagged
Sgt2-TPR-C/TA
complex
imidazole
Elution
cMyc-tag
BRIL Linker TMD (Leu/Ala)
A
B
C
37
20
15
(kDa)
10
11[L8]
A272C
L327C
I286C
M323C
M289C
A319C
M289C
N322C
N285C
G329C
WT
37
20
15
10