1
Supplementary Materials
Structure determination of Ser24Ala, Tyr51Phe, and Thr95Ala
Crystals were prepared under conditions close to those developed for wild-type
RNase Sa. 1 The crystals grew in the hanging-drop vapor-diffusion experiments at room
temperature from a solution of 10mg/mL protein in 0.1 M phosphate buffer at pH 7.2 and
room temperature with 0.7-0.9 M ammonium sulfate as precipitant. The mutant T95A
was crystallized in cacodylate buffer in the same conditions.
The structure of all three mutants was solved and refined by a procedure used for
most of RNase Sa structures, their complexes with mononucleotides and their mutants
(see ref. 2 ). In brief, data from crystals of all three mutants were collected using
synchrotron radiation at EMBL Hamburg. Data were processed with DENZO and
SCALEPACK. 2 A summary of data collection and processing is given in Table 1. S24A
structure was solved by AMORE 3 using as the starting model RNase Sa 1.2 Å coordinate
set 1RGG and refined with the program REFMAC. 4 The structures of Y51F and T95A
were solved without molecular replacement. X-ray data were directly used in REFMAC
refinement with 1RGG and 1T2H coordinates, respectively. Refinement parameters are
given in Table 2.
In the structure of Y51F, 13 amino acids were modeled with two alternative
conformations of main chain in molecule A (Asp1,Thr5,Val6, Ser24, Asp25, Gly26,
Pro27, Glu41, Glu54, Glu74, Ala75, Thr76, Glu77) and 7 in molecule B (Asp1, Val2,
Ser3, Gly4, Thr5, Arg65, Tyr86). The structure T95A contains 4 residues modeled with
two alternative conformations of main chain in molecule A (Ser42, Arg63, Thr64, Arg65)
and 2 in molecule B (Tyr86 and Ala87). Molecules A and B in structures of mutants were
2
superposed with corresponding molecules in 1RGG wt RNase Sa structure using the
program LSQAB
5,6
, Table 3. Residues with alternative main chain conformations were
excluded from superposition in the figures.
3
Table 1. Data collection statistics. a
S24A (4GHO)
Y51F (4J5K)
T95A (4J5G)
X-ray source
BW7B
X31
X31
Wavelength (Ǻ)
0.834
1.1
1.1
Temperature (K)
298.0
100
100.0
Resolution range (Ǻ)
1.1-20.0
1.2-20.0
1.3-19.6
Space group
P212121
P212121
P212121
38.92 64.69 78.28
38.30 63.81 77.62
38.12 64.19 78.23
Last resolution shell (Ǻ)
1.10-1.11
1.23-1.24
1.31-1.32
Matthews coef. (Ǻ /Da)
2.33
2.24
2.27
47
45.2
45.8
80 959 (5 117)
54 845 (3 366)
46 925 (1 240)
Completeness (%)
98.2 (96.7)
97.8 (82.6)
93.6 (80.8)
I/σ(I)
18.5 (3.3)
29.7 (4.9)
28.5 (4.6)
1.2 (1.0)
2.3 (1.8)
3.5 (3.2)
14.5
12.0
7.5
Unit cell a, b, c (Ǻ)
3
Solvent content (%)
No of unique reflections
Redundancy
Wilson B-factors (Ǻ )
2
a
Values in parentheses refer to the outer shell.
4
Table 2. Refinement statistics. a
S24A (4GHO)
Y51F (4J5K)
T95A (4J5G)
Resolution (Ǻ)
1.1-14.5
1.2-18.6
1.3-18.7
Last resolution shell (Ǻ)
1.10-1.13
1.23-1.26
1.31-1.34
R factor (%)
9.8 (15.7)
10.6 (14.6)
11.4 (17.4)
Rfree factor (%)
11.7 (18.6)
13.6 (18.0)
15.0 (21.9)
80 959 (5 117)
52 028 (3 366)
41 707 (2 696)
3 991
2 778
2 204
based on R
0.019
0.033
0.046
based on Rfree
0.021
0.034
0.046
Protein molecules in asymm. unit
2
2
2
SO4 2-
1
1
3
Glycerol
-
2
2
Cacodylate anion
-
-
1
Water molecules
341
416
462
1 482
1 490
1 488
5
17
26
14.5
12.0
10.7
13.78/15.47
9.7 / 11.0
8.1 / 9.2
water molecules
37.1
27.4
26.4
SO42-
14.0
8.5
14.6
glycerol
-
17.4
18.9
cacodylate anion
-
-
28.3
bond lenghts (Ǻ)
0.028
0.023
0.023
bond angles (°)
2.140
2.108
2.217
0.362
0.173
0.156
0.011
0.012
0.012
most favourable (%)
97.5
97.7
97.5
additionally allowed (%)
0.5
0.3
0.5
No of reflections
No of reflections, test set
Coordinates ESU (Ǻ)
Protein non-H atoms
Ligand atoms
Average B factors (Ǻ2)
protein atoms molecule A/B
RMSD from ideal values
chiral centers (Ǻ )
3
planar groups (Ǻ)
Ramachandran profile
a
b
b
Values in parentheses refer to the outer shell.
Ramachandran profile was calculated using the Molprobity server
(http://molprobity.biochem.duke.edu/).
5
Table 3. Superposition of mutant and wt RNase Sa structures. a
S24A_1RGG
Y51F_1RGG
T95A_1RGG
A mol
B mol
A mol
B mol
A mol
B mol
96
96
83
89
92
94
rms (Å)
0.101
0.100
0.245
0.201
0.238
0.337
average (Å)
0.075
0.077
0.214
0.178
0.205
0.282
maximum (Å)
0.444
0.374
0.578
0.452
0.579
0.889
(Pro27)
(Ser3)
(Arg40)
(Ala87)
(Thr76)
(Gly4)
No of atoms
XYZ
displacement
a
1RGG wt RNase Sa structure was used in superposition as a standard molecule.
Amino-acid residues with two CA conformations were excluded from superposition.
6
Analysis of the 59 Ser to Ala mutations included in Table VIII
For the 59 Ser to Ala mutations, 44 of the Ser residues were hydrogen bonded and
15 were not. This was determined using pfis. 7 Protherm was used to find Ser to Ala
mutants where (G) values had been measured. 8 The original reference was then
consulted to confirm the results. Twelve of the (G) values for Ser to Ala mutants were
from Table VII in this paper. The (G) values for the other 47 Ser to Ala mutants are
given below. The name of the protein is followed (in parentheses) by the Ser residue that
was mutated, and its Δ(ΔG) value in kcal/mol.
The results for the mutants where the Ser was hydrogen bonded in the wild type
protein include: Staph nuclease (128, 0.7 and 141, -0.4), 9 barnase (85, 0.1, 91, -1.9, 92,
-2.8), 10,11 human lysozyme (24, -0.5, 36, -1.1, 51, -0.2, 61, -1.4), 12 chymotrypsin
inhibitor-2 (12, -0.9, 31, -0.9), 13 apoflavodoxin (71, -0.7, 110, -0.6), 14 human growth
hormone (71,-1.0), 15 HPr (31, -0.5, 46, -1.0), 16 SH3 domain from Fyn tyrosine kinase
(41, -0.7), 17 RNase H1 (68, -0.8), 18 T4 lysozyme (38, -0.8, 117, 1.3), 19,20 Pho PQ
activated gene P from E. coli (58, -1.3, 130, -1.3), 21 Succ 1from the cyclin-dependent
kinase (cks) family (28, -0.3), 22 Cks-1 (9, -0.3), 23 DHFR (148, -0.4), 24 Arc repressor
(32, -3.8, 35, 0.2, 44, -1.6), 25 FF domain from human HYPA/FBP11 (32, 0, 35, -1.0, 56,
-0.5), 26 ribose binding protein (9, -1.3), 27 RNase T1 (12, -1.2, 17, 0.6, 64, -1.5), 28 and
RNase A (75, -2.5). 29
The results for the mutants where the Ser was not hydrogen bonded in the wild
type protein include: Staph nuclease (59, 0.4), 9 barnase (28, 0.4, 31, 0.1), 30 human
lysozyme (80, 0.5, 82, 0.4), 12 T4 lysozyme (44, 0.4), 31 Cks-1 (39, -0.8), 23 myoglobin
7
(117, -0.3), 32 hen egg white lysozyme (91, -0.2), 33 Arc repressor (5, 0.1), 25 and the FF
domain from human HYPA/FBP11 (50, -0.2). 26
For the 44 hydrogen bonded residues, the average ∆(∆G) = -0.82 with a standard
deviation of 0.89. For the 15 non hydrogen bonded residues, the average ∆(∆G) = 0.10
with a standard deviation of 0.39. These are the values given in Table VIII in the
manuscript.
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