Task 6: Sequence-based mutation analysis

Task description

A detailed task description can be found here.

Mutation selection

We selected the following ten mutations:

• I65T
• R71H
• R158Q
• R261Q
• T266A
• P275S
• T278N
• P281L
• G312D
• R408W

5 of these mutations are associated with our disease phenylketonuria the other 5 not. The 5 disease causing mutations are the most frequent missense/nonsense mutations of people who suffer from phenylketonuria. These numbers were taken from pahDB. However, at this point we are not going to tell which of these are the associated and which are not. We are going to lift this "secret" after our sequence based mutation analysis in order to validate our in silico generated predictions.

To keep our association secret we encrypted the file which contains the disease association for each mutation with the following linux command: "vim -x selected_mutations_and_association.txt"

The encrypted output is as follows:

The decryption can be only performed with the correct password.

Physicochemical properties and changes

For the annotation of the physicochemical properties of our amino acids we used the following venn diagram from [1]:

Additionally to this we used also the table of this Wikipedia page as a reference.

I65T

I: aliphatic, hydrophob, large, unpolar, neutral
T: small, hydrophob, polar, neutral

R71H

R: polar, aliphatic, positive charged, hydrophob, large, neutral
H: positive charged, polar, aromatic, hydrophob, basic

R158Q

R: polar, aliphatic, positive charged, hydrophob, large, neutral

Q: polar, aliphatic, hydrophob, neutral

R261Q

R: polar, aliphatic, positive charged, hydrophob, large, neutral
Q: polar, aliphatic, hydrophob, neutral

T266A

T: small, hydrophob, polar, neutral

A: tiny, hydrophobic, nonpolar, neutral

P275S

P: small, proline, nonpolar, neutral

S: tiny, polar, neutral

T278N

T: small, hydrophob, polar, neutral

N: small, polar, neutral

P281L

P: small, proline, nonpolar, neutral

L: hydrophobic, aliphatic, nonpolar, neutral

G312D

G: very small, unpolar, neutral
D: polar, small, negative charged, acidic

R408W

R: polar, aliphatic, positive charged, hydrophob, large, neutral
W: polar, aromatic, large, hydrophob, neutral

Visualization of the changed Amino Acid

We used the structure 1J8U to visualize our mutations with Pymol. This structure is only solved from residue 118 to residue 424. Hence we could not visualize the mutations at position 65 and 71.

I65T

Was not included in the used structure.

R71H

Was not included in the used structure.

R158Q

The following picture shows the mutation from the wild type (WT) Arginine (R, seen in orange) to the mutant Glutamine (Q, seen in yellow) at position 158. Furthermore, the seen spheres in the background belong to the ligands FE2 and 5,6,7,8-TETRAHYDROBIOPTERIN (HB4) which are bound to the catalytic site and are required for the reaction.

We observed that the WT has polar contacts to:

• E280
• Y154
• E141

We think that these polar contact get lost in the mutant.

R261Q

Visualization of the mutated residue Q (white) versus the wildtype residue R (green) in the structure of PAH:

T266A

The following picture shows the mutation from the wild type (WT) Threonine (T, seen in orange) to the mutant Alanine (A, seen in yellow) at position 266. Furthermore, the seen spheres in the background belong to the ligands FE2 and 5,6,7,8-TETRAHYDROBIOPTERIN (HB4) which are bound to the catalytic site and are required for the reaction.

We observed that the WT has polar contacts to:

• E286

We think that these polar contact get lost in the mutant.

P275S

The following picture shows the mutation from the wild type (WT) Proline (P, seen in orange) to the mutant Serine (S, seen in yellow) at position 275. Furthermore, the seen spheres in the background belong to the ligands FE2 and 5,6,7,8-TETRAHYDROBIOPTERIN (HB4) which are bound to the catalytic site and are required for the reaction.

We observed that the WT has polar contacts to:

• E270

We think that this polar contact get's not lost. Since this is a polar contact from the backbone and not side chain.

T278N

The following picture shows the mutation from the wild type (WT) Threonine (T, seen in orange) to the mutant Asparagine (N, seen in yellow) at position 278. Furthermore, the seen spheres in the background belong to the ligands FE2 and 5,6,7,8-TETRAHYDROBIOPTERIN (HB4) which are bound to the catalytic site and are required for the reaction.

We observed that the WT has polar contacts to:

• E280

We think that this polar contact get's not lost. Because the mutated residue also has a uncharged polar side chain which points with its O end in the same direction as our WT side chain. However, this is only a guess since we did not calculate the most probable rotamer for the mutant.

P281L

The following picture shows the mutation from the wild type (WT) Proline (P, seen in orange) to the mutant Leucine (L, seen in yellow) at position 281. Furthermore, the seen spheres in the background belong to the ligands FE2 and 5,6,7,8-TETRAHYDROBIOPTERIN (HB4) which are bound to the catalytic site and are required for the reaction.

We observed that the WT has polar contacts to:

• E268

We think that this polar contact get's not lost. Since the polar contact is between the backbone of the residue.

G312D

Visualization of the wildtype residue G (blue) in the structure of PAH.

Visualization of the mutated residue D (white) in the structure of PAH.

R408W

Visualization of the mutated residue W (white) versus the wildtype residue R (green) in the structure of PAH.

Mutations compared to BLOSUM62, PAM(1/250), PSSM and conservation of MSA with all mammalian homologous

BLOSUM 62 matrix

BLOSUM 62 Matrix
The BLOSUM 62 was calculated from blocks of clusters with a sequence identity of 62%. A positive score is given to the more likely substitutions while a negative score is given to the less likely substitutions. Source: [2] Disclaimer: This file is redistributed from Wikimedia and copyrighted under the Creative Commons licence.

PAM 1/250 matrix

We took the values for our PAM1 and PAM 250 matrix from this page.

PAM1 Matrix

	A	R	N	D	C	Q	E	G	H	I	L	K	M	F	P	S	T	W	Y	V
A	9867	2	9	10	3	8	17	21	2	6	4	2	6	2	22	35	32	0	2	18
R	1	9913	1	0	1	10	0	0	10	3	1	19	4	1	4	6	1	8	0	1
N	4	1	9822	36	0	4	6	6	21	3	1	13	0	1	2	20	9	1	4	1
D	6	0	42	9859	0	6	53	6	4	1	0	3	0	0	1	5	3	0	0	1
C	1	1	0	0	9973	0	0	0	1	1	0	0	0	0	1	5	1	0	3	2
Q	3	9	4	5	0	9876	27	1	23	1	3	6	4	0	6	2	2	0	0	1
E	10	0	7	56	0	35	9865	4	2	3	1	4	1	0	3	4	2	0	1	2
G	21	1	12	11	1	3	7	9935	1	0	1	2	1	1	3	21	3	0	0	5
H	1	8	18	3	1	20	1	0	9912	0	1	1	0	2	3	1	1	1	4	1
I	2	2	3	1	2	1	2	0	0	9872	9	2	12	7	0	1	7	0	1	33
L	3	1	3	0	0	6	1	1	4	22	9947	2	45	13	3	1	3	4	2	15
K	2	37	25	6	0	12	7	2	2	4	1	9926	20	0	3	8	11	0	1	1
M	1	1	0	0	0	2	0	0	0	5	8	4	9874	1	0	1	2	0	0	4
F	1	1	1	0	0	0	0	1	2	8	6	0	4	9946	0	2	1	3	28	0
P	13	5	2	1	1	8	3	2	5	1	2	2	1	1	9926	12	4	0	0	2
S	28	11	34	7	11	4	6	16	2	2	1	7	4	3	17	9840	38	5	2	2
T	22	2	13	4	1	3	2	2	1	11	2	8	6	1	5	32	9871	0	2	9
W	0	2	0	0	0	0	0	0	0	0	0	0	0	1	0	1	0	9976	1	0
Y	1	0	3	0	3	0	1	0	4	1	1	0	0	21	0	1	1	2	9945	1
V	13	2	1	1	3	2	2	3	3	57	11	1	17	1	3	2	10	0	2	9901

PAM250 Matrix

	A	R	N	D	C	Q	E	G	H	I	L	K	M	F	P	S	T	W	Y	V
A	13	6	9	9	5	8	9	12	6	8	6	7	7	4	11	11	11	2	4	9
R	3	17	4	3	2	5	3	2	6	3	2	9	4	1	4	4	3	7	2	2
N	4	4	6	7	2	5	6	4	6	3	2	5	3	2	4	5	4	2	3	3
D	5	4	8	11	1	7	10	5	6	3	2	5	3	1	4	5	5	1	2	3
C	2	1	1	1	52	1	1	2	2	2	1	1	1	1	2	3	2	1	4	2
Q	3	5	5	6	1	10	7	3	7	2	3	5	3	1	4	3	3	1	2	3
E	5	4	7	11	1	9	12	5	6	3	2	5	3	1	4	5	5	1	2	3
G	12	5	10	10	4	7	9	27	5	5	4	6	5	3	8	11	9	2	3	7
H	2	5	5	4	2	7	4	2	15	2	2	3	2	2	3	3	2	2	3	2
I	3	2	2	2	2	2	2	2	2	10	6	2	6	5	2	3	4	1	3	9
L	6	4	4	3	2	6	4	3	5	15	34	4	20	13	5	4	6	6	7	13
K	6	18	10	8	2	10	8	5	8	5	4	24	9	2	6	8	8	4	3	5
M	1	1	1	1	0	1	1	1	1	2	3	2	6	2	1	1	1	1	1	2
F	2	1	2	1	1	1	1	1	3	5	6	1	4	32	1	2	2	4	20	3
P	7	5	5	4	3	5	4	5	5	3	3	4	3	2	20	6	5	1	2	4
S	9	6	8	7	7	6	7	9	6	5	4	7	5	3	9	10	9	4	4	6
T	8	5	6	6	4	5	5	6	4	6	4	6	5	3	6	8	11	2	3	6
W	0	2	0	0	0	0	0	0	1	0	1	0	0	1	0	1	0	55	1	0
Y	1	1	2	1	3	1	1	1	3	2	2	1	2	15	1	2	2	3	31	2
V	7	4	4	4	4	4	4	4	5	4	15	10	4	10	5	5	5	72	4	17

PSSM

The readable PSSM file of PSI-Blast can be generated by the -Q option:
blastpgp -d '/data/nr/nr' -i './reference.fasta' -o './reference_psi_e10E-6_i5.blast' -h 10E-6 -j 5 -Q reference_i5_e10E-6.txt

Position	AA	A	R	N	D	C	Q	E	G	H	I	L	K	M	F	P	S	T	W	Y	V			A	R	N	D	C	Q	E	G	H	I	L	K	M	F	P	S	T	W	Y	V
1	M	-3	-4	-5	-5	-3	-3	-4	-5	-4	0	5	-4	7	-1	-5	-4	-3	-3	-3	-1			0	0	0	0	0	0	0	0	0	0	66	0	34	0	0	0	0	0	0	0	1.13	0.65
2	S	4	-3	1	-3	-3	-3	-2	1	1	-4	-4	-3	-3	-5	-3	3	-1	-5	-4	-3			46	0	8	0	0	0	2	11	4	0	0	0	0	0	0	28	1	0	0	0	0.75	0.91
3	T	2	-3	2	-3	-4	-3	-3	2	2	-5	-5	-3	-4	-4	-2	4	1	3	-3	-4			19	0	9	0	0	0	0	13	6	0	0	0	0	0	1	39	9	5	0	0	0.68	1.04
4	A	2	-3	0	-1	-4	-3	-3	-1	-4	0	1	1	-2	-4	-4	2	-2	-5	-4	3			18	0	5	3	0	0	1	4	0	4	14	9	0	0	0	17	1	0	0	25	0.35	1.10
5	V	-1	-5	-4	-5	3	-1	-3	-5	-5	5	0	-4	0	-3	-3	0	2	-5	-4	3			4	0	0	0	5	3	1	0	0	37	6	0	1	0	1	6	16	0	0	18	0.67	1.15
6	L	4	-1	-4	-2	-4	1	0	-2	-4	0	2	2	-2	-3	-4	-2	-3	-5	0	-1			33	3	0	2	0	7	6	2	0	5	20	13	0	1	0	1	0	0	3	4	0.39	1.19
7	E	-2	-1	3	3	-6	0	5	-2	-3	-3	-5	0	-5	-6	-1	-1	-3	-6	-5	-5			3	3	17	17	0	2	42	2	0	2	0	5	0	0	2	4	1	0	0	0	0.86	1.21
8	N	-3	1	4	4	-2	-3	-1	-1	0	-2	-5	1	-3	-5	0	2	-2	-6	-4	-5			0	9	24	24	1	0	2	4	2	3	0	7	1	0	5	15	1	0	0	0	0.65	1.26
9	P	1	-3	-1	-4	-4	-4	-4	-2	-1	4	-3	-1	-3	-2	5	0	-2	-5	1	-2			15	1	3	0	0	0	0	3	2	29	0	4	0	1	26	9	1	0	6	0	0.67	1.28
10	G	1	-2	1	-2	-5	0	3	2	0	-3	-1	-2	-4	-5	1	1	-1	-5	-3	-3			10	1	6	1	0	4	24	17	2	1	6	1	0	0	7	11	4	0	1	2	0.36	1.25
11	L	-1	-4	2	4	-5	-3	0	-3	-4	-2	3	-3	-3	-2	-2	1	-2	-5	-1	-3			4	0	10	26	0	0	4	1	0	2	34	1	0	1	2	10	2	0	2	0	0.54	1.30
12	G	0	0	2	0	-5	0	1	2	1	-5	-4	0	-4	-5	-3	1	3	-5	-2	-3			8	5	11	6	0	4	11	13	3	0	1	6	0	0	1	10	19	0	1	1	0.35	1.31
13	R	-2	3	0	-3	-5	2	3	-4	-3	-5	-3	2	-4	-5	4	1	1	-6	-5	-4			2	17	4	0	0	8	17	0	0	0	3	11	0	0	18	12	7	0	0	1	0.58	1.34
14	K	-3	1	4	1	-4	1	2	-4	1	3	-3	2	-4	-2	-4	1	-2	-5	-2	-2			1	8	18	7	0	7	14	0	3	16	1	11	0	2	0	10	1	0	1	2	0.42	1.36
15	L	-3	-3	4	0	-5	-1	3	-1	-2	1	1	-2	-3	4	-5	-2	-3	-4	-2	-3			1	1	19	3	0	2	23	6	0	10	14	2	0	16	0	2	0	0	1	0	0.49	1.33
16	S	-3	-3	2	4	-5	-1	0	-4	-1	-1	-1	0	0	-1	-2	2	1	-5	-1	1			0	0	10	23	0	2	6	0	1	3	6	6	2	2	2	13	10	0	2	10	0.35	1.34
17	D	-3	0	2	4	-5	-2	1	-1	0	0	-3	0	-1	-3	-4	-1	3	-5	-2	0			1	6	10	23	0	1	8	4	3	5	2	5	2	1	0	3	18	0	1	8	0.39	1.33
18	F	-1	3	-4	-3	-3	-2	1	-1	1	-1	1	-1	-3	3	1	-1	-2	-4	0	-1			6	20	0	1	1	1	10	5	4	4	15	3	0	11	7	6	1	0	3	3	0.26	1.31
19	G	-1	-3	3	1	-5	0	-3	3	0	-4	-2	-2	0	5	-5	-1	-2	-4	-3	-4			4	1	16	8	0	4	1	25	2	1	4	2	3	22	0	3	2	0	0	1	0.56	1.34
20	Q	-3	2	5	0	-5	1	0	-2	-1	-1	2	2	-3	-1	-4	-3	-3	-5	-4	-2			1	10	28	5	0	5	5	2	1	4	18	13	0	3	1	0	0	0	0	3	0.48	1.37
21	E	0	-2	-1	1	-4	0	3	-3	-3	0	-3	1	-3	1	-4	1	1	-5	-1	1			6	1	3	7	0	3	21	2	0	6	1	10	0	6	0	10	10	0	2	11	0.27	1.35
22	T	1	-2	-1	-2	-3	-3	-2	1	-4	2	-1	-1	-2	-3	-3	1	4	-4	-3	0			11	1	3	2	0	0	2	11	0	15	4	3	1	1	1	11	26	0	1	7	0.36	1.30
23	S	0	3	-1	0	-4	-2	-2	1	-3	1	-2	-1	-3	-3	-2	3	-1	-4	-4	-1			8	16	3	5	0	1	1	12	0	8	4	4	0	1	2	28	2	0	0	4	0.35	1.28
24	Y	-2	-1	-2	-2	-2	0	2	-3	0	1	-2	0	-2	5	-4	-1	-3	-2	5	-2			3	3	1	3	1	4	13	2	2	8	2	6	1	23	0	4	1	0	20	2	0.55	1.30
25	I	-2	-1	-1	-2	-3	1	-1	-3	1	3	1	0	-1	-2	0	1	-1	-5	-3	2			3	3	4	3	1	6	3	2	4	17	15	6	1	1	5	10	4	0	1	14	0.19	1.31
26	E	0	-2	-1	1	-4	0	3	-3	-3	-3	-3	2	-1	1	1	0	-3	-2	-4	1			8	2	3	8	0	4	21	1	0	1	2	15	1	6	7	8	1	1	0	11	0.30	1.33
27	D	-2	0	0	3	-2	-1	4	-3	-2	0	-3	-1	-3	-3	1	0	0	-5	-4	0			3	5	4	16	1	1	27	2	1	6	3	4	0	1	8	6	6	0	0	6	0.38	1.33
28	N	1	-3	3	2	-5	-1	3	2	1	-5	-4	0	-4	-5	1	0	-1	-5	-4	-2			12	1	12	10	0	2	18	17	4	0	2	5	0	0	7	5	3	0	0	3	0.39	1.33
29	C	-1	1	0	-1	2	-1	2	-4	1	-1	-3	2	-3	0	4	0	-2	-2	-4	0			5	8	4	3	4	2	13	1	4	4	1	12	0	5	17	7	2	1	0	7	0.30	1.33
30	N	-1	-2	3	3	-3	0	0	-3	0	-4	-4	0	-3	-4	-3	3	3	-5	-3	-3			5	2	12	16	0	4	6	1	3	0	1	5	1	1	1	26	16	0	1	1	0.51	1.33
31	Q	-1	-2	0	-1	-4	0	1	5	-3	0	-4	-2	-3	-4	-1	0	-2	-2	0	-1			5	1	4	2	0	4	11	44	0	7	1	2	0	0	3	6	1	0	4	4	0.59	1.28
32	N	0	2	3	2	-1	-1	-1	-2	1	-2	-3	3	-2	-5	0	0	-2	-6	-4	0			6	12	15	13	1	2	2	3	3	2	1	19	1	0	5	6	1	0	0	7	0.36	1.33
33	G	1	-3	-1	5	-1	2	0	0	0	-1	-4	0	-2	-4	-4	-1	-2	-5	0	-2			13	1	2	36	1	9	4	8	2	4	0	6	1	0	0	3	1	0	4	3	0.54	1.28
34	A	-2	3	0	-3	-1	0	0	-4	-3	-2	-1	5	-2	-5	-4	-1	0	-5	-4	-1			2	17	5	0	1	4	6	1	0	2	7	39	1	0	0	5	6	0	0	5	0.62	1.31
35	I	1	-4	-3	-4	-4	-1	-3	-4	-4	2	0	-2	1	0	-4	-1	6	-5	-4	0			10	0	1	0	0	3	1	0	0	11	10	2	3	3	0	1	50	0	0	5	0.76	1.33
36	S	2	-3	1	-1	2	-3	-2	-3	-4	-3	-5	-2	-4	-5	-3	5	2	-3	-4	-3			13	1	5	4	4	0	1	0	0	1	0	2	0	0	1	53	12	0	0	1	0.85	1.34
37	L	-4	-5	-6	-6	-1	-5	-5	-6	-5	5	4	-5	1	0	-5	-5	-3	-5	-4	3			0	0	0	0	1	0	0	0	0	31	41	0	3	3	0	0	0	0	0	18	1.05	1.36
38	I	-1	-2	-4	-5	-2	0	-2	-6	-5	4	3	-4	2	0	-5	-4	-3	-5	-4	3			4	2	1	0	1	4	2	0	0	28	23	0	5	4	0	0	1	0	0	24	0.70	1.36
39	F	-3	-5	-6	-6	-1	-5	-6	-6	-5	2	1	-5	1	7	-6	-5	-3	-3	-1	2			1	0	0	0	1	0	0	0	0	10	14	0	3	54	0	0	1	0	0	15	1.28	1.36
40	S	0	1	-3	-3	-2	-2	-2	-2	2	0	-4	-2	-3	-2	-4	5	3	-5	-2	-3			8	6	0	0	1	1	2	2	5	6	0	2	0	1	0	49	15	0	1	1	0.71	1.36
41	L	0	-4	-4	-5	-2	-2	-5	-4	-5	1	4	-4	2	-2	1	-1	1	-5	-4	2			7	0	0	0	1	2	0	1	0	7	42	0	4	1	7	5	10	0	0	12	0.58	1.40
42	K	-2	4	1	-1	-3	0	1	-4	2	-4	-4	4	-4	-5	2	0	-1	-6	-4	-3			3	21	8	3	1	3	7	0	5	1	1	25	0	0	9	7	3	0	0	2	0.58	1.39
43	E	-3	-1	5	5	-6	-2	2	-4	2	-6	-6	-2	-5	-5	-4	-1	-2	-6	0	-4			1	3	31	36	0	0	11	0	5	0	0	1	0	0	1	3	2	0	4	1	1.06	1.39
44	E	-3	3	0	-2	-6	2	3	1	-1	-5	-5	4	-3	-5	-2	-1	-3	-5	1	-3			1	16	4	1	0	9	20	10	1	0	0	24	0	0	2	4	1	0	5	2	0.59	1.40
45	V	1	-4	-5	-5	2	-3	-4	-5	-5	1	-2	-1	3	-2	5	-1	0	-1	-2	3			10	0	0	0	4	1	0	0	0	7	2	3	7	1	28	5	7	1	1	23	0.67	1.38
46	G	-2	-4	1	-3	-5	-1	-2	6	-4	-4	-6	-3	-5	-6	0	0	-1	-5	-5	-5			1	0	7	1	0	3	3	69	0	1	0	0	0	0	5	5	4	0	0	0	1.29	1.36
47	A	4	-2	-1	-1	-1	-3	-1	1	-3	-2	-4	-3	-2	0	-1	3	0	-5	-4	-2			40	1	2	3	1	0	4	9	0	2	1	0	1	4	3	23	5	0	0	2	0.57	1.36
48	L	-4	-5	-6	-6	-4	-5	-6	-6	-6	0	6	-5	2	0	-6	-5	-4	-4	-4	0			1	0	0	0	0	0	0	0	0	4	83	0	4	2	0	0	0	0	0	6	1.41	1.38
49	A	3	-1	-4	-4	1	-2	-2	-1	3	-2	-2	-1	-2	1	-2	2	-1	-4	4	1			21	3	0	0	3	1	2	3	8	1	3	4	1	5	2	19	2	0	14	9	0.39	1.38
50	K	-2	4	2	2	-5	0	2	-4	2	-5	-5	3	1	-5	-4	0	-1	-6	-4	-5			2	24	9	10	0	4	13	0	5	0	0	18	3	0	0	7	3	0	0	0	0.63	1.38
51	V	3	-5	-4	-5	-1	-5	-5	-5	-5	4	2	-5	2	-2	-5	-2	0	-5	-2	2			25	0	1	0	1	0	0	0	0	27	19	0	4	1	0	2	5	0	2	14	0.66	1.39
52	L	-2	-5	-6	-6	-1	-5	-6	-4	-6	1	6	-5	0	-1	-6	-5	-4	-4	-2	-1			3	0	0	0	1	0	0	1	0	6	84	0	1	2	0	0	0	0	1	2	1.36	1.39
53	R	1	2	0	0	-3	2	2	-1	-3	-5	-5	4	-2	-6	-4	1	-1	-6	-3	-3			12	9	5	4	0	7	12	3	0	0	0	31	1	0	0	9	2	0	1	2	0.52	1.39
54	L	-1	-5	1	-4	-3	-4	-1	-5	-2	3	1	-3	-1	-3	4	-1	0	-3	-1	3			5	0	6	0	0	0	4	0	1	17	12	1	1	0	20	5	5	0	2	21	0.50	1.41
55	F	-5	-5	-6	-6	-5	-6	-6	-6	-4	1	1	-5	1	8	-6	-5	-5	-2	1	-2			0	0	0	0	0	0	0	0	0	9	14	1	3	70	0	0	0	0	2	2	1.71	1.41
56	E	2	0	-2	0	-5	2	3	-3	1	-4	-5	3	-4	-4	-2	1	-1	-1	-2	-5			17	4	1	5	0	11	23	1	3	1	0	16	0	1	1	12	2	1	1	0	0.52	1.42
57	E	-1	2	1	2	-5	0	3	-4	-3	-2	-3	2	-1	-6	-1	0	2	-6	-5	-4			4	11	7	13	0	3	19	0	0	2	2	14	2	0	3	7	13	0	0	0	0.45	1.41
58	N	0	2	2	-4	-2	-2	-1	-4	7	-5	-2	1	-3	3	-5	-2	-3	-1	3	-5			8	10	10	0	1	1	5	0	26	0	5	8	0	14	0	1	1	1	10	0	0.73	1.41
59	D	-3	-1	2	2	-4	0	1	4	4	-6	-6	2	-5	-6	-4	-1	-4	-6	-3	-6			1	3	10	11	0	3	10	32	9	0	0	15	0	0	0	4	0	0	1	0	0.70	1.40
60	V	0	-5	-5	-6	-4	-5	-5	-1	-6	6	-1	-5	-2	-3	-5	-4	-1	-6	-4	4			8	0	0	0	0	0	0	6	0	43	3	0	0	0	0	0	3	0	0	35	1.03	1.41
61	N	-3	-1	7	0	0	0	-1	-3	-1	-4	-6	1	-5	-6	-2	2	0	-6	-5	-5			1	3	51	4	2	4	2	1	1	1	0	9	0	0	2	16	4	0	0	0	1.04	1.44
62	L	-4	-5	-6	-6	-4	-5	-6	-6	-6	3	5	-5	5	-3	-6	-5	-3	-5	-4	2			0	0	0	0	0	0	0	0	0	18	49	0	17	0	0	0	1	0	0	15	1.16	1.44
63	T	-2	-2	-1	-4	3	0	-3	-4	2	0	1	-2	-1	-4	-5	2	5	-5	-2	0			1	1	3	0	6	3	1	1	6	4	13	2	1	0	0	15	36	0	1	6	0.58	1.49
64	H	-4	3	-2	-1	-6	0	-1	-5	9	-2	-4	2	-2	-2	-5	-3	-4	-1	-1	-5			0	14	1	4	0	4	4	0	52	3	1	13	1	2	0	1	0	1	1	0	1.43	1.49
65	I	-4	-6	-6	-6	-4	-6	-6	-7	-6	6	2	-6	0	1	-6	-5	-4	-5	-4	3			0	0	0	0	0	0	0	0	0	58	21	0	1	4	0	0	0	0	0	17	1.34	1.49
66	E	-2	-1	-3	1	-6	0	7	-5	-3	-6	-6	-2	-5	-6	-4	1	-4	-6	-5	-5			3	2	0	5	0	3	79	0	0	0	0	0	0	0	0	8	0	0	0	0	1.62	1.53
67	S	-2	-1	2	-3	-4	-3	-3	-4	-4	-3	-2	-2	-4	-5	-2	6	4	-6	-5	-4			0	3	8	0	0	0	0	0	0	1	5	1	0	0	2	58	22	0	0	0	1.06	1.56
68	R	-4	8	-2	-4	-6	-2	-2	-5	-3	-2	-3	1	-1	-4	-5	-1	-4	-5	-4	-5			0	82	1	0	0	0	1	0	0	3	2	5	1	1	0	3	0	0	0	0	1.79	1.56
69	P	-3	-2	-4	-2	-2	-2	-3	-3	-4	-4	0	2	-4	-5	7	-1	-2	-5	-5	-3			1	2	0	2	1	2	1	2	0	0	9	13	0	0	58	5	1	0	0	1	1.37	1.50
70	S	1	-1	0	-2	-1	-2	-2	-3	-4	-3	0	-3	-2	0	-2	5	-1	-5	-4	-2			12	3	5	1	1	1	1	1	0	1	9	0	1	4	1	52	3	0	0	3	0.69	1.50
71	R	-1	4	2	-3	-5	2	0	-3	0	-5	-2	4	-4	-5	1	-2	-2	-5	-4	-3			4	27	10	1	0	8	6	2	2	0	5	24	0	0	7	2	2	0	0	1	0.64	1.49
72	L	0	4	0	-1	-3	0	-1	-2	-1	-2	-1	3	0	-1	-3	0	0	2	-1	-2			7	21	4	3	0	3	4	2	1	2	7	17	3	2	1	7	6	3	3	3	0.28	1.52
73	K	-1	2	1	-1	-2	-1	1	2	0	-1	-2	2	0	0	-1	0	0	-5	-4	-2			5	9	6	3	1	2	11	16	2	3	4	13	3	4	3	8	5	0	0	2	0.19	1.50
74	K	0	0	2	1	0	-2	-3	1	-2	-3	-1	3	-2	-4	2	1	0	-5	-4	-3			8	6	11	8	2	1	1	10	1	1	7	16	1	1	10	9	7	0	0	1	0.27	1.50
75	D	-2	-2	1	2	-5	-2	0	2	-2	-3	-3	1	-3	-2	-1	0	-2	8	0	0			2	2	8	10	0	1	6	14	1	1	2	8	1	1	3	7	2	20	4	8	0.59	1.50
76	E	-1	-1	1	2	-3	3	4	-1	0	-5	-4	0	-2	-5	-3	1	0	-5	-3	-2			5	2	5	10	1	13	28	4	2	0	1	6	1	0	1	11	7	0	1	3	0.49	1.48
77	Y	-1	-4	-3	-2	-5	0	-4	-5	3	-2	1	-4	1	3	-2	-1	-4	-2	7	-1			5	0	1	3	0	5	0	0	7	1	11	0	3	9	3	4	0	0	43	4	0.96	1.53
78	E	-2	-1	1	3	1	-1	4	-4	-2	0	0	-2	-1	-2	-4	-2	-2	-5	-1	1			3	3	6	19	3	2	27	1	1	7	8	1	1	2	0	2	2	0	2	9	0.43	1.53
79	F	-2	-2	-5	-5	1	-4	-5	-5	-4	2	-1	-4	1	7	-5	-4	-1	-3	2	2			3	3	0	0	3	0	0	0	0	11	4	0	3	45	0	0	4	0	5	16	0.97	1.33
80	F	-4	-4	-5	-5	-4	-4	-5	-5	1	0	2	-4	3	6	-5	-2	0	3	4	-1			0	0	0	0	0	0	0	0	3	5	17	0	7	35	0	2	6	3	14	4	0.90	1.33
81	T	-2	-2	-1	-5	-4	-4	-4	-5	-5	4	1	-4	1	0	-5	-4	0	-5	-3	5			3	2	4	0	0	0	0	0	0	23	11	0	3	3	0	0	5	0	0	43	0.79	1.33
82	H	-2	1	0	5	-5	0	1	-1	0	-2	-5	1	-4	-1	-4	0	0	-5	-1	-2			2	8	4	36	0	3	10	4	2	2	1	10	0	3	0	5	5	0	2	2	0.57	1.33
83	L	-2	-5	-5	-5	7	-4	-5	-5	-4	2	2	-5	1	3	-5	-2	-3	-4	-1	2			2	0	0	0	27	0	0	0	0	11	21	0	4	14	0	2	0	0	2	13	0.92	1.32
84	D	-1	-1	-1	4	-5	0	5	-4	-2	0	-3	-1	-2	-3	-1	0	0	-5	-3	-3			4	3	2	21	0	2	37	0	1	5	2	2	1	1	3	6	6	0	1	2	0.61	1.33
85	K	0	-2	-1	-3	3	-2	-2	4	-4	-1	-2	1	2	1	1	-2	-2	-4	-4	1			7	1	3	1	7	2	2	29	0	3	2	8	5	7	7	2	2	0	0	10	0.37	1.31
86	R	-2	1	2	1	-4	-1	2	-2	7	-2	-3	0	-4	-4	0	-1	1	-5	-3	-2			3	8	10	7	0	2	12	3	25	2	3	6	0	0	5	3	7	0	0	3	0.54	1.29
87	S	-1	2	0	0	-1	0	3	1	1	-4	-4	1	-3	-5	-2	3	-1	-5	-4	-3			3	11	4	6	1	3	17	9	3	1	2	7	1	0	2	25	4	0	0	2	0.38	1.46
88	L	-1	0	1	4	0	0	0	-2	-2	-1	0	1	-1	0	-4	0	0	-4	0	-1			4	6	5	21	2	3	6	2	1	4	11	8	2	4	1	7	5	0	3	5	0.18	1.36
89	P	-1	-1	1	2	-4	2	3	0	0	-1	-4	0	-1	-4	2	0	-3	2	-1	-2			5	4	6	12	0	8	21	8	2	4	1	4	2	0	10	6	1	3	2	2	0.32	1.38
90	A	0	1	3	1	-4	1	2	-4	-1	-1	-2	2	-1	-3	0	-1	-1	-5	-4	0			8	7	15	7	0	6	12	1	1	5	4	14	1	1	4	3	4	0	0	6	0.26	1.43
91	L	-3	-4	-2	-3	-2	-3	-5	-4	-5	2	4	-3	1	0	-5	-1	-2	-4	-1	3			2	1	2	2	1	1	0	1	0	11	40	1	3	4	0	6	2	0	2	21	0.62	1.59
92	T	0	0	2	1	-5	1	1	-1	1	1	0	2	1	-4	-2	0	-2	-5	-3	0			9	5	8	6	0	5	7	4	3	7	10	11	3	1	2	7	2	0	1	6	0.12	1.59
93	N	1	1	2	2	-2	3	2	-2	0	-4	-2	1	-3	-5	-3	0	-1	-5	-4	-4			11	6	9	10	1	16	15	2	2	1	4	10	1	0	1	7	4	0	0	1	0.38	1.59
94	I	3	-4	-3	-5	-2	-4	-2	-3	-2	2	2	-1	0	-2	-5	-1	-1	-5	-2	2			28	0	1	0	1	0	3	2	1	12	21	3	2	1	0	5	3	0	2	15	0.44	1.62
95	I	-2	-4	-3	-5	0	-1	-2	-5	-5	4	3	-2	1	0	-5	-3	-1	-5	-2	1			4	1	2	0	2	2	3	1	0	26	33	3	3	3	0	1	4	0	1	10	0.60	1.62
96	K	-1	1	-1	1	-1	1	3	-2	-1	-3	-1	3	-3	-3	-2	0	-1	-5	-3	-4			6	8	3	7	1	7	20	3	1	1	8	20	0	1	2	6	3	0	1	1	0.35	1.62
97	I	0	0	0	1	0	-1	2	-2	-1	-1	1	-1	2	-1	-4	2	-2	-5	-1	-2			9	6	5	7	2	2	15	3	1	3	15	4	4	2	0	15	2	0	2	2	0.19	1.59
98	L	-2	-2	-3	-2	-4	-1	-3	-6	-2	1	5	-2	2	1	-5	-4	-2	-5	-4	1			3	3	1	2	0	3	1	0	1	6	56	3	4	5	0	0	2	0	0	9	0.76	1.62
99	R	-1	4	-1	-1	-2	2	0	-1	-4	1	-1	3	0	-5	-4	0	-1	-5	-4	-1			4	20	2	3	1	10	5	5	0	7	5	21	2	0	0	6	4	0	0	5	0.39	1.60
100	H	-1	2	2	1	-5	3	3	-1	2	-4	-4	1	-4	-5	-2	0	0	-5	-3	-4			4	12	8	7	0	12	21	4	5	1	1	7	0	0	1	7	6	0	1	1	0.44	1.58
101	D	-3	2	1	2	0	2	2	-2	4	0	-2	1	-3	-3	-3	1	-1	-4	0	-3			1	11	7	10	2	8	12	3	9	6	4	8	0	1	1	9	4	0	4	1	0.28	1.53
102	I	1	-4	-2	-2	4	-3	-2	-3	1	1	0	-4	-3	0	-2	0	1	-2	-1	3			14	1	2	2	10	1	2	2	3	8	8	1	0	4	2	6	9	1	2	24	0.35	1.52
103	G	2	0	-1	0	1	1	-2	1	0	0	-2	0	-1	-4	-1	3	1	-4	-4	-2			15	6	3	4	3	6	1	10	2	5	5	6	1	0	3	20	7	0	0	3	0.20	1.52
104	A	1	-2	-1	-1	-4	-1	0	-2	-4	-2	-1	-2	2	4	-1	3	-1	-2	0	-1			11	2	2	4	0	2	5	3	0	2	6	2	5	20	4	21	4	1	3	4	0.31	1.56
105	T	-2	-1	0	-2	-4	-2	-3	-1	0	0	0	-1	0	2	-1	0	2	-3	4	2			3	3	5	2	0	1	1	5	2	5	8	4	2	7	4	6	13	0	13	16	0.23	1.56
106	V	-2	0	0	-1	2	-1	-2	-3	-4	0	0	3	-1	2	1	-2	-1	-2	-3	2			2	5	4	3	5	3	3	2	0	6	10	18	1	9	6	2	3	1	0	17	0.26	1.61
107	H	-2	0	3	-1	-1	-1	-1	-3	3	0	-1	2	-1	-2	-2	0	-1	-5	-1	3			4	5	12	3	1	3	4	2	8	5	5	12	1	2	2	6	3	0	2	21	0.26	1.60
108	E	-1	-4	-2	-1	-4	-2	2	-4	-3	3	3	-2	-2	0	-5	-2	-1	-5	-3	1			6	0	2	4	0	1	14	1	1	15	32	2	0	3	0	3	4	0	1	9	0.42	1.66
109	L	-2	-3	-1	0	-4	-1	-2	3	-4	1	2	-1	-1	-1	-1	-1	0	3	-4	-1			3	1	3	5	0	3	2	21	0	7	23	5	1	3	3	3	7	4	0	5	0.27	1.67
110	S	-1	-1	1	0	-5	0	-1	0	-4	0	-2	1	1	-5	1	2	1	-5	0	1			5	4	7	5	0	5	4	9	0	6	4	8	3	0	7	13	8	0	3	10	0.13	1.86
111	R	-2	1	1	0	-5	1	1	-1	1	-1	-2	1	1	0	-1	0	0	3	2	-1			3	6	7	6	0	6	9	4	3	4	4	10	3	4	3	8	5	4	6	4	0.14	1.83
112	D	-1	-4	3	2	-5	0	3	-2	1	-2	-2	0	-4	-1	1	0	0	-5	2	-2			5	0	12	11	0	3	17	3	3	3	4	5	0	2	8	7	7	0	6	3	0.27	1.78
113	K	-1	0	1	0	3	1	0	-1	2	0	-1	0	2	1	0	0	0	-5	1	-4			6	6	5	6	5	5	6	6	5	6	6	6	5	5	6	6	6	0	5	0	0.11	1.89
114	K	0	0	0	2	1	0	0	1	-4	0	-2	3	1	-5	0	1	0	-5	-5	0			6	4	4	10	3	4	6	11	0	5	4	15	3	0	4	10	4	0	0	6	0.18	1.80
115	K	0	-4	1	0	-5	1	0	0	3	0	-1	0	2	1	-4	0	0	-4	2	0			6	0	6	6	0	6	6	6	6	6	6	6	6	6	0	6	6	0	6	6	0.13	1.67
116	D	-1	0	1	0	3	1	0	-1	2	0	-4	0	-4	1	0	0	0	4	-4	0			6	6	6	6	6	6	6	6	6	6	1	6	0	6	6	6	6	6	0	6	0.17	1.78
117	T	0	0	1	0	3	1	0	0	-4	0	-1	0	2	-4	-4	0	0	-5	1	0			6	6	6	6	6	6	6	6	0	6	6	6	6	0	0	6	6	0	6	6	0.13	1.67
118	V	0	-4	0	0	-4	0	-1	-1	2	1	-1	-1	2	0	-4	-1	0	-5	-4	4			6	0	4	6	0	4	4	6	5	9	4	4	5	4	0	6	5	0	0	27	0.28	1.63
119	P	1	-1	-2	-2	-3	-1	2	-1	1	0	-2	-2	-3	-3	6	-1	-1	-4	-3	-1			11	3	1	1	0	2	15	4	4	7	3	1	0	1	39	3	3	0	0	3	0.69	0.70
120	W	-3	-3	-3	-1	-3	-3	-3	-3	-3	0	-3	-3	-2	-1	-3	0	2	11	0	-3			1	1	1	4	0	1	1	1	0	8	1	1	0	1	1	10	14	54	0	1	1.66	0.71
121	F	1	1	-3	-3	-3	-3	-3	-3	-2	-1	-2	-3	-2	6	-3	0	0	-1	3	-2			14	8	1	1	0	1	1	1	0	3	1	1	0	48	1	6	6	0	7	1	0.73	0.71
122	P	-2	-2	-3	-3	-4	-2	-2	-3	-3	-4	-4	2	-3	-4	8	-2	-2	-4	-4	-3			1	1	1	1	0	1	1	1	0	1	1	14	0	1	75	1	1	0	0	1	1.81	0.71
123	R	-3	1	1	-3	4	2	1	0	3	-4	0	1	3	-5	1	-3	1	-5	-4	-4			0	8	8	0	8	8	8	8	8	0	8	8	8	0	8	0	8	0	0	0	0.37	1.22
124	T	-3	2	0	2	-1	0	0	-4	5	-5	-4	4	1	-5	-4	2	-1	-5	-2	-3			0	10	4	10	2	2	4	0	14	0	0	29	5	0	0	15	4	0	1	1	0.58	1.04
125	I	0	-2	-4	1	-4	-4	-1	-5	-5	6	0	-4	-2	-3	0	-2	-3	-5	-4	2			9	3	0	9	0	0	3	0	0	51	5	0	0	0	4	3	0	0	0	12	0.72	1.04
126	Q	1	2	1	-3	-2	0	1	-1	1	-4	-4	0	-4	2	-4	3	-1	-4	0	-4			10	10	8	0	1	5	8	5	3	0	0	6	0	12	0	29	3	0	2	0	0.39	1.04
127	E	-4	0	-1	6	-6	1	4	0	4	-6	-6	-2	-5	-5	-4	-2	-3	-6	-4	-5			0	5	0	49	0	5	25	6	10	0	0	0	0	0	0	0	0	0	0	0	1.13	0.98
128	L	-1	-4	-5	-5	-3	-4	-5	-5	-5	0	5	-4	-1	1	-5	-1	1	2	-3	0			4	0	0	0	0	0	0	0	0	4	68	0	0	4	0	5	8	3	0	5	0.87	0.98
129	D	-3	-3	0	7	-5	3	1	-4	-3	-5	-6	1	-5	-6	-4	0	-3	-6	-5	-5			0	0	3	64	0	14	4	0	0	0	0	7	0	0	0	8	0	0	0	0	1.29	0.98
130	R	-3	3	0	-1	-4	1	0	-3	1	-2	-1	3	-3	-1	0	0	0	-4	2	0			0	14	3	2	0	7	4	1	4	1	7	24	0	2	5	6	5	0	7	8	0.29	0.96
131	F	-3	-4	-4	-4	7	-4	-4	-4	-4	2	1	-4	-2	2	-4	1	0	-3	1	0			1	0	0	0	29	0	0	1	0	14	12	0	0	11	0	12	6	0	6	7	0.77	1.03
132	A	0	1	1	1	4	1	-3	0	3	-4	-4	-3	-3	-3	-4	0	1	-4	2	0			8	8	8	8	8	8	0	8	8	0	1	0	0	0	0	8	8	0	8	8	0.30	1.23
133	N	-2	-1	4	1	-2	2	0	1	7	-6	-5	-1	-1	-5	1	-1	-3	-5	-3	-5			2	3	18	8	1	12	5	11	25	0	0	4	1	0	6	3	1	0	0	0	0.72	1.21
134	Q	-3	2	-1	-1	2	2	2	-5	3	-3	2	-1	-2	-4	-4	-1	0	-5	-4	1			0	13	2	3	4	11	16	0	8	0	23	3	0	0	0	3	5	0	0	9	0.38	1.15
135	I	0	-4	-1	-5	0	-4	-4	-5	2	4	2	-2	2	-3	-5	-3	1	-5	-4	4			8	0	4	0	2	0	0	0	5	21	16	2	6	0	0	0	7	0	0	29	0.57	1.13
136	L	-3	1	-4	-1	-4	-3	-2	-1	-4	0	3	-3	5	-3	-5	-1	3	-5	-4	-1			0	7	0	5	0	0	3	6	0	3	30	0	18	0	0	3	23	0	0	3	0.58	1.19
137	S	0	0	0	0	-5	0	1	-2	2	-1	-2	3	4	-5	-4	2	-1	-5	-4	-1			7	5	5	4	0	4	11	2	5	4	3	22	11	0	0	13	2	0	0	3	0.34	1.26
138	Y	0	1	0	-4	-4	-3	-4	-5	2	0	-1	-3	2	3	-5	0	0	-3	4	2			7	9	4	0	0	0	0	0	4	4	4	0	6	14	0	9	5	0	13	19	0.39	1.11
139	G	-3	-3	2	2	-4	2	1	1	-3	1	-4	1	-3	-5	-4	1	1	-5	-4	1			0	0	10	10	0	10	10	10	0	10	0	10	0	0	0	10	10	0	0	10	0.30	1.00
140	A	2	-2	-2	-2	-4	-3	1	0	-4	-1	-4	-2	-4	-5	5	1	-1	-6	-5	-1			22	1	1	2	0	0	9	7	0	5	0	2	0	0	32	13	2	0	0	4	0.71	1.18
141	E	1	-3	1	4	-5	1	4	-3	4	-5	-5	-3	-4	-5	-4	-1	1	-6	-4	-3			14	0	5	21	0	5	28	2	11	0	0	0	0	0	0	3	10	0	0	2	0.70	1.17
142	L	-1	-4	1	-4	-4	0	-4	-5	-4	-2	3	-2	0	0	4	-2	2	-5	-4	-3			4	0	7	0	0	4	0	0	0	0	36	1	2	4	19	3	13	0	0	0	0.49	1.17
143	D	0	2	1	4	2	2	-1	-4	-3	-5	-5	-2	-1	-5	-4	2	0	-5	-5	-4			6	11	7	29	4	9	3	0	0	0	0	0	2	0	0	14	7	0	0	0	0.50	1.22
144	A	1	-2	-3	1	1	0	2	-4	1	0	0	0	3	-4	0	0	0	-5	-4	-2			12	2	0	7	3	5	12	0	3	7	9	6	8	0	5	7	4	0	0	1	0.11	1.22
145	D	1	2	-1	3	-4	1	-1	-2	-3	1	0	-1	-3	0	-4	-1	-3	-4	-1	-3			12	13	3	19	0	8	3	2	0	9	8	4	0	4	0	3	0	0	2	0	0.19	1.11
146	H	-3	2	-3	2	-4	0	-2	-4	5	-4	-1	-2	-3	2	3	1	-3	-3	3	-4			0	11	0	11	0	5	0	0	13	0	5	0	0	8	14	12	0	0	11	0	0.39	0.94
147	P	1	-3	-3	-3	-4	2	1	1	-3	-2	0	-3	-2	-4	2	-2	1	-4	-4	1			11	0	0	0	0	11	11	11	0	0	11	0	0	0	11	0	11	0	0	11	0.19	0.89
148	G	1	-3	-2	0	0	-1	1	6	-3	-5	-4	-1	-4	-5	-3	-2	0	-4	-4	-4			9	0	0	5	2	3	9	56	0	0	1	4	0	0	0	1	6	0	0	1	0.88	1.01
149	F	0	-1	-4	-4	-4	-3	-2	-4	0	-1	-1	-1	5	6	-4	-3	-3	-2	3	-1			7	3	0	0	0	0	3	1	2	2	4	4	19	41	0	1	0	0	8	4	0.86	1.04
150	K	1	-1	0	-2	-4	-1	-2	0	3	-4	-3	3	-3	-4	3	2	2	-4	-3	-3			11	2	3	2	0	3	0	6	7	0	2	17	0	0	15	14	16	0	0	0	0.42	1.04
151	D	0	-4	0	6	-4	-3	0	1	-3	-4	-3	-3	-4	-5	0	-2	1	-5	-4	-4			6	0	3	57	0	0	3	11	0	0	2	0	0	0	4	0	11	0	0	0	1.00	1.04
152	P	0	-1	0	1	-4	1	1	-1	-2	0	-1	1	-1	-4	2	-1	2	-5	-4	1			7	2	5	7	0	7	9	4	1	4	5	8	1	0	12	2	13	0	0	13	0.20	1.04
153	V	1	-4	-1	-2	-4	-2	2	-4	-4	0	-1	-2	0	-2	-4	0	0	-5	-4	4			15	0	3	2	0	1	13	0	0	4	4	1	2	1	0	7	6	0	0	41	0.48	1.05
154	Y	-3	-3	-3	-4	-4	2	-3	-4	-2	-4	-3	-3	-3	3	-4	1	3	-1	7	-3			0	0	0	0	0	13	0	0	0	0	0	0	0	10	0	11	22	0	44	0	1.07	1.02
155	R	1	5	-2	-1	-5	0	-3	-2	-3	-1	-4	0	-1	-5	2	-3	1	4	-4	-2			14	37	2	3	0	3	0	3	0	4	0	5	2	0	9	0	11	6	0	2	0.63	1.18
156	A	1	0	-2	-1	3	2	2	-4	-3	-3	-3	5	0	-5	2	-1	-2	-5	-4	-2			11	4	1	2	6	7	13	0	0	1	2	36	2	0	8	2	2	0	0	2	0.56	1.18
157	R	-3	6	2	-3	-5	1	-2	-4	-3	-4	1	2	-3	-5	2	-1	-3	-5	-4	-4			0	45	11	0	0	5	0	0	0	0	14	9	0	0	11	5	0	0	0	0	0.82	1.00
158	R	-3	6	-3	0	-4	1	-2	-4	-3	-3	-1	1	2	-4	-4	-3	0	-4	-3	0			1	50	0	6	0	6	1	1	0	0	7	7	6	0	0	1	6	0	0	7	0.80	1.06
159	K	0	1	1	1	-4	1	1	0	-3	-4	-4	1	3	-4	-4	0	1	-4	2	0			8	8	7	8	0	7	8	8	0	0	1	8	7	0	0	8	8	0	7	8	0.22	1.34
160	Q	-2	0	-3	2	-5	3	2	-4	2	0	-2	-1	1	0	-4	0	-2	1	3	-1			4	4	0	14	0	13	14	1	5	6	2	3	4	4	0	8	1	2	12	3	0.36	1.46
161	F	-4	-5	0	-5	-4	-4	-5	-2	0	4	0	-4	-2	5	1	-4	-4	3	4	-1			0	0	6	0	0	0	0	3	3	29	8	0	0	21	8	0	0	3	13	3	0.75	1.45
162	A	4	-4	-1	0	0	1	-2	-2	-4	-4	-2	-3	-1	-1	-2	2	1	-5	-4	0			42	0	3	5	2	6	2	3	0	0	3	0	1	3	2	14	7	0	0	6	0.54	1.56
163	D	0	2	0	3	-5	2	2	-2	5	-5	-3	0	0	-5	0	-1	-1	-5	-4	-3			6	10	3	18	0	9	15	3	13	0	3	5	2	0	4	3	4	0	0	2	0.45	1.60
164	I	0	-1	-4	-2	1	0	-4	-5	-5	3	2	-1	1	-1	-1	-1	3	-5	-4	1			8	4	0	3	3	4	0	0	0	14	20	4	3	3	3	4	18	0	0	9	0.33	1.61
165	A	4	1	0	0	-4	0	-1	0	1	-5	-2	0	-4	-5	1	0	0	-5	-4	-4			36	8	5	5	0	5	4	6	4	0	4	4	0	0	7	7	5	0	0	0	0.43	1.64
166	Y	-1	0	1	0	-5	1	0	-1	2	0	-1	0	2	1	0	0	0	-5	1	0			6	6	5	6	0	5	6	6	5	6	6	6	5	5	6	6	6	0	5	6	0.08	1.89
167	N	-1	0	2	2	-5	1	-1	-1	3	-2	0	1	-4	2	-1	-1	-1	-5	-1	0			5	6	9	13	0	5	3	4	7	2	11	9	0	8	3	4	4	0	2	6	0.18	1.75
168	Y	1	-2	-4	-5	0	-1	-4	-5	4	-4	-2	-1	-4	1	0	-1	-4	4	6	1			12	2	0	0	2	3	0	0	10	0	3	4	0	6	6	4	0	6	30	10	0.76	1.75
169	R	-2	4	0	-1	-5	0	-3	-2	2	-1	-2	3	1	-5	-1	-1	2	-5	0	-1			4	19	3	4	0	4	0	4	6	4	5	17	3	0	4	4	12	0	3	4	0.34	1.89
170	H	-1	0	0	0	2	0	0	-1	2	0	-1	0	2	1	0	-1	0	4	1	-1			5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	0.09	2.12
171	G	0	-3	1	1	-4	1	0	0	3	0	-4	0	2	-4	1	0	0	4	-4	0			7	0	7	7	0	7	7	7	6	7	1	7	6	0	7	7	7	6	0	7	0.23	1.56
172	Q	-3	-1	1	5	0	4	1	-2	-1	-2	-3	0	-1	-5	-4	0	-1	-5	-5	0			2	3	5	28	2	21	7	2	1	2	2	5	1	0	0	7	3	0	0	7	0.58	1.60
173	P	0	-1	-2	1	-5	1	1	-2	0	-2	-3	0	-4	-5	6	0	-1	-5	-5	-3			9	3	2	6	0	7	10	3	3	2	2	5	0	0	39	5	3	0	0	2	0.77	1.58
174	I	-3	-1	0	0	-4	4	-1	-2	1	4	0	-1	1	-4	0	-2	-1	-5	-4	0			1	3	4	5	0	21	4	3	3	22	9	3	4	0	4	3	3	0	0	7	0.39	1.56
175	P	-1	-2	0	0	-5	-1	-1	4	2	-5	-5	-1	-4	-1	5	-3	-2	-5	-4	-2			5	3	4	4	0	3	4	28	4	0	1	3	0	3	29	0	3	0	0	3	0.70	1.60
176	R	-4	0	1	0	3	1	0	-5	2	0	-1	0	2	1	-5	0	0	4	2	0			0	6	6	6	6	6	6	0	6	6	6	6	6	6	0	6	6	6	6	6	0.23	1.78
177	V	-1	-2	-2	0	-3	-2	0	-4	-1	4	-1	-3	-1	-3	-2	-2	-1	-4	-1	4			5	2	2	6	0	2	7	1	1	27	3	1	1	0	2	2	3	0	2	31	0.54	1.30
178	E	0	-1	0	2	-4	0	4	-2	2	-2	-2	-1	-3	-4	-1	-1	0	-4	-3	-2			7	3	4	13	0	4	34	3	6	2	4	3	0	1	3	3	7	0	0	3	0.45	1.12
179	Y	-4	-3	-4	-4	-4	-3	-4	-4	3	-3	-3	-4	-3	3	-4	-3	-3	8	8	-3			1	1	1	1	0	0	1	1	7	1	1	1	0	9	1	1	1	18	56	1	1.70	1.05
180	M	-1	-4	0	0	2	-3	-3	0	-4	-4	-2	-1	1	0	1	3	4	-5	-4	-3			5	0	4	5	4	0	0	8	0	0	5	4	4	4	6	21	29	0	0	0	0.47	1.45
181	E	0	-1	0	2	-5	1	3	-1	1	-1	-2	0	-4	-4	2	-1	-1	-5	0	-2			7	4	3	10	0	7	23	4	3	3	4	7	0	0	9	5	4	0	3	3	0.29	1.45
182	E	1	-1	-1	1	-1	2	5	-2	-1	-1	-4	-2	-3	-4	-2	0	0	-1	-4	0			11	3	2	7	1	10	35	2	1	5	1	1	0	0	2	6	6	1	0	6	0.46	1.17
183	E	-3	-2	1	3	-5	1	6	-3	-2	-4	-4	-2	-4	-4	-3	-2	-3	-4	-4	-4			1	1	8	18	0	4	61	1	0	1	1	1	0	0	1	1	1	0	0	1	1.15	1.04
184	K	-3	-1	2	0	-4	2	-1	-3	7	1	-2	0	-3	-1	-3	-2	0	-4	-2	-1			1	2	10	4	0	8	3	1	35	9	3	6	0	2	1	1	7	0	0	5	0.71	1.04
185	K	3	0	0	2	-1	0	2	-1	-1	-3	-3	1	-1	-4	-3	-1	-1	4	-3	-2			25	4	5	10	1	4	15	3	2	2	2	10	2	0	1	3	2	5	0	2	0.34	1.18
186	T	-1	-1	-3	-4	-3	-3	-3	-4	-4	3	1	-3	1	-3	-4	-2	5	-4	-3	2			4	4	0	1	0	0	1	1	0	17	10	1	4	0	1	1	39	0	0	17	0.68	1.18
187	W	-4	0	-5	-5	-4	-4	-4	-4	-4	-4	-3	-4	-3	1	-5	-4	-4	12	2	-4			1	5	0	1	0	0	1	1	0	1	1	1	0	5	1	1	1	76	6	1	2.92	1.18
188	G	-1	3	0	1	-5	1	-1	2	3	-1	-4	1	1	-4	-4	0	-1	-5	0	-4			5	18	4	7	0	7	4	19	6	4	1	8	4	0	0	6	4	0	4	0	0.37	1.62
189	T	0	3	-1	1	2	1	1	-4	1	0	-1	-1	1	0	-4	-1	2	-5	0	0			6	14	3	7	4	5	8	0	4	6	6	4	3	3	0	3	14	0	4	6	0.19	1.62
190	V	-1	-5	-5	-5	2	-4	-5	-5	-5	2	3	-5	-2	-3	-2	-4	-3	-5	-4	5			4	0	0	0	5	0	0	0	0	8	29	0	0	0	2	0	0	0	0	47	0.91	1.36
191	F	-2	-4	-5	-5	0	-4	-5	-5	1	0	0	-5	4	5	-5	-2	-2	4	6	0			2	0	0	0	2	0	0	0	4	4	7	0	11	24	0	3	3	5	27	6	0.94	1.48
192	K	0	4	1	1	-5	0	2	-1	0	-3	-3	0	-4	-5	-1	0	1	-5	-4	-2			8	25	7	7	0	4	12	5	2	1	3	5	0	0	3	6	10	0	0	3	0.37	1.48
193	T	-2	5	0	-2	-1	2	2	-4	-1	-2	-2	2	-1	-5	-4	0	1	-5	-4	-3			2	28	5	2	1	7	14	0	2	3	5	13	1	0	0	6	9	0	0	2	0.52	1.48
194	L	-4	1	1	-4	-5	5	-3	-5	1	0	3	-3	2	-4	-5	-1	-1	-5	-4	-1			0	8	8	0	0	26	0	0	4	4	33	0	4	0	0	3	3	0	0	5	0.68	1.56
195	K	0	0	-1	-4	0	1	-1	-3	3	-1	0	2	3	-1	-5	0	1	1	-1	1			7	5	3	0	2	5	3	2	7	2	11	14	6	2	0	6	8	2	2	12	0.20	1.56
196	S	-1	0	1	0	3	1	0	-1	2	0	-1	0	2	-5	1	0	0	-6	-5	0			6	6	6	6	6	6	6	6	6	6	6	6	6	0	6	6	6	0	0	6	0.14	1.78
197	L	-1	-1	0	-6	1	-1	-5	-3	0	0	4	0	2	0	-6	-4	-2	-5	1	1			4	3	4	0	3	3	0	2	2	4	45	6	5	3	0	0	2	0	4	9	0.54	1.61
198	Y	-1	-5	-2	-6	-5	-4	-5	-3	3	1	3	-3	1	0	-6	-2	-2	3	5	0			5	0	2	0	0	0	0	2	8	9	31	2	4	2	0	3	2	3	22	6	0.67	1.61
199	K	0	2	0	-1	-5	1	1	-2	1	-3	-3	1	1	-6	4	0	-1	-6	-5	-2			6	10	5	2	0	7	9	2	4	2	2	9	3	0	24	7	4	0	0	3	0.45	1.61
200	T	-1	0	-1	2	-5	0	-1	3	-1	-4	-3	2	-4	-5	-1	0	3	-6	-3	-3			4	5	2	12	0	4	2	20	2	1	3	16	0	0	2	5	20	0	1	1	0.42	1.51
201	H	-3	4	-1	-2	-1	-3	-4	-5	7	-2	0	0	-1	-1	-5	-3	-1	-4	3	1			1	20	2	2	2	0	0	0	28	1	9	4	2	3	0	1	3	0	11	11	0.78	1.51
202	A	6	-5	-5	-5	2	-4	-4	-3	-5	-4	-1	-4	0	-2	-4	-1	-1	-5	-4	-1			77	0	0	0	4	0	0	0	0	0	5	0	2	2	0	3	4	0	0	3	1.25	1.51
203	C	0	-5	-2	-5	10	-5	-5	-5	4	-2	-4	-5	-4	0	-1	0	-1	-5	-2	-1			9	0	1	0	55	0	0	0	10	2	0	0	0	4	3	7	3	0	1	4	1.65	1.48
204	Y	-1	0	1	0	-5	1	0	-1	2	0	-1	0	2	-4	0	0	0	-5	1	0			6	6	6	6	0	6	6	6	6	6	6	6	6	0	6	6	6	0	6	6	0.10	1.78
205	E	0	0	-3	0	1	1	5	-2	1	-4	-2	-1	-4	0	0	0	-3	-5	-4	-1			8	4	0	4	4	6	36	4	4	0	4	4	0	4	4	7	0	0	0	5	0.48	1.62
206	Y	-3	-4	-4	-5	-5	-1	-4	-5	4	-2	-2	-4	-3	5	-5	-4	-4	-1	7	-2			2	0	0	0	0	2	0	0	11	2	3	0	0	27	0	0	0	0	47	3	1.46	1.32
207	N	-3	-1	3	-1	0	0	-4	-5	-2	2	4	-2	3	-1	-5	-2	-3	-5	-4	0			1	4	14	3	2	4	0	0	1	10	40	3	6	2	0	3	0	0	0	6	0.53	1.36
208	H	0	2	1	3	0	2	3	-3	3	-4	-5	0	-1	-3	-2	-2	-2	-5	1	-3			7	10	5	15	2	10	22	1	7	1	0	5	2	1	1	2	2	0	5	1	0.46	1.46
209	I	2	-5	2	-1	2	-4	-4	5	0	-1	-3	-4	0	-3	-5	-1	-2	-5	-2	0			18	0	12	3	4	0	0	38	2	3	2	0	2	1	0	4	1	0	2	7	0.64	1.46
210	F	-2	-5	-5	-6	-4	-5	-5	-6	-5	1	4	-2	1	5	-3	-5	-2	4	0	0			2	0	0	0	0	0	0	0	0	8	39	3	3	28	2	0	2	5	2	6	0.89	1.42
211	P	0	0	-1	1	-2	1	2	-1	1	-3	-3	1	0	-5	4	-1	-2	-5	-4	-3			8	4	3	7	1	6	14	6	3	1	3	10	3	0	21	4	2	0	0	2	0.37	1.36
212	L	-2	1	-3	0	-4	1	-1	-2	0	0	4	3	0	-3	-4	-2	-2	-4	-4	-2			3	9	0	6	0	6	3	3	2	5	36	16	2	0	0	2	3	0	0	2	0.40	1.34
213	L	-1	-4	-4	-4	-3	-4	-4	-1	-4	1	5	-4	2	2	-4	-4	-3	-3	-3	-1			5	1	1	1	0	1	1	5	0	7	58	1	4	9	1	1	1	0	0	3	0.81	1.27
214	E	-1	-1	-1	1	-1	3	3	-1	-2	0	-1	0	-1	-3	-1	1	-1	-3	-3	0			4	3	2	8	1	12	23	4	1	5	6	6	2	1	3	9	4	0	1	6	0.25	1.14
215	K	-1	1	-1	2	-3	0	2	-1	-1	-1	-1	3	-1	-2	-2	-1	-1	-3	-2	-2			4	8	3	13	1	4	16	5	1	3	6	18	1	2	2	3	3	0	1	4	0.26	1.02
216	Y	1	-1	2	0	-1	-1	2	-2	3	-2	-1	-1	-2	0	-2	-1	0	-2	3	-2			13	3	10	4	2	2	12	3	8	2	6	4	1	4	2	5	6	0	9	3	0.20	1.02
217	C	-1	0	-1	-1	7	-1	-2	0	-3	-1	1	-1	0	-2	-2	0	0	-3	-3	-1			5	5	2	3	26	2	2	7	1	4	17	4	2	1	2	6	6	0	1	3	0.51	1.10
218	G	0	0	1	-1	-4	-3	-3	5	0	1	-2	-2	-3	-4	-3	-2	-3	-4	-4	-1			7	5	6	3	0	1	2	49	2	8	4	2	0	1	2	2	1	0	0	5	0.71	1.34
219	F	-2	-2	-5	-5	-1	-4	-5	-4	-3	1	3	-4	3	4	-5	-3	-4	-2	6	-1			3	3	0	0	1	0	0	1	0	9	27	0	6	16	0	1	0	0	27	2	0.85	1.46
220	H	0	3	0	-1	0	-1	0	0	-1	-4	-2	-1	-4	-3	3	2	0	-5	-4	-1			6	17	5	3	2	2	5	8	1	0	4	3	0	1	14	19	5	0	0	4	0.32	1.46
221	E	1	2	-2	0	-3	0	4	-1	-1	0	-2	1	-4	-5	3	-1	-1	-5	-5	-2			11	9	1	5	1	3	25	5	2	5	4	7	0	0	12	3	3	0	0	2	0.36	1.52
222	D	-3	-2	1	5	-4	0	3	0	1	-4	-4	-1	-3	-1	0	0	1	-4	-1	-4			1	2	6	32	0	4	16	7	3	1	1	2	0	3	4	6	8	0	2	1	0.52	0.98
223	N	-1	4	2	-1	-1	1	0	1	3	-3	-4	1	-1	-1	-3	0	-1	-4	-1	-3			4	23	9	4	1	6	6	12	6	2	1	7	1	2	1	7	3	0	2	2	0.32	0.99
224	I	-1	-4	-4	-4	-3	-4	-4	-4	-4	6	1	-4	2	-2	-4	-3	-1	-4	-3	3			6	1	0	1	0	0	1	1	0	53	9	1	4	0	1	1	3	0	0	18	0.90	1.01
225	P	-1	-4	-4	-3	-4	-3	-1	-4	-4	0	-4	-3	-4	-4	8	-3	-3	-5	-4	-3			4	1	0	1	0	0	5	1	0	5	1	1	0	0	78	1	1	0	0	1	1.97	1.01
226	Q	-3	1	1	3	-1	6	0	-3	0	-4	-4	1	-3	-5	-3	0	-2	-4	-4	-2			1	6	7	19	2	41	3	1	2	0	1	7	0	0	0	6	2	0	0	3	0.78	1.05
227	L	-3	-4	-4	-5	-4	-2	-4	-5	1	1	4	-2	2	5	-2	-4	-3	-3	0	0			1	0	0	0	0	2	0	1	5	8	39	2	6	25	3	1	0	0	2	4	0.72	1.05
228	E	0	1	-1	3	-4	3	3	-1	-1	-4	-3	-1	-1	-4	0	1	-2	-4	0	-2			7	8	3	15	0	12	23	4	1	0	2	3	1	0	4	8	2	0	3	3	0.38	1.19
229	D	0	0	-1	5	-5	1	3	-1	1	-3	-3	-1	-4	-3	-4	-2	0	-5	-4	-2			7	5	1	36	0	5	20	4	3	1	3	2	0	1	0	1	6	0	0	3	0.62	1.19
230	V	-1	-4	-4	-4	-3	-4	-4	-4	-4	3	1	-4	3	-3	-4	-1	0	-4	-3	5			5	0	0	0	0	0	1	1	0	19	13	1	8	0	0	5	6	0	0	38	0.71	1.21
231	S	-1	-3	4	0	1	-3	-3	-1	1	-2	-4	-3	-4	-4	-1	4	0	-4	0	-4			4	0	23	5	3	0	0	3	3	3	1	0	0	0	3	39	6	0	4	0	0.66	1.24
232	Q	-1	0	1	0	3	1	0	-1	2	0	-1	0	2	-5	0	0	0	4	-4	0			6	6	5	6	5	5	6	6	5	6	6	6	5	0	6	6	6	5	0	6	0.14	1.89
233	F	-2	0	-1	-5	1	-1	-1	-2	1	-1	-2	0	-4	6	0	-2	-1	2	2	1			4	5	4	0	3	3	3	3	3	4	3	5	0	29	6	3	4	3	6	9	0.45	1.89
234	L	-4	-5	-5	-5	-4	-4	-5	-5	-5	1	6	-5	2	1	-1	-5	-4	-4	-3	0			1	0	0	0	0	0	0	0	0	5	72	0	4	5	4	0	0	0	0	4	1.20	1.53
235	Q	0	1	-1	-1	-5	2	0	-1	2	-3	-2	4	2	-2	-4	1	-1	-5	-1	-3			6	6	2	3	0	9	6	4	4	2	4	30	6	2	0	10	2	0	2	2	0.41	1.56
236	T	2	1	-2	1	0	0	3	-2	-3	-4	-2	2	-3	-5	1	0	0	-5	-4	-3			17	9	1	7	2	4	19	2	0	0	5	12	0	0	8	7	5	0	0	2	0.34	1.17
237	C	1	3	-2	-3	2	0	1	-4	1	0	0	1	0	-3	-4	0	1	-4	-3	-2			12	19	1	0	5	3	11	1	4	5	10	9	3	1	0	5	9	0	0	2	0.27	1.15
238	T	-2	-3	-2	-3	-3	-2	-3	-1	-3	-3	-2	-3	-3	-4	-3	2	7	-4	-3	-2			2	1	1	1	0	1	1	4	0	1	2	1	0	0	1	12	71	0	0	2	1.18	1.11
239	G	-2	-2	1	-1	-4	0	-3	6	-1	-5	-5	-3	-4	-5	-4	-1	-3	-4	-4	-5			1	2	5	3	0	4	0	75	2	0	1	0	0	0	0	4	0	0	0	0	1.35	1.03
240	F	-4	-4	-5	-5	-4	-4	-4	-4	-4	-3	-2	-4	2	6	-5	-4	-4	10	1	-3			1	0	0	0	0	0	0	1	0	0	2	0	6	37	0	1	0	47	2	0	2.09	1.03
241	R	0	3	-1	-2	-2	3	1	-1	1	-3	-3	0	-1	-4	-3	1	3	-4	-3	-3			9	15	2	1	1	14	10	5	3	1	2	5	1	0	1	8	22	0	0	1	0.39	1.00
242	L	0	-4	-4	-4	1	-4	-4	-4	-4	3	4	-4	0	-1	-4	-2	0	-3	0	3			8	1	0	1	3	0	1	1	0	16	36	1	1	2	1	2	4	0	4	19	0.54	1.00
243	R	0	4	-3	-3	0	1	-1	-3	-3	-1	-2	0	-2	-3	-3	-1	-1	-4	0	3			8	29	0	1	2	6	4	2	0	2	3	4	0	0	1	3	3	0	4	28	0.47	1.00
244	P	2	-2	-2	-1	-2	-2	-2	-1	-3	-3	-4	-1	-2	-4	7	-1	-1	-2	-4	-2			18	2	1	4	1	1	1	4	0	1	1	4	1	0	50	2	3	1	0	3	1.05	1.00
245	V	2	-4	-4	-4	0	-3	-4	-2	-4	0	-1	-4	-2	-3	-4	-3	0	-4	-3	6			15	1	0	1	2	0	1	3	0	2	3	1	0	0	1	1	4	0	0	64	0.89	1.00
246	A	2	-2	-3	1	-4	1	0	0	1	-4	-4	-1	-3	-2	5	0	-1	-4	-3	-2			22	2	0	7	0	6	7	6	4	1	1	3	0	2	30	5	2	0	0	2	0.58	0.96
247	G	1	-4	0	0	-4	-3	-1	6	-3	-4	-4	-3	0	-4	-3	-2	-3	-4	-4	-4			11	1	3	5	0	0	3	67	0	1	1	1	3	0	1	1	1	0	0	1	1.15	0.96
248	L	-2	-4	-4	-4	-3	-1	-4	-2	-3	0	4	-4	1	3	-4	-3	-3	1	3	-1			4	1	0	1	0	3	1	3	0	5	50	1	3	11	1	1	1	2	11	2	0.65	0.96
249	L	-3	-4	-4	-4	2	-4	-4	-5	-4	4	4	-4	2	-2	-4	-4	-3	-4	-3	3			1	1	0	1	4	0	1	1	0	30	38	1	5	0	1	1	1	0	0	16	0.79	0.96
250	S	-1	1	1	0	-4	-2	-2	-2	1	-3	-4	-1	-3	-4	6	3	0	-4	-4	-3			2	7	5	5	0	0	1	3	4	2	1	2	0	0	37	23	6	0	0	1	0.80	0.96
251	S	1	-3	0	2	-4	1	0	-1	-1	-3	-2	-3	-3	2	4	0	-3	-3	0	-2			12	1	4	14	0	7	5	5	1	1	3	1	0	11	23	6	1	0	3	2	0.43	0.96
252	R	0	5	0	2	-4	0	0	-2	3	-4	-2	0	-3	-2	-1	-1	0	-4	-3	-2			8	34	3	12	0	3	6	3	6	1	3	3	0	2	3	3	6	0	0	4	0.47	1.08
253	D	0	1	0	4	-4	0	2	-4	1	-1	-1	-1	-3	-4	0	-2	0	-4	-4	1			7	9	3	23	0	4	14	1	4	3	5	2	0	0	4	2	6	0	0	12	0.32	1.08
254	F	-4	-4	-4	-4	-4	-4	-4	-4	-3	-2	-2	-4	-2	8	-4	-4	-4	2	3	-3			1	1	0	1	0	0	1	1	0	1	1	1	0	80	1	1	1	3	7	1	1.77	0.97
255	L	-3	-4	-4	-4	0	-4	-4	-4	-3	0	3	-4	3	6	-4	-4	-2	-2	3	-2			1	1	0	1	2	0	1	1	0	3	27	1	8	42	1	1	3	0	8	1	0.94	0.97
256	G	2	-1	1	2	-4	0	4	0	-3	-4	-4	-1	-3	0	-3	1	-1	2	-2	-3			18	2	6	11	0	3	27	8	0	1	1	3	0	4	1	8	3	3	1	1	0.40	0.97
257	G	-1	-3	-3	-3	0	-2	-1	2	5	-2	2	-3	2	2	-4	1	-3	-3	0	-2			4	1	0	1	2	1	4	14	16	1	24	1	6	8	1	10	1	0	3	1	0.38	0.97
258	L	-3	-3	-4	-4	-3	2	-3	-4	-4	1	5	-3	1	-1	-4	-4	-3	-1	-3	-1			1	1	0	1	0	12	1	1	0	5	71	1	2	1	1	1	1	1	0	1	0.94	0.97
259	A	6	-1	-2	-3	-3	-1	-2	0	-3	-3	-2	-2	-3	-4	-3	1	-2	-4	-1	-2			68	3	1	1	0	2	2	5	0	1	3	1	0	0	1	10	1	0	2	1	0.87	0.97
260	F	-2	-1	4	1	-4	1	0	-2	2	-3	-2	-1	-1	4	-4	-1	-2	-3	2	-3			3	3	25	9	0	6	6	3	6	1	3	2	1	19	1	2	1	0	8	1	0.49	0.97
261	R	-2	6	0	-3	-1	0	-2	0	0	-4	-4	3	-3	-4	-2	-1	-3	-4	-3	-4			2	47	5	1	2	3	1	8	2	1	1	22	0	0	2	3	1	0	0	1	0.87	0.94
262	V	-2	3	-2	-3	-3	1	-1	-3	-1	0	0	1	-1	-2	-3	-2	-1	-4	-1	4			2	19	2	1	0	6	4	2	1	3	8	7	1	1	1	2	4	0	3	35	0.41	0.95
263	F	-4	-4	-4	-2	-4	-4	-5	-5	0	-2	1	-5	-2	8	-5	-4	-4	-1	2	-3			0	0	0	2	0	0	0	0	2	0	16	0	0	71	0	0	0	0	4	0	1.51	1.00
264	H	-2	-2	-2	-3	0	1	-3	-4	5	-3	-4	-3	-3	-2	7	-3	-3	-5	-1	1			3	2	2	0	2	6	0	0	15	0	0	0	0	2	52	0	0	0	2	12	1.24	1.00
265	C	1	-4	-3	-4	6	-3	-4	-4	2	3	-1	-4	-2	-3	-4	2	0	-4	-3	3			13	0	0	0	17	0	0	0	5	20	3	0	0	0	0	17	5	0	0	18	0.60	1.00
266	T	4	0	-3	-4	-3	-3	-3	0	-4	-4	-4	-3	-3	-4	-3	0	5	-4	1	-3			40	4	0	0	0	0	0	6	0	0	0	0	0	0	0	5	40	0	5	0	0.83	1.06
267	Q	-1	0	0	0	2	2	-3	-4	-4	1	0	-3	1	-4	-4	2	3	4	-4	-1			5	6	4	5	4	11	0	0	0	8	9	0	4	0	0	15	18	6	0	4	0.34	1.22
268	Y	-4	0	0	2	-5	0	0	-5	2	-4	-4	-4	-4	5	-5	-4	0	7	3	-1			0	5	5	13	0	5	7	0	5	0	0	0	0	23	0	0	6	16	11	5	0.81	1.22
269	I	-3	-5	-5	-2	-4	-4	-5	-6	-5	6	2	-5	5	-3	-5	-4	-1	-5	-4	2			0	0	0	3	0	0	0	0	0	51	14	0	16	0	0	0	3	0	0	13	1.06	1.11
270	R	-4	8	-3	-4	-6	-1	-3	-5	-3	-6	-5	0	-4	-6	-5	-3	-4	-5	-4	-5			0	100	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2.22	1.22
271	H	-2	1	-1	-1	0	1	-1	-2	8	-5	-5	1	-4	-4	-1	0	1	-5	0	-2			2	8	3	3	2	5	3	3	41	0	0	7	0	0	3	7	8	0	2	2	0.89	1.22
272	G	0	2	-2	-2	0	-1	1	0	2	-1	-1	-1	0	-2	3	0	-2	1	-1	-1			6	14	2	2	2	2	8	8	6	3	5	4	2	2	17	8	2	2	2	5	0.23	1.22
273	S	0	-1	1	2	-5	0	4	-1	2	-5	-5	-1	-4	-2	-2	4	0	-6	-4	-5			5	2	6	12	0	2	24	3	4	0	0	3	0	2	2	29	5	0	0	0	0.59	1.31
274	K	-1	-1	0	3	-1	3	3	-4	3	-5	-5	0	-1	-5	-4	2	-1	0	-2	-2			4	3	4	17	1	12	22	0	6	0	0	6	2	0	0	14	4	1	2	2	0.52	1.31
275	P	-3	-4	-4	2	-4	-4	-2	-5	-4	2	1	-4	1	2	5	-3	-2	-4	-3	2			0	0	0	12	0	0	2	0	0	13	13	0	3	9	29	0	2	0	0	17	0.64	0.83
276	M	-3	-1	2	5	-5	-1	2	0	0	-3	0	-2	3	0	-2	-1	-2	-4	-1	-4			1	4	8	33	0	1	13	7	2	0	10	1	9	4	1	2	1	0	2	0	0.47	0.83
277	Y	-4	-4	-1	-5	-4	-3	-4	-5	5	-3	-2	-4	3	4	-5	-4	-4	-1	8	-3			0	0	3	0	0	0	0	0	14	0	0	0	10	18	0	0	0	0	55	0	1.40	0.83
278	T	-1	-3	-1	-3	-3	-2	1	-3	-3	0	1	-3	-2	-4	-3	3	4	-5	-4	-1			4	0	2	0	0	0	10	0	0	7	14	0	0	0	0	28	33	0	0	3	0.52	0.83
279	P	0	0	-3	2	-4	2	0	-4	-3	-1	-2	-1	-3	-5	6	-2	-1	-5	-4	-1			7	5	0	13	0	12	6	0	0	4	4	3	0	0	38	0	3	0	0	4	0.77	0.83
280	E	0	-2	-2	0	-5	-1	6	-4	-2	-4	-1	0	-3	0	-3	-2	-1	-4	-3	-4			7	0	0	2	0	0	70	0	0	0	9	3	0	4	0	0	4	0	0	0	0.99	0.83
281	P	-3	-4	-4	-3	-5	-3	0	-4	-3	-5	-5	-3	-4	-4	8	-3	-3	-4	2	-4			0	0	0	0	0	0	6	0	0	0	0	0	0	0	84	0	0	0	10	0	2.19	0.83
282	D	0	-3	3	7	-5	-2	-1	-3	-3	-5	-5	-2	-5	-5	2	-2	-3	-6	-5	-5			8	0	16	65	0	0	0	0	0	0	0	0	0	0	11	0	0	0	0	0	1.26	0.78
283	I	-1	-4	-3	2	4	-3	-3	-2	-4	2	-1	-3	1	0	2	-1	2	-4	-1	1			3	0	0	16	10	0	0	3	0	15	5	0	4	4	11	3	13	0	3	12	0.34	0.78
284	C	1	-5	-5	-5	7	-4	-5	-4	-4	2	0	-4	-2	5	0	-3	-3	-3	-1	1			16	0	0	0	24	0	0	0	0	16	5	0	0	27	5	0	0	0	0	7	0.79	0.78
285	H	-3	-2	-1	-3	-4	-1	-2	-4	9	-5	-4	-2	-3	-3	-4	1	2	-4	-1	-4			0	0	0	0	0	0	0	0	72	0	0	0	0	0	0	14	13	0	0	0	1.61	0.75
286	E	-3	-3	-2	4	-5	-1	5	2	-3	-4	0	-1	-3	-4	-3	-2	-3	-5	-4	-4			0	0	0	23	0	0	47	14	0	0	13	1	0	0	0	0	0	0	0	0	0.80	0.75
287	L	-1	-2	-3	-3	0	-2	-3	-2	-3	2	3	2	0	-1	-3	-1	-2	-3	-1	2			5	1	1	1	2	1	1	3	0	10	33	14	1	2	1	5	2	0	2	15	0.29	0.65
288	L	0	-3	-3	-4	-3	-3	-3	-3	-3	2	2	-3	0	6	-3	-2	-2	-2	1	0			9	1	1	1	0	1	1	1	0	10	23	1	2	39	1	2	1	0	2	4	0.62	0.65
289	G	-1	-3	-2	-2	0	-2	-3	6	4	-4	-4	-3	-3	-3	-3	-2	-3	-3	-3	-4			3	1	1	1	3	1	1	69	14	1	1	1	0	1	1	1	1	0	0	1	1.15	0.65
290	H	-3	-2	-1	-3	-3	-1	-2	-3	9	-3	1	-2	-2	-2	-3	-2	-3	-3	0	-3			1	1	1	1	0	1	1	1	72	1	15	1	0	1	1	1	1	0	0	1	1.48	0.65
291	V	1	-3	-3	-3	4	-3	-3	-1	-3	1	2	-3	3	-2	-3	-2	-1	-3	-3	4			14	1	1	1	9	1	1	5	0	4	17	1	7	1	1	2	3	0	0	33	0.39	0.65
292	P	-1	-2	0	-3	5	-3	-3	2	-4	-2	-4	-3	-4	-5	7	-1	-3	-5	-5	-4			5	2	4	0	13	0	0	14	0	2	0	0	0	0	56	2	0	0	0	0	1.35	0.75
293	L	-2	-4	-5	-5	3	-1	-4	-5	-4	2	3	-4	6	1	2	-4	-1	3	0	-1			3	0	0	0	6	3	0	0	0	10	29	0	22	5	10	0	3	4	3	3	0.64	1.00
294	F	-3	-4	-5	-5	-4	-4	-5	-5	2	2	4	-4	2	2	-5	0	-3	-4	-2	1			0	0	0	0	0	0	0	0	5	11	53	0	5	10	0	10	0	0	0	6	0.76	1.00
295	S	3	-4	-4	-1	-3	-3	-4	-1	-4	0	1	-3	4	0	-4	0	1	-4	0	1			33	0	0	3	0	0	0	3	0	4	11	0	14	3	0	6	9	0	3	9	0.43	1.00
296	D	-3	-3	5	5	-5	0	0	-3	4	-1	-5	-3	-4	-5	-4	1	-1	-5	-4	-4			0	0	30	34	0	3	5	0	9	4	0	0	0	0	0	10	2	0	0	0	0.94	1.12
297	R	-1	1	-4	0	-6	1	-1	-1	-5	-1	-2	0	-4	-6	6	-1	-4	-6	-5	-1			5	8	0	5	0	6	4	5	0	4	4	6	0	0	44	5	0	0	0	4	0.89	1.61
298	S	-1	1	1	2	-5	-1	1	-2	0	-2	-1	-1	0	-1	-1	2	0	2	0	-1			5	8	7	11	0	3	8	3	3	3	7	3	3	3	3	15	7	3	3	4	0.14	1.61
299	F	-4	1	-5	-6	-5	-5	-5	-6	-3	0	-1	-4	1	7	-6	-4	-1	-2	5	-1			0	7	0	0	0	0	0	0	0	4	4	0	3	57	0	0	3	0	17	4	1.36	1.19
300	A	6	-4	-1	-4	2	-3	-4	-1	-4	-3	0	-3	1	-4	-4	0	-3	-5	-4	0			69	0	4	0	4	0	0	3	0	0	7	0	3	0	0	5	0	0	0	5	0.96	1.19
301	Q	0	1	2	1	-5	2	1	0	3	-5	-1	1	-4	-5	-4	1	1	-5	-4	-4			8	8	8	8	0	8	8	8	8	0	8	8	0	0	0	8	8	0	0	0	0.29	1.22
302	F	-3	-5	-5	-6	-5	-5	-5	-6	0	0	0	-5	0	7	-1	-5	-2	-2	5	-1			2	0	0	0	0	0	0	0	2	4	8	0	2	54	4	0	3	0	18	4	1.32	1.36
303	S	-1	-1	-1	-5	1	-4	-4	-5	-4	-1	1	-2	3	2	-2	2	3	-5	0	2			5	3	3	0	3	0	0	0	0	3	14	3	7	9	2	17	16	0	4	12	0.34	1.52
304	Q	-1	2	-3	-4	0	5	1	-5	5	-2	-5	0	-4	-1	-5	-1	-1	2	0	-2			6	11	0	0	2	33	9	0	13	3	0	4	0	3	0	5	3	3	3	3	0.73	1.52
305	E	-1	1	1	0	-5	1	0	-5	3	0	-1	0	2	1	-5	0	0	-5	2	0			6	6	6	6	0	6	6	0	6	6	6	6	6	6	0	6	6	0	6	6	0.18	1.67
306	I	-2	-5	-5	-6	0	-5	-5	-1	-3	3	1	-5	1	4	-5	-3	-4	-2	6	-1			4	0	0	0	2	0	0	5	0	21	10	0	4	16	0	2	0	0	34	2	0.92	1.25
307	G	0	-5	-3	-4	3	-4	-1	7	-5	-6	-6	-4	-5	-6	-5	-1	-4	-5	-6	-5			6	0	0	0	6	0	5	79	0	0	0	0	0	0	0	5	0	0	0	0	1.60	1.25
308	L	0	2	-2	-2	2	2	2	-2	-4	-2	2	2	-1	-2	-2	-2	-3	-5	-4	-2			7	11	1	2	5	10	14	2	0	1	25	12	1	1	2	2	0	0	0	3	0.33	1.25
309	A	3	-4	-2	-5	1	-4	-4	0	-5	2	2	-1	0	-4	-4	-1	-1	-5	-4	1			31	0	1	0	2	0	0	6	0	11	22	5	2	0	0	5	3	0	0	11	0.45	1.25
310	S	2	0	-2	-4	-1	-4	-4	4	-4	-1	-3	-3	-4	0	-2	3	-3	-5	0	-1			15	6	1	0	1	0	0	36	0	4	2	0	0	4	1	21	0	0	3	5	0.58	1.25
311	L	0	1	-4	-5	1	-1	-3	-5	-4	0	4	-2	1	1	-4	-1	-2	0	-1	1			9	9	0	0	3	3	1	0	0	5	40	3	2	6	0	4	2	1	2	8	0.42	1.20
312	G	2	2	-1	-1	-2	0	-1	3	-1	-2	-4	3	-1	-5	-4	-1	1	-5	-4	-4			16	12	2	4	1	4	2	20	1	3	0	21	2	0	0	4	7	0	0	0	0.42	1.27
313	A	5	-4	-2	0	0	-1	0	-2	3	-2	-2	-3	-4	0	-4	-1	-1	-5	-2	-1			54	0	2	7	2	2	5	2	7	1	3	0	0	4	0	3	2	0	1	5	0.66	1.32
314	P	-1	0	-1	1	1	0	2	-2	1	1	-2	-1	1	-1	0	1	1	2	-4	-2			5	4	3	8	3	4	14	3	4	7	4	3	4	3	5	13	7	3	0	3	0.15	1.67
315	D	0	1	0	3	-6	1	2	0	2	-6	-2	2	-5	-6	2	0	0	-6	-5	-5			6	7	4	15	0	5	14	8	4	0	4	12	0	0	10	5	5	0	0	0	0.38	1.67
316	E	-1	-1	1	2	-5	0	4	2	-3	-5	-5	1	-5	-5	-3	1	-3	-6	-1	-2			4	3	5	12	0	4	27	18	0	0	1	8	0	0	1	12	0	0	2	3	0.53	1.43
317	Y	2	1	-2	4	-5	1	3	-3	-2	-1	-4	-2	0	-2	-4	-2	-2	-1	1	-1			19	6	1	22	0	6	20	2	1	3	1	1	3	1	0	2	1	1	5	6	0.45	1.43
318	I	-3	2	-5	-5	0	-1	-5	-4	-4	4	3	-2	3	1	-5	-2	-2	1	-1	1			2	11	0	0	2	3	0	1	0	23	27	1	7	6	0	3	2	2	2	8	0.50	1.32
319	E	0	-1	-2	1	-5	2	3	-3	-1	-2	-2	2	-2	-5	0	1	-2	3	-4	-1			9	3	1	8	0	8	20	1	1	2	5	11	1	0	4	13	2	5	0	6	0.31	1.33
320	K	0	2	0	-4	-5	1	-1	-4	2	-4	1	3	1	2	-4	0	-2	3	1	-4			8	12	4	0	0	8	4	0	6	0	12	17	3	11	0	5	2	4	4	0	0.34	1.17
321	L	-1	-4	-5	-6	-4	-4	-5	-5	-5	3	5	-1	2	1	-5	-4	-3	-4	-3	1			7	0	0	0	0	0	0	0	0	17	53	5	5	6	0	0	0	0	0	8	0.81	1.07
322	A	3	1	-4	-4	-4	1	-1	1	-4	-1	-1	-1	1	0	-1	1	0	-5	-4	-1			28	8	0	0	0	6	4	10	0	4	6	4	4	4	4	10	5	0	0	5	0.27	1.28
323	T	-1	5	2	0	-5	2	-3	-1	2	-5	-5	1	-4	-5	-4	0	1	-5	-4	-1			5	38	8	5	0	8	0	5	5	0	0	6	0	0	0	6	10	0	0	5	0.65	1.28
324	I	-3	-4	-5	-5	4	-4	-5	-5	-4	2	3	-4	3	3	1	-4	-1	-4	-2	2			0	0	0	0	11	0	0	0	0	9	31	0	9	12	8	0	4	0	0	15	0.61	0.81
325	Y	1	-3	0	-4	-4	-3	-3	-4	5	-3	-1	-3	-3	3	-4	-1	-3	4	7	-3			12	0	5	0	0	0	0	0	13	0	4	0	0	14	0	5	0	6	40	0	0.96	0.81
326	W	-2	-4	-5	-5	1	1	-4	-4	-4	-3	-1	-4	1	2	1	-4	-4	11	0	-1			4	0	0	0	4	7	0	0	0	0	5	0	4	8	8	0	0	55	0	6	1.76	0.81
327	F	-4	1	-4	-5	-4	-4	-4	-5	1	-2	0	-3	0	6	-5	-2	-3	5	5	-3			0	9	0	0	0	0	0	0	3	0	7	0	3	48	0	4	0	9	17	0	1.07	0.76
328	T	1	-3	-2	-3	1	-3	-3	-3	-4	-1	-3	-3	-2	-4	-3	2	6	-4	-3	1			11	0	0	0	3	0	0	0	0	3	0	0	0	0	0	13	61	0	0	9	0.84	0.76
329	V	-1	-4	-4	-5	-3	0	-4	-5	-4	3	1	-4	2	0	-4	-3	-1	5	-2	5			4	0	0	0	0	5	0	0	0	15	9	0	6	3	0	0	2	8	0	48	0.69	0.71
330	E	-2	-2	-2	0	-4	1	6	-3	-2	-4	-4	-1	-3	-4	-3	2	-2	-4	-3	-4			0	0	0	0	0	4	75	0	0	0	0	0	0	0	0	16	0	0	0	0	1.15	0.68
331	F	-3	-4	-2	-4	1	-4	-4	-4	-2	-2	-2	-4	-2	7	-4	-2	-1	-1	4	-1			0	0	2	0	4	0	0	0	0	0	1	0	0	68	0	2	4	0	13	3	1.24	0.68
332	G	-1	-3	-2	-2	-3	-3	-3	7	-3	-4	-4	-3	-4	-4	-3	-2	-3	-3	-4	-4			1	1	0	1	0	0	1	90	0	1	1	1	0	0	1	1	1	0	0	1	1.51	0.58
333	L	1	-3	-3	-3	-2	-2	-3	-3	-3	2	2	-3	4	-2	-3	-2	2	-3	-2	2			15	1	1	1	0	1	1	1	0	15	16	1	15	1	1	1	15	0	0	15	0.36	0.57
334	C	-3	-4	-4	-4	7	-4	-4	-4	-4	3	1	-4	3	-3	-4	-1	0	-4	1	3			1	1	0	1	26	0	1	1	0	14	12	1	9	0	1	5	5	0	5	18	0.77	1.06
335	K	0	3	0	-1	0	1	-1	0	0	-2	-2	4	0	-3	-3	-1	-1	1	1	-2			7	15	4	4	2	7	4	7	2	3	3	26	2	0	1	3	3	2	5	3	0.31	1.06
336	Q	-2	-1	0	0	-5	4	4	-2	0	-5	-5	1	-4	-6	-4	-1	3	-5	-4	-5			2	2	4	4	0	22	32	3	2	0	0	10	0	0	0	3	17	0	0	0	0.77	1.32
337	G	-1	-1	3	3	-5	0	0	2	1	-2	-3	-1	-4	-5	2	-2	-2	-6	-5	-2			5	3	17	19	0	3	6	15	3	2	3	4	0	0	11	2	2	0	0	3	0.44	1.32
338	D	-1	-4	2	2	-5	0	2	5	-1	-6	-6	-1	-5	-5	-2	-2	-2	4	-5	-5			4	0	10	12	0	4	12	43	1	0	0	3	0	0	2	2	1	6	0	0	0.82	1.32
339	S	-1	0	1	0	-5	1	0	-1	2	0	-1	0	2	1	1	0	0	-6	-4	0			6	6	6	6	0	6	6	6	6	6	6	6	6	6	6	6	6	0	0	6	0.10	1.78
340	I	-4	1	1	-4	3	1	-4	-5	-4	1	0	0	3	1	1	0	0	-4	2	0			0	7	7	0	7	7	0	0	0	7	7	7	7	7	7	7	7	0	7	7	0.27	1.44
341	K	-2	5	-2	-2	1	0	2	-4	-2	-4	-4	5	-2	-5	-3	-2	-2	-5	-4	-3			1	30	1	0	3	2	12	0	0	0	0	47	1	0	0	1	1	0	0	1	0.92	0.77
342	A	4	-4	-4	-4	0	-3	-4	1	-4	4	-1	-3	0	-3	-4	-1	-2	-4	-4	1			44	0	0	0	2	0	0	13	0	26	3	0	2	0	0	1	0	0	0	10	0.60	0.77
343	Y	-4	-4	2	-4	-1	-3	-4	-5	0	-2	-2	-4	-3	3	-5	-3	-3	0	8	-3			0	0	11	0	2	0	0	0	0	2	3	0	0	9	0	0	0	0	74	0	1.53	0.77
344	G	-2	1	-2	-3	-5	-3	-4	7	-4	-6	-5	-3	-4	-5	-4	-2	-3	-4	-5	-5			0	11	0	0	0	0	0	89	0	0	0	0	0	0	0	0	0	0	0	0	1.64	0.77
345	A	5	2	-3	-3	-3	-2	-3	1	-3	-3	-4	-2	-3	-4	-3	2	-2	-5	-4	-1			58	15	0	0	0	0	0	11	0	0	0	0	0	0	0	13	0	0	0	3	0.74	0.75
346	G	0	-4	-2	2	-4	-3	-3	6	3	-5	-5	-3	-4	-5	-4	-1	-2	-5	-4	-3			6	0	0	13	0	0	0	66	8	0	0	0	0	0	0	2	2	0	0	2	1.11	0.75
347	L	-1	-4	-5	-5	5	-4	-5	-5	-5	5	3	-4	3	-2	-5	-4	-3	-4	-3	1			3	0	0	0	13	0	0	0	0	38	32	0	8	0	0	0	0	0	0	6	0.84	0.75
348	L	-1	-4	-4	-4	5	-3	-4	-4	-4	3	4	-4	1	-2	-4	-3	-2	-3	-3	2			4	0	0	0	13	0	1	1	0	22	41	0	2	0	0	1	0	0	0	11	0.68	0.68
349	S	-1	-2	-1	-2	-3	-2	-2	0	4	-4	-4	-2	-3	-3	-2	5	1	-4	-2	-3			1	0	0	0	0	0	1	5	13	0	1	0	0	0	0	67	8	0	0	1	0.87	0.68
350	S	0	2	-1	-2	3	-2	-2	-1	-2	-4	-4	-1	-3	-4	-2	5	-1	-4	-3	-3			4	14	0	0	6	0	1	3	0	0	1	0	0	0	0	68	0	0	0	1	0.82	0.68
351	F	-1	1	-2	-1	-1	-2	-2	-3	1	2	-2	-1	-2	1	3	1	-1	-3	3	-1			3	9	1	4	1	1	1	2	3	13	2	4	0	6	15	15	3	0	14	4	0.27	0.68
352	G	-1	-1	1	0	-4	0	2	5	-1	-4	-4	1	-3	-4	-3	0	0	-4	-4	-4			5	2	7	4	0	3	15	42	1	1	1	9	0	0	1	5	4	0	0	1	0.58	0.71
353	E	-2	-2	-2	-1	-4	1	6	-3	-2	-4	-4	-1	-3	0	-3	1	-2	-4	-3	-3			1	1	0	1	0	4	71	1	0	1	1	1	0	5	1	12	1	0	0	1	1.04	0.71
354	L	1	-3	-3	-3	6	-3	-3	-2	-3	-1	3	-3	0	-2	-3	1	1	-3	-3	-1			11	1	0	1	17	0	1	2	0	3	32	1	1	0	1	14	11	0	0	3	0.47	0.71
355	Q	-2	3	-2	0	-2	1	1	-3	-1	-1	0	1	-1	-4	-2	0	2	-5	-4	1			2	16	1	5	1	6	11	1	1	3	11	10	1	0	1	8	13	0	0	9	0.23	0.88
356	Y	-3	-3	-1	-2	-2	-1	0	-4	8	-5	-5	-4	-4	2	-5	-2	-4	2	7	-5			1	1	3	2	1	3	5	1	35	0	0	0	0	6	0	3	0	3	36	0	1.43	1.48
357	C	4	-5	-4	-5	6	-4	-4	-3	-5	-2	-3	-4	0	3	-4	2	-1	-5	-4	0			40	0	0	0	17	0	0	1	0	2	1	0	3	12	0	16	2	0	0	7	0.87	1.48
358	L	-2	0	-5	-5	1	-3	-5	-6	0	1	4	-2	4	1	-5	-3	-1	-4	-1	1			2	6	0	0	3	1	0	0	2	6	42	2	10	7	0	2	4	0	2	10	0.62	1.44
359	S	-1	-2	2	-1	-2	0	1	-2	-1	-4	-5	-1	-4	-5	-2	5	2	-5	-4	-3			2	1	9	3	1	3	8	2	1	1	1	4	0	0	1	50	11	0	0	1	0.73	1.40
360	E	1	-2	1	5	-5	0	0	3	-4	-5	-5	-2	-1	-5	2	-1	-2	-6	-5	-3			10	2	5	32	0	4	4	19	0	0	0	2	2	0	10	3	3	0	0	1	0.65	1.43
361	K	0	-1	1	1	-1	-1	2	-1	-1	0	-3	4	-2	-5	-1	0	0	-6	-5	-2			7	1	6	6	1	2	16	4	2	5	2	28	1	0	3	6	7	0	0	3	0.38	1.46
362	P	2	-5	-3	-2	2	-3	-1	-4	-5	-1	-3	-4	0	-5	6	-1	-2	-6	-5	2			21	0	1	2	4	1	4	0	0	2	2	0	2	0	41	3	1	0	0	16	0.96	1.45
363	K	-3	1	3	-1	-1	1	4	-5	-1	-1	-2	3	-2	-2	0	-3	-2	-5	-4	-1			1	6	15	2	1	5	28	0	1	4	3	17	1	2	6	0	2	0	0	5	0.50	1.45
364	L	-4	4	-4	-5	-2	-2	-4	-6	3	3	0	2	-1	1	-5	-4	-2	-5	2	2			0	22	0	0	1	1	0	0	7	20	7	13	1	6	0	0	3	0	6	14	0.59	1.48
365	L	-3	4	-3	-3	-5	0	1	-4	0	2	1	2	0	0	-5	-2	-2	-5	0	0			1	24	1	1	0	4	9	1	2	12	12	15	2	4	0	3	1	0	3	7	0.43	1.48
366	P	0	1	-2	1	-6	-1	1	-1	-3	-4	-5	0	-2	-6	6	-2	-3	-1	-2	-4			7	7	1	8	0	2	8	5	1	1	1	6	1	0	46	2	1	1	1	1	0.99	1.48
367	L	-5	-5	-6	-6	-5	-5	-6	-6	-1	-1	2	-6	0	8	-6	-4	-5	4	3	-3			0	0	0	0	0	0	0	0	1	2	18	0	2	61	0	1	0	6	8	0	1.63	1.48
368	E	-3	-2	2	6	-6	-1	2	-3	-2	-4	-6	-1	-5	-6	-4	1	-1	-6	-5	-3			1	2	8	53	0	2	12	1	1	1	0	3	0	0	0	11	3	0	0	2	1.09	1.48
369	L	-2	-5	-5	-5	-5	-5	-5	-6	-5	3	2	-5	1	-2	6	-4	-1	-6	-5	1			3	0	0	0	0	0	0	0	0	14	23	0	3	2	42	0	4	0	0	10	1.13	1.49
370	E	0	-2	-1	1	-2	1	4	-4	1	-1	-1	0	1	1	-3	0	-2	-5	-4	-1			8	2	3	8	1	5	30	1	3	4	7	5	3	7	1	5	1	0	0	5	0.34	1.43
371	K	1	2	-2	3	-5	0	0	-4	1	1	-2	1	-2	-4	-3	-1	1	-1	-4	1			13	13	1	16	0	4	6	0	3	7	3	8	1	1	1	4	9	1	0	10	0.27	1.46
372	T	3	-5	-4	-3	2	-4	-4	-5	0	2	-2	-3	2	-4	-1	-3	3	-6	-4	4			22	0	0	1	4	0	0	0	2	10	2	1	6	0	2	0	22	0	0	27	0.65	1.47
373	A	3	-1	-5	-5	5	-2	-5	0	-5	1	2	-4	5	-1	-5	-1	-2	-5	-4	-1			24	3	0	0	11	2	0	7	0	9	18	0	15	2	0	4	1	0	0	3	0.57	1.46
374	I	-3	4	2	2	-2	1	0	-3	-1	-3	-2	1	-3	-3	-1	-3	0	-6	-2	0			1	26	8	12	1	6	6	1	1	1	5	9	1	1	4	1	6	0	2	8	0.40	1.47
375	Q	-3	2	-3	-4	-5	5	1	-5	-1	-2	-3	0	0	-5	-4	-2	5	-5	-2	-2			1	9	0	0	0	30	7	0	1	2	2	4	2	0	0	1	39	0	1	2	0.91	1.47
376	N	-2	0	-1	1	-6	-2	3	-4	0	-5	-4	2	1	-6	5	-1	-1	-6	-5	-2			3	5	3	8	0	1	21	1	2	0	1	11	4	0	30	4	4	0	0	3	0.73	1.47
377	Y	-2	-5	-5	-6	5	-4	-5	-4	-1	-2	-4	-5	-4	3	-4	-2	-4	-1	8	-2			2	0	0	0	13	1	0	1	1	2	0	0	0	11	1	3	0	0	64	2	1.76	1.47
378	T	-3	4	0	4	-6	1	0	-4	-1	0	-2	-1	-3	-5	3	-1	-3	-3	0	-2			2	22	3	21	0	7	4	0	2	7	4	3	0	0	14	3	1	0	4	2	0.51	1.48
379	V	-3	-5	-5	2	-4	-5	-5	-6	-6	7	-1	0	-2	-3	-1	-4	-1	-6	-3	1			1	0	0	11	0	0	0	0	0	63	3	6	0	1	3	0	4	0	1	8	1.12	1.48
380	T	-3	-4	0	5	-3	2	-1	-3	-3	-5	-4	-3	-4	-4	-4	0	5	-2	-1	-3			0	0	3	32	0	10	2	2	0	0	1	0	0	1	0	4	40	1	2	1	0.96	1.48
381	E	-1	0	-1	0	-2	0	1	0	2	2	-4	2	-2	-5	-4	1	3	-6	-4	-3			5	4	3	6	1	4	10	7	6	13	0	12	1	0	0	9	18	0	0	1	0.32	1.48
382	F	-4	-5	-1	-6	-5	-3	-4	-6	-3	-1	0	-4	5	5	0	-4	-3	-2	6	-1			0	0	3	0	0	0	1	0	0	3	8	1	17	23	5	0	1	0	34	3	1.13	1.43
383	Q	-3	-2	-2	-3	-5	8	-1	-4	4	-5	-5	-1	-3	-5	2	-3	-3	-5	-3	-5			0	0	0	0	0	76	0	0	12	0	0	0	0	0	12	0	0	0	0	0	1.62	1.04
384	P	-1	-1	1	1	-5	1	4	-4	1	-5	-3	1	-4	-5	4	-1	-1	-5	-4	-4			3	3	7	5	0	6	32	0	3	0	3	8	0	0	23	4	3	0	0	0	0.70	1.04
385	L	0	-1	3	-1	0	0	-1	0	3	0	-2	-1	0	-4	-4	-1	3	-5	-4	1			10	3	14	2	2	4	3	7	8	5	3	3	2	0	0	3	20	0	0	12	0.27	1.04
386	Y	-4	-4	-5	-5	-4	-4	-4	-5	-1	-2	2	-4	-2	3	-5	-4	-4	0	8	-3			0	0	0	0	0	0	0	0	0	0	22	0	0	10	0	0	0	0	67	0	1.52	0.83
387	Y	-4	-4	-5	-5	1	-5	-5	-5	-2	-2	-1	-5	1	7	-5	-4	-4	4	5	-3			0	0	0	0	3	0	0	0	0	0	3	0	4	67	0	0	0	5	18	0	1.49	0.78
388	V	0	-3	-5	-5	0	-4	-2	-5	-4	2	-1	-4	0	-2	-5	-2	-1	-4	4	5			6	1	0	0	2	0	3	0	0	9	3	0	2	0	0	2	3	0	15	56	0.86	1.10
389	A	2	-4	-4	-4	2	-4	-4	-4	-4	4	1	-4	-2	0	-4	1	2	-4	-3	0			18	0	0	0	4	0	0	0	0	27	15	0	0	4	0	10	16	0	0	4	0.50	1.06
390	E	-3	0	1	5	-5	-1	4	-3	-1	-5	-5	0	-4	-5	2	-1	-2	-5	-4	-5			1	6	7	32	0	2	28	1	1	0	0	3	0	0	11	4	2	0	0	0	0.80	1.06
391	S	-1	-3	1	3	-4	-1	-1	-1	0	-5	-5	-1	-4	-5	-3	6	0	-5	-4	-4			1	0	5	15	0	1	2	4	3	0	0	2	0	0	0	63	3	0	0	0	0.93	1.06
392	F	-4	-5	-5	-5	0	-5	-5	-5	-4	2	2	-5	0	7	-5	-4	-3	-2	2	-1			0	0	0	0	2	0	0	0	0	10	20	0	2	52	0	0	1	0	6	4	1.16	1.06
393	N	0	-1	-1	3	-2	1	4	-2	-1	-4	-3	1	-4	-3	-2	1	-1	-5	-4	-3			8	2	2	18	1	6	34	2	1	0	2	9	0	1	1	10	2	0	0	1	0.56	1.05
394	D	-2	-1	-1	4	-5	3	3	-4	4	-3	-4	0	-2	-1	-3	-1	-1	-4	0	-4			3	3	1	21	0	16	23	0	10	1	0	5	1	3	0	5	3	0	3	1	0.60	1.05
395	A	3	-4	-4	-5	2	-4	-4	-3	-4	0	4	-4	3	-3	-4	-2	-3	-4	-3	0			27	0	0	0	4	0	0	2	0	3	50	0	6	0	0	1	0	0	0	4	0.74	1.05
396	K	-2	0	-2	0	-4	0	0	-4	-2	0	0	3	0	3	-4	0	-1	-3	2	-2			3	5	1	6	0	3	6	1	1	6	10	23	3	14	0	6	4	0	8	2	0.29	1.08
397	E	-1	0	0	4	-3	3	5	-3	-1	-3	-3	0	-4	-5	-4	0	-2	-5	-3	-3			4	6	3	23	0	11	35	1	1	1	2	3	0	0	0	7	1	0	1	1	0.72	1.13
398	K	0	0	-2	-1	-4	1	-1	-4	-4	3	1	4	-1	-4	-2	-1	-2	-5	-4	2			6	4	1	4	0	5	4	1	0	17	12	25	1	0	1	3	1	0	0	14	0.35	1.12
399	V	1	-4	-4	-4	0	-3	-3	-4	-4	0	3	-4	5	2	-4	-1	0	-4	-3	1			12	0	0	0	1	0	2	1	0	4	33	0	16	7	0	6	6	0	0	10	0.52	1.10
400	R	-1	5	-1	1	-5	1	2	-1	1	-1	-4	2	-2	-4	-2	-2	-3	-4	0	-2			4	33	2	9	0	6	12	5	3	5	1	9	1	0	1	2	1	0	4	2	0.50	1.12
401	N	0	0	0	1	-2	2	3	-1	-3	-1	-2	2	3	-4	-3	0	1	-1	-2	-1			9	5	3	7	1	8	18	4	0	3	4	11	7	0	0	7	7	1	1	3	0.23	1.14
402	F	-3	-4	-3	3	-4	-3	-3	-4	-3	0	0	-4	0	6	-4	-3	-3	4	4	-3			2	1	0	19	0	0	1	1	0	4	8	1	2	36	1	1	1	5	17	1	0.90	1.09
403	A	4	-3	-3	-3	0	-2	-1	0	-2	2	1	-3	0	0	-3	-1	-1	2	-3	0			34	1	0	1	2	2	4	6	1	13	12	1	2	4	1	4	4	3	0	6	0.35	1.10
404	A	2	0	2	1	-2	1	1	-1	-2	-3	-2	1	5	-4	-3	0	-2	-4	-4	-3			21	4	8	7	1	5	11	4	1	1	2	10	15	0	0	8	1	0	0	1	0.36	1.14
405	T	1	0	-1	-2	-4	2	-2	-1	1	-3	0	1	-2	-3	-2	2	4	-2	-3	-3			12	6	3	2	0	8	2	5	3	0	9	7	1	1	1	14	25	1	0	0	0.32	1.17
406	I	0	-3	-4	-5	-1	-2	-3	-1	-4	4	2	-3	6	0	-4	-3	-1	-4	-1	0			7	1	0	0	1	1	1	4	0	27	21	1	19	3	0	2	3	0	2	5	0.59	1.17
407	P	-1	-1	-2	0	0	3	0	-2	1	-2	-1	2	-3	-3	3	-1	-2	-5	-2	1			5	3	2	6	2	15	6	3	3	2	5	13	0	1	13	4	2	0	1	13	0.26	1.21
408	R	0	6	-3	0	-4	-1	-1	-2	0	-1	-1	3	-3	-4	-1	-3	-1	-5	-4	-3			7	40	0	4	0	2	2	2	2	5	7	18	0	0	3	0	4	0	0	2	0.65	1.20
409	P	-3	-1	-2	4	-5	1	2	-4	-4	-4	-3	-1	-4	-6	6	-2	-3	-6	-5	-5			1	3	1	23	0	5	12	1	0	1	3	3	0	0	45	3	0	0	0	0	1.19	1.24
410	F	-3	-5	-3	-3	-5	-5	-4	-5	2	-3	0	-5	0	8	-4	-2	-2	-2	0	-3			1	0	1	2	0	0	1	0	6	0	6	0	2	73	1	3	3	0	1	1	1.55	1.22
411	S	3	-3	-2	-1	-4	-1	1	2	-1	0	-3	-1	-2	-3	0	2	0	-5	-3	-2			25	1	1	4	0	2	11	15	1	6	1	3	1	1	5	13	6	0	1	2	0.31	1.27
412	V	-1	-3	-5	-3	-1	-4	-4	-4	-5	0	0	-4	-3	-3	6	-3	0	-5	-4	4			5	1	0	1	1	0	0	1	0	4	11	1	0	1	37	1	5	0	0	30	0.93	1.27
413	R	-1	5	-2	-2	0	-3	-3	-5	1	0	1	2	-1	-3	-4	-3	-1	1	1	-1			5	31	1	2	2	0	0	0	3	5	17	13	1	0	0	1	4	2	5	5	0.48	1.27
414	Y	-4	-4	-3	-3	-5	-4	-3	-2	1	-3	-3	-4	-3	5	-5	-4	-4	-1	8	-4			0	0	1	1	0	0	1	3	3	0	1	0	0	26	0	0	0	0	61	0	1.72	1.23
415	D	0	-2	4	5	-5	1	2	-4	-3	-5	-5	-2	-4	-5	2	-2	-1	-6	-5	-5			8	1	25	30	0	7	12	0	0	0	1	1	0	0	12	0	3	0	0	0	0.85	1.23
416	P	0	0	-1	-3	-5	-2	-2	-4	-2	-4	-4	-2	-4	0	7	-1	-3	-6	-4	-2			9	6	3	0	0	1	2	0	1	1	1	1	0	5	63	4	0	0	0	2	1.50	1.24
417	Y	0	-2	-2	-2	-5	-2	0	-4	1	-1	0	3	-2	1	-4	-3	-2	-2	6	-2			8	1	2	2	0	1	7	1	3	4	11	18	0	6	1	1	2	0	33	1	0.62	1.24
418	T	0	-2	-2	-2	-4	-2	0	-4	1	-2	-3	-2	-3	-1	-2	0	6	-4	3	-2			9	2	1	2	0	1	5	0	3	1	2	1	0	2	2	5	50	0	11	1	0.72	1.25
419	Q	1	1	-2	0	-1	5	2	0	-1	-5	-2	2	-2	-5	-2	-1	-3	-5	-4	-4			12	6	1	4	1	31	13	6	1	0	5	14	1	0	2	3	0	0	0	0	0.56	1.25
420	R	3	1	-1	-1	-1	0	-1	-2	-4	-3	-4	-2	-3	-5	-2	4	3	-5	-4	-3			25	7	2	2	1	5	3	1	0	1	1	1	0	0	2	31	16	0	0	1	0.56	1.24
421	I	0	-4	-2	-2	-4	-4	-4	2	-5	4	0	-3	-2	-3	-2	-2	-1	-5	-4	4			7	0	2	2	0	0	0	16	0	24	6	1	0	0	2	3	3	0	0	32	0.55	1.16
422	E	1	-3	-1	4	-3	1	5	-4	3	-3	-4	-1	-4	-5	-3	-2	-2	-5	-4	-3			12	0	1	21	1	5	42	0	6	2	1	2	0	0	0	2	2	0	0	1	0.80	1.12
423	V	0	-4	-5	-5	-3	-3	-4	-3	-5	4	0	-3	1	-3	-3	-3	-2	-5	-3	6			6	0	0	0	0	1	0	2	0	20	5	2	2	0	1	0	2	0	0	58	0.93	1.10
424	L	-1	-4	-5	-5	-4	-2	-5	-5	-5	2	5	-3	0	-1	-5	-2	-2	-4	-3	0			4	0	0	0	0	2	0	0	0	10	70	2	1	2	0	4	2	0	0	3	0.96	1.15
425	D	-2	-2	0	6	-5	-2	1	-3	-4	-5	-5	1	-2	-6	3	0	0	-6	-4	-5			3	2	4	42	0	1	6	1	0	0	0	10	1	0	17	5	6	0	1	0	0.86	1.19
426	N	-1	-2	2	2	-2	-1	0	2	-1	-5	-5	-1	-4	-3	-2	3	2	-5	-3	-2			3	2	12	12	1	2	6	13	2	0	0	3	0	1	2	26	14	0	1	3	0.44	1.19
427	T	-2	-2	-2	5	-5	-1	-1	-4	-4	0	-2	1	-1	-2	2	-2	2	-6	-5	0			3	2	1	37	0	2	3	0	0	5	3	9	1	2	10	2	11	0	0	9	0.56	1.20
428	Q	-1	5	-1	1	-4	3	0	-2	2	-4	-3	1	-4	-3	0	-1	-2	-5	-2	-2			5	32	2	7	0	11	6	3	6	1	3	9	0	1	5	5	1	0	1	2	0.50	1.20
429	Q	1	0	-2	-1	-3	2	-3	-3	-2	0	-3	2	0	-4	-4	1	-2	-5	-4	4			12	5	1	3	0	10	0	1	1	4	0	16	2	0	0	13	1	0	0	31	0.44	1.19
430	L	-3	-2	-2	-5	-4	-4	-3	-3	-5	5	3	-3	1	0	-5	-4	-3	-4	-1	2			1	2	2	0	0	0	2	2	0	34	32	1	2	4	0	0	0	0	2	15	0.75	1.19
431	K	0	0	2	-2	0	3	1	-4	2	-2	-3	2	-1	-5	-1	-1	3	-5	-4	-2			9	4	9	2	2	13	9	0	6	3	2	11	2	0	3	2	24	0	0	2	0.39	1.20
432	I	0	5	2	0	-3	-1	0	-2	1	-2	-1	-1	-2	-2	-4	0	1	-5	-4	-1			6	32	12	6	0	1	5	2	3	2	6	2	1	1	0	6	8	0	0	5	0.41	1.20
433	L	0	-5	-4	-5	-2	-4	-4	4	1	-2	3	-4	-1	-2	-5	0	-3	-5	-4	1			9	0	0	0	1	0	1	36	4	1	28	0	1	1	0	7	1	0	0	12	0.63	1.20
434	A	1	-4	-2	-3	-2	-4	-3	-3	-5	0	2	-4	1	-1	-1	0	4	-5	-3	2			10	0	1	1	1	0	1	1	0	5	19	0	3	2	3	6	31	0	1	17	0.48	1.20
435	D	-1	3	2	0	-3	3	1	3	1	-5	-5	0	-4	-5	-4	1	-2	-2	-3	-5			3	14	11	5	1	15	10	20	3	1	0	5	0	0	0	10	1	1	1	0	0.50	1.19
436	S	-3	0	2	3	-2	2	5	-2	1	-5	-5	0	-4	-5	-4	0	-1	-3	-4	-3			1	4	10	17	1	9	36	3	4	0	0	3	0	0	0	8	4	0	0	1	0.69	1.13
437	I	-3	-4	-5	-5	-3	-4	-5	-6	-4	4	3	-4	3	0	-5	-4	0	-4	2	3			0	0	0	0	0	0	0	0	0	24	34	0	6	2	0	0	5	0	7	22	0.76	0.93
438	N	-3	4	3	-1	-5	3	-1	-3	2	-5	-5	4	-4	-5	-4	1	-2	-5	-4	-5			0	22	17	3	0	16	1	0	5	0	0	24	0	0	0	11	0	0	0	0	0.77	0.92
439	S	-1	-3	1	2	-3	0	2	2	4	-4	-1	-3	-3	-2	-4	2	0	-5	-2	-3			3	0	7	11	0	4	12	18	11	1	6	0	0	2	0	17	6	0	1	1	0.37	0.91
440	E	-1	-3	0	4	-5	1	5	-1	-2	-5	-5	-2	1	-5	-3	-2	-3	-5	-3	-4			5	0	3	27	0	5	50	5	0	0	0	0	4	0	0	0	0	0	1	0	0.98	0.89
441	I	-1	-4	-5	-5	-3	-4	-5	-5	-5	2	4	-4	4	1	-5	-3	-2	-4	-3	2			5	0	0	0	0	0	0	0	0	13	49	0	13	7	0	1	1	0	0	12	0.80	0.89
442	G	-2	-2	3	2	1	2	0	0	2	-4	-2	0	-3	-5	-4	2	0	-5	-4	-2			2	1	16	14	4	12	3	8	4	0	4	6	0	0	0	18	5	0	0	3	0.37	0.89
443	I	0	-4	0	-4	-3	0	-3	-4	4	4	0	-2	-1	-3	-4	0	2	-5	-3	1			9	0	4	0	0	4	0	0	11	32	7	1	1	0	0	7	15	0	0	9	0.47	0.89
444	L	-3	-4	-5	-5	-3	-4	-1	-6	-5	2	5	-4	1	0	-5	-4	0	-4	-3	2			0	0	0	0	0	0	4	0	0	10	61	0	2	3	0	0	5	0	0	14	0.83	0.89
445	C	0	-2	1	0	5	1	-1	0	-1	-2	-3	-1	1	-3	-4	2	3	-5	-4	-1			7	1	7	4	13	5	4	7	2	1	1	4	3	1	0	14	23	0	0	3	0.37	0.89
446	S	-2	-3	4	4	0	-1	1	-2	3	-4	-3	-1	-4	-3	-4	1	2	-5	-1	-3			2	0	21	26	2	2	7	2	7	0	2	3	0	1	0	10	12	0	2	1	0.56	0.89
447	A	6	-4	-3	-4	-3	-3	-3	-2	-4	-3	-1	-3	-3	1	-3	1	-1	-4	-3	-1			77	0	0	0	0	0	0	0	0	0	4	0	0	6	0	8	2	0	0	2	0.97	0.85
448	L	0	-4	-5	-5	-3	-4	-5	-5	-5	1	5	-4	2	0	-5	-4	-3	-4	-3	1			9	0	0	0	0	0	0	0	0	6	71	0	5	2	0	0	0	0	0	7	0.94	0.85
449	Q	0	1	3	1	-2	0	2	0	0	-5	-3	2	-4	-5	-3	2	-2	-5	-4	-3			6	9	18	8	1	3	15	8	2	0	2	12	0	0	0	15	1	0	0	1	0.39	0.85
450	K	0	3	-1	-3	-5	0	-1	-4	-3	-4	-4	6	-2	-5	-3	-1	-1	-5	-1	-1			6	12	2	0	0	1	2	0	0	0	0	66	1	0	0	2	2	0	2	5	0.95	0.83
451	I	-3	-4	-5	-4	-1	-4	-5	-5	-5	4	4	-4	6	-1	-5	-4	-3	-4	-3	1			0	0	0	1	1	0	0	0	0	32	39	0	23	1	0	0	0	0	0	4	0.95	0.83
452	K	-2	2	-2	-2	-5	4	2	-3	-2	-4	-4	5	-3	-5	-3	-2	-2	-4	-3	-4			0	11	0	0	0	24	10	0	0	0	0	55	0	0	0	0	0	0	0	0	0.91	0.53

MSA with all mammalian homologous

We identified homologous sequences from mammalians with the help of HSSP. We used the entry 1J8U. Then we checked whether the homologous sequences of this entry are from mammalians and whether they are not only a "Fragment". The reason for this is that fragment sequences are very short and may not be very helpful for our alignment. The following entries fulfilled these requirements:

FASTA Header
PDBID|CHAIN|SEQUENCE
P00439|PH4H_HUMAN Phenylalanine-4-hydroxylase OS=Homo sapiens GN=PAH PE=1 SV=1
Q16265|Q16265_HUMAN PAH protein (Fragment) OS=Homo sapiens GN=PAH PE=4 SV=1
Q16021|Q16021_HUMAN PAH protein (Fragment) OS=Homo sapiens GN=PAH PE=2 SV=2
Q8TEY0|Q8TEY0_HUMAN Phenylalanine hydroxylase OS=Homo sapiens GN=PAH PE=2 SV=1
Q16266|Q16266_HUMAN PAH protein (Fragment) OS=Homo sapiens GN=PAH PE=4 SV=1
Q91WV1|Q91WV1_MOUSE Phenylalanine hydroxylase OS=Mus musculus GN=Pah PE=2 SV=1
B2R8I4|B2R8I4_HUMAN cDNA, FLJ93916, highly similar to Homo sapiens phenylalanine hydroxylase (PAH), mRNA OS=Homo sapiens PE=2 SV=1
Q8TEY0|Q8TEY0_HUMAN Phenylalanine hydroxylase OS=Homo sapiens GN=PAH PE=2 SV=1
Q5RDX0|Q5RDX0_PONAB Putative uncharacterized protein DKFZp469G0337 OS=Pongo abelii GN=DKFZp469G0337 PE=2 SV=1
E2R3S1|E2R3S1_CANFA Uncharacterized protein OS=Canis familiaris GN=PAH PE=4 SV=1
E2R366|E2R366_CANFA Uncharacterized protein OS=Canis familiaris GN=PAH PE=4 SV=1
Q91WV1|Q91WV1_MOUSE Phenylalanine hydroxylase OS=Mus musculus GN=Pah PE=2 SV=1
Q3UEH8|Q3UEH8_MOUSE Putative uncharacterized protein OS=Mus musculus GN=Pah PE=2 SV=1
P16331|PH4H_MOUSE Phenylalanine-4-hydroxylase OS=Mus musculus GN=Pah PE=1 SV=3
Q2KIH7|PH4H_BOVIN Phenylalanine-4-hydroxylase OS=Bos taurus GN=PAH PE=2 SV=1
D3Z270|D3Z270_MOUSE Uncharacterized protein OS=Mus musculus GN=Pah PE=4 SV=1
P04176|PH4H_RAT Phenylalanine-4-hydroxylase OS=Rattus norvegicus GN=Pah PE=1 SV=3
Q8HXR0|Q8HXR0_MACMU Tryptophan hydroxylase (Fragment) OS=Macaca mulatta GN=TPH PE=2 SV=1
Q6AYW2|Q6AYW2_RAT Phenylalanine hydroxylase OS=Rattus norvegicus GN=Pah PE=2 SV=1
B4DPN2|B4DPN2_HUMAN cDNA FLJ56667, highly similar to Phenylalanine-4-hydroxylase (EC 1.14.16.1) OS=Homo sapiens PE=2 SV=1
Q14CC5|Q14CC5_HUMAN TPH2 protein OS=Homo sapiens GN=TPH2 PE=2 SV=1
Q8IWU9|TPH2_HUMAN Tryptophan 5-hydroxylase 2 OS=Homo sapiens GN=TPH2 PE=1 SV=1
Q2HZ26|TPH2_MACMU Tryptophan 5-hydroxylase 2 OS=Macaca mulatta GN=TPH2 PE=2 SV=1
Q8CGU9|TPH2_RAT Tryptophan 5-hydroxylase 2 OS=Rattus norvegicus GN=Tph2 PE=2 SV=1
P17752|TPH1_HUMAN Tryptophan 5-hydroxylase 1 OS=Homo sapiens GN=TPH1 PE=1 SV=4
Q86Y20|Q86Y20_HUMAN Tryptophan hydroxylase (Fragment) OS=Homo sapiens PE=2 SV=1
Q86WC5|Q86WC5_HUMAN Tryptophan hydroxylase (Fragment) OS=Homo sapiens PE=2 SV=1
Q86Y19|Q86Y19_HUMAN Tryptophan hydroxylase (Fragment) OS=Homo sapiens PE=2 SV=1
Q9JHZ8|Q9JHZ8_MOUSE Tryptophan hydroxylase (Fragment) OS=Mus musculus GN=Tph1 PE=2 SV=1
Q8K3R1|Q8K3R1_MESAU Tryptophane hydroxylase OS=Mesocricetus auratus PE=2 SV=1
E1BGX6|E1BGX6_BOVIN Uncharacterized protein OS=Bos taurus GN=TPH2 PE=4 SV=1
Q0EAB8|TPH2_HORSE Tryptophan 5-hydroxylase 2 OS=Equus caballus GN=TPH2 PE=2 SV=1
Q0VBT4|Q0VBT4_MOUSE Tryptophan hydroxylase 2 OS=Mus musculus GN=Tph2 PE=2 SV=1
B6EY10|B6EY10_CANFA Tryptophan hydroxylase 1 isoform 1 OS=Canis familiaris GN=TPH1 PE=2 SV=1
Q8CGV2|TPH2_MOUSE Tryptophan 5-hydroxylase 2 OS=Mus musculus GN=Tph2 PE=2 SV=1
P09810|TPH1_RAT Tryptophan 5-hydroxylase 1 OS=Rattus norvegicus GN=Tph1 PE=2 SV=2
P17290|TPH1_RABIT Tryptophan 5-hydroxylase 1 OS=Oryctolagus cuniculus GN=TPH1 PE=1 SV=2
P17532|TPH1_MOUSE Tryptophan 5-hydroxylase 1 OS=Mus musculus GN=Tph1 PE=2 SV=1
Q3UK52|Q3UK52_MOUSE Tryptophan hydroxylase 1 OS=Mus musculus GN=Tph1 PE=2 SV=1
Q76IQ3|TY3H_CANFA Tyrosine 3-monooxygenase OS=Canis familiaris GN=TH PE=2 SV=1
P04177|TY3H_RAT Tyrosine 3-monooxygenase OS=Rattus norvegicus GN=Th PE=1 SV=3
Q8IZE2|Q8IZE2_HUMAN Putative tyrosine hydroxylase variant (Fragment) OS=Homo sapiens PE=2 SV=1
Q8IZE1|Q8IZE1_HUMAN Truncated tyrosine hydroxylase (Fragment) OS=Homo sapiens PE=2 SV=1
Q8N1X9|Q8N1X9_HUMAN cDNA FLJ37295 fis, clone BRAMY2015311, moderately similar to TRYPTOPHAN 5-MONOOXYGENASE (EC 1.14.16.4) OS=Homo sapiens PE=2 SV=1
Q8MJD2|Q8MJD2_RABIT Tyrosine hydroxylase (Fragment) OS=Oryctolagus cuniculus PE=2 SV=1
Q8HY50|Q8HY50_RABIT Tyrosine hydroxylase (Fragment) OS=Oryctolagus cuniculus PE=2 SV=1
Q95LQ6|Q95LQ6_HORSE Tyrosine hydroxylase (Fragment) OS=Equus caballus GN=TH PE=2 SV=1
Q8HZ55|Q8HZ55_PANTR Tyrosine hydroxylase (Fragment) OS=Pan troglodytes PE=4 SV=1
Q8HZ54|Q8HZ54_9PRIM Tyrosine hydroxylase (Fragment) OS=Gorilla gorilla PE=4 SV=1
Q8MIU1|Q8MIU1_PIG Tyrosine hydroxylase (Fragment) OS=Sus scrofa PE=4 SV=1
Q8HZ53|Q8HZ53_PONPY Tyrosine hydroxylase (Fragment) OS=Pongo pygmaeus PE=4 SV=1
Q8HXJ0|Q8HXJ0_MACFA Putative uncharacterized protein OS=Macaca fascicularis PE=2 SV=1
P97517|P97517_PHOSU Tyrosine hydroxylase (Fragment) OS=Phodopus sungorus PE=2 SV=1
P07101|TY3H_HUMAN Tyrosine 3-monooxygenase OS=Homo sapiens GN=TH PE=1 SV=5
Q2M3B4|Q2M3B4_HUMAN Tyrosine hydroxylase OS=Homo sapiens GN=TH PE=2 SV=1
B6EY12|B6EY12_CANFA Tryptophan hydroxylase 2 OS=Canis familiaris GN=TPH2 PE=2 SV=1
B7ZL73|B7ZL73_HUMAN TH protein OS=Homo sapiens GN=TH PE=2 SV=1
E0V877|E0V877_MICOH Tyrosine hydroxylase OS=Microtus ochrogaster GN=TH PE=4 SV=1
Q3UTB3|Q3UTB3_MOUSE Putative uncharacterized protein OS=Mus musculus GN=Th PE=2 SV=1
Q76IQ3|TY3H_CANFA Tyrosine 3-monooxygenase OS=Canis familiaris GN=TH PE=2 SV=1
Q8K3R1|Q8K3R1_MESAU Tryptophane hydroxylase OS=Mesocricetus auratus PE=2 SV=1
P24529|TY3H_MOUSE Tyrosine 3-monooxygenase OS=Mus musculus GN=Th PE=1 SV=3
B3RFH4|B3RFH4_SORAR Tyrosine 3-monooxygenase (Predicted) OS=Sorex araneus GN=TH PE=4 SV=1
B7NZU5|B7NZU5_RABIT Tyrosine hydroxylase isoform b (Predicted) OS=Oryctolagus cuniculus GN=TH PE=4 SV=1
Q0PWM3|Q0PWM3_HUMAN Tyrosine hydroxylase isoform D1b,2,8,9 OS=Homo sapiens GN=TH PE=2 SV=1
Q0PWM2|Q0PWM2_HUMAN Tyrosine hydroxylase isoform D2,8,9 OS=Homo sapiens GN=TH PE=2 SV=1
B2KIN8|B2KIN8_RHIFE Tyrosine hydroxylase (Predicted) OS=Rhinolophus ferrumequinum GN=TH PE=4 SV=1
P17289|TY3H_BOVIN Tyrosine 3-monooxygenase OS=Bos taurus GN=TH PE=1 SV=5
Q8HXJ0|Q8HXJ0_MACFA Putative uncharacterized protein OS=Macaca fascicularis PE=2 SV=1
B6EY11|B6EY11_CANFA Tryptophan hydroxylase 1 isoform 2 OS=Canis familiaris GN=TPH1 PE=2 SV=1
Q3TZH2|Q3TZH2_MOUSE Putative uncharacterized protein OS=Mus musculus GN=Tph1 PE=2 SV=1
Q8N1X9|Q8N1X9_HUMAN cDNA FLJ37295 fis, clone BRAMY2015311, moderately similar to TRYPTOPHAN 5-MONOOXYGENASE (EC 1.14.16.4) OS=Homo sapiens PE=2 SV=1
Q08EN2|Q08EN2_HUMAN TPH1 protein OS=Homo sapiens GN=TPH1 PE=2 SV=1

After we had all our FASTA sequences we used t-coffee 3D to create our MSA. We utilized the following command to create our MSA with the structure of 1J8U as reference:

• t_coffee ../homo_seq.fasta -method sap_pair,slow_pair -template_file 1J8U

The resulting MSA is as follows:

I65T

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 65

Substitution Scores:
Blosum 62: -1 (max: 4, max/dia: 3, min: -4)
PAM 1: 11 (max: 9872, max/dia: 57, min: 0)
PAM 250: 6 (max: 15, max/dia: 15, min: 0)
PSI-PSSM(I): score 6/weighted observed percentages 58 (max-score: 6, min-score: -6, max-weighted observed percentages: 58)
PSI-PSSM(T): score -4/weighted observed percentages 0 (max-score: 6, min-score: -6, max-weighted observed percentages: 58)
Multiple Alignment(I): 1/74
Multiple Alignment(T): 0/74

R71H

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 71

Substitution Scores:
BLOSUM62: 0 (max: 5, max/dia: 2, min: -3)
PAM1: 10 (max: 9917, max/dia: 37, min: 0)
PAM250: 5 (max: 18, max/dia: 18, min: 1)
PSI-PSSM(R): score -2/weighted observed percentages 27 (max-score: 5, min-score: -5, max-weighted observed percentages: 27)
PSI-PSSM(H): score -2/weighted observed percentages 2 (max-score: 5, min-score: -5, max-weighted observed percentages: 27)
Multiple Alignment(R): 1/74
Multiple Alingment(H): 0/74

R158Q

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 158

Substitution Scores:
BLOSUM62: 1 (max: 5, max/dia: 2, min: -3)
PAM1: 9 (max: 9917, max/dia: 37, min: 0)
PAM250: 5 (max: 18, max/dia: 18, min: 1)
PSI-PSSM(R): score 6/weighted observed percentages 50% (max-score: 6, min-score: -4, max-weighted observed percentages: 50)
PSI-PSSM(Q): score 1/weighted observed percentages 6% (max-score: 6, min-score: -4, max-weighted observed percentages: 50)
Multiple Alignment(R): 62/74
Multiple Alingment(Q): 0/74

R261Q

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 261

Substitution Scores:
BLOSUM62: 1 (max: 5, max/dia: 2, min: -3)
PAM1: 9 (max: 9917, max/dia: 37, min: 0)
PAM250: 5 (max: 18, max/dia: 18, min: 1)
PSI-PSSM(R): score 6/weighted observed percentages 47% (max-score: 6, min-score: -4, max-weighted observed percentages: 47)
PSI-PSSM(Q): score 0/weighted observed percentages 0% (max-score: 6, min-score: -4, max-weighted observed percentages: 47)
Multiple Alignment(R): 1/74
Multiple Alignment(Q): 0/74

T266A

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 266

Substitution Scores:
BLOSUM62: 0 (max: 5, max/dia: 1, min: -2)
PAM1: 32 (max: 9871, max/dia: 38, min: 0)
PAM250: 11 (max: 11, max/dia: 11, min: 0)
PSI-PSSM(T): score 5/weighted observed percentages 40% (max-score: 5, min-score: -4, max-weighted observed percentages: 40)
PSI-PSSM(A): score 4/weighted observed percentages 40% (max-score: 5, min-score: -4, max-weighted observed percentages: 40)
Multiple Alignment(T): 48/74
Multiple Alingment(A): 0/74

P275S

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 275

Substitution Scores:
BLOSUM62: -1 (max: 7, max/dia: -1, min: -4)
PAM1: 17 (max: 9926, max/dia: 22, min: 0)
PAM250: 9 (max: 20, max/dia: 11, min: 0)
PSI-PSSM(P): score 5/weighted observed percentages 29% (max-score: 5, min-score: -5, max-weighted observed percentages: 29)
PSI-PSSM(S): score -3/weighted observed percentages 0% (max-score: 5, min-score: -5, max-weighted observed percentages: 29)
Multiple Alignment(P): 48/74
Multiple Alingment(S): 0/74

T278N

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 278

Substitution Scores:
BLOSUM62: 0 (max: 5, max/dia: 1, min: -2)
PAM1: 9 (max: 9871, max/dia: 38, min: 0)
PAM250: 4 (max: 11, max/dia: 11, min: 0)
PSI-PSSM(T): score 4/weighted observed percentages 33% (max-score: 4, min-score: -5, max-weighted observed percentages: 33)
PSI-PSSM(N): score -1/weighted observed percentages 2% (max-score: 4, min-score: -5, max-weighted observed percentages: 33)
Multiple Alignment(T): 34/74
Multiple Alingment(N): 0/74

P281L

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 281

Substitution Scores:
BLOSUM62: -3 (max: 7, max/dia: -1, min: -4)
PAM1: 3 (max: 20, max/dia: 11, min: 0)
PAM250: 5 (max: 20, max/dia: 11, min: 0)
PSI-PSSM(P): score 8/weighted observed percentages 84% (max-score: 8, min-score: -5, max-weighted observed percentages: 84)
PSI-PSSM(L): score -5/weighted observed percentages 0% (max-score: 8, min-score: -5, max-weighted observed percentages: 84)
Multiple Alignment(P): 48/74
Multiple Alingment(L): 0/74

G312D

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 312

Substitution Scores:
BLOSUM62: -1 (max: 6, max/dia: 0, min: -4)
PAM1: 6 (max: 9925, max/dia: 21, min: 0)
PAM250: 11 (max: 27, max/dia: 12, min: 0)
PSI-PSSM(G): score 3/weighted observed percentages 20% (max-score: 5, min-score: -5, max-weighted observed percentages: 21)
PSI-PSSM(D): score -1/weighted observed percentages 0% (max-score: 5, min-score: -5, max-weighted observed percentages: 21)
Multiple Alignment(G): 1/74
Multiple Alignment(D): 0/74

R408W

Figure 3. Histogram of PSI-Blast PSSM conservation scores for position 408

Substitution Scores:
BLOSUM62: -3 (max: 5, max/dia: 2, min: -3)
PAM1: 2 (max: 9917, max/dia: 37, min: 0)
PAM250: 2 (max: 18, max/dia: 18, min: 1)
PSI-PSSM(R): score 6/weighted observed percentages 40% (max-score: 6, min-score: -5, max-weighted observed percentages: 40)
PSI-PSSM(W): score -5/weighted observed percentages 0% (max-score: 6, min-score: -5, max-weighted observed percentages: 40)
Multiple Alignment(R): 1/74
Multiple Alignment(W): 0/74

Mutations and secondary structure

Predicted secondary structure

We took the secondary structure prediction from Task 3:

Reference	MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAK
DSSP	--------------------------------------------------
Reference	VLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRH
DSSP	--------------------------------------------------
Reference	DIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFK
DSSP	-----------------.....SBGGGGGGTT.S.------..TTSTTTT
Reference	DPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKT
DSSP	.HHHHHHHHHHHHHHHH..TTS........HHHHHHHHHHHHHHHHHHHH
Reference	HACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLS
DSSP	HB.HHHHHHHHHHHHHS..BTTB...HHHHHHHHHHHT..EEEE.SS...
Reference	SRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFA
DSSP	HHHHHHHHTTTEEEE......TT.TT..SS..HHHHHTTTTTTTTSHHHH
Reference	QFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSS
DSSP	HHHHHHHHHHTT..HHHHHHHHHHHHTTTTT.EEEETTEEEE..HHHHT.
Reference	FGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVR
DSSP	HHHHHHTTSSSS..EE..HHHHTT....SSS..S..EEES.HHHHHHHHH
Reference	NFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQK
DSSP	HHHHTS..SSEEEEETTTTEEEEE.HHHHHHHHHHHHHHHHHHHHHHHHH
Reference	IK
DSSP	T.

I65T

Not covered by the structure used for DSSP.

R71H

Not covered by the structure used for DSSP.

R158Q

The mutation affects a helix.

R261Q

The mutation affects a turn.

T266A

The mutation affect an unstructured region.

P275S

The mutation affects a turn.

T278N

The mutation affects an unstructured region.

P281L

The mutation affects an unstructured region.

G312D

The mutation affects a turn.

R408W

The mutation affects an unstructured region.

Catalytic sites and mutations

We identifier the following catalytic sites with the help of Catalytic Site Atlas. We looked for catalytic sites in the structure of 1J8U.

We identified the following catalytic sites:

• HIS 285, functional part: side chain
• SER 349, functional part: side chain

In the following picture we can see the location of the mutated residues in orange and the two identified catalytic sites in yellow:

I65T

Probably no effect on catalytic site because it is too far away.

R71H

Probably no effect on catalytic site because it is too far away.

R158Q

Probably no effect on catalytic site because it is too far away.

R261Q

Probably no effect on catalytic site because it is too far away.

T266A

No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center.

P275S

Probably no effect on catalytic site because it is too far away.

T278N

No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center. ´

P281L

Probably direct influence on catalytic site residue HIS 285. In addition, this residue is located what we would define as the catalytic center.

G312D

Probably no effect on catalytic site because it is too far away.

R408W

Probably no effect on catalytic site because it is too far away.

Predicting the effect of mutations with SNAP, SIFT and Polyphen2

SNAP

Execution

snapfun -i reference.fasta -m mutations.txt -o snap.txt

Result

mutation	effect	Reliability	Expected Accuracy
R408W	Non-neutral	4	82%
I65T	Non-neutral	5	87%
R261Q	Non-neutral	5	87%
G312D	Non-neutral	1	63%
R71H	Neutral	0	53%
P281L	Non-neutral	3	78%
R158Q	Non-neutral	6	93%
P275S	Non-neutral	2	70%
T266A	Non-neutral	2	70%
T278N	Non-neutral	4	82%

SIFT

Execution

To run our predictions with SIFT we used the webservice. We had the following input (See also figure):

SIFT input

• Sequence: Fasta sequence from the Uniprot entry P00439.
• Substitutions: our ten selected substitutions
• Database to search: UniRef90_2011_Apr
• Median conservation of sequences: 3.00
• Removed sequences: If sequence identity is greater than 90%

Results

Position	Reference AA	Mutated AA	Predicted effect	Score	Median sequence conservation	Sequences represented at this position
65	I	T	AFFECT PROTEIN FUNCTION	0	3.03	64
71	R	H	AFFECT PROTEIN FUNCTION	0.05	3.03	64
158	R	Q	AFFECT PROTEIN FUNCTION	0	3	64
261	R	Q	AFFECT PROTEIN FUNCTION	0	3.01	65
266	T	A	AFFECT PROTEIN FUNCTION	0	3.01	65
275	P	S	AFFECT PROTEIN FUNCTION	0	3.01	65
278	T	N	AFFECT PROTEIN FUNCTION	0	3.01	65
281	P	L	AFFECT PROTEIN FUNCTION	0	3.01	65
312	G	D	AFFECT PROTEIN FUNCTION	0	3.01	65
408	R	W	AFFECT PROTEIN FUNCTION	0	3	64

Polyphen2

Execution

To run our predictions with Polyphen2 we used the webservice. We had the following input (See also figure):

Polyphen2 input

• Sequence: Fasta sequence from the Uniprot entry P00439.
• Substitutions: our ten selected substitutions
• Classifier model: HumDiv
• Genome assembly: GRCh37/hg19
• Transcripts: canonical
• Annotations: missense

Results

#o_snp_id	snp_id	acc	pos	aa1	aa2	prediction	pph2_prob	pph2_FPR	ph2_TPR
P00439_I65T	P00439_I65T	P00439	65	I	T	probably damaging	1	0.0001	0.0002
P00439_R71H	P00439_R71H	P00439	71	R	H	possibly damaging	0.936	0.0539	0.796
P00439_R158Q	P00439_R158Q	P00439	158	R	Q	probably damaging	1	0.0001	0.0002
P00439_R261Q	rs5030849	P00439	261	R	Q	probably damaging	1	0.0001	0.0002
P00439_T266A	P00439_T266A	P00439	266	T	A	probably damaging	1	0.0001	0.0002
P00439_P275S	P00439_P275S	P00439	275	P	S	probably damaging	1	0.0001	0.0002
P00439_T278N	P00439_T278N	P00439	278	T	N	probably damaging	0.998	0.0114	0.269
P00439_P281L	P00439_P281L	P00439	281	P	L	probably damaging	1	0.0001	0.0002
P00439_G312D	P00439_G312D	P00439	312	G	D	probably damaging	1	0.0001	0.0002
P00439_R408W	P00439_R408W	P00439	408	R	W	probably damaging	1	0.0001	0.0002

Discussion

Some mutations can cause severe malfunctions if they occur at the wrong place. Using only sequence based predictions probably fail to predict the effect of such a mutation. Such a mutation might seem to be non-harmful regarding only general substitution scores or physico-chemical properties.

Mutations in Detail

I65T

The mutation changes the residue from unpolar to polar. The region, where the residue is located, is presumably a disordered region. Therefore it probably folds with the right binding partner. Even in PAM1 this mutation seems to take place quite often. Therefore theres seem to be some proteins, where this kind of mutation is allowed. In the PSSM of PSI-BLAST the mutated residue is not recorded in any of the sequences. The secondary structure of this residue could not be determined by DSSP, because the used structure does not contain it. SNAP predicts the mutation to be damaging with high reliability and accuracy. SIFT and Polyphen2 agree with this prediction.

Depending on the fold of the region, where the mutated residue is located, the mutation can cause severe results during the folding and therefore the binding properties of the disordered region. Therefore we would predict it to be probably damaging to the function of the protein.

R71H

The residues differ in their physiko-chemical properties. H has an aromatic group and it is basic. The residue is probably at the border of a disordered region. The general substitution matrices differ in their scoring. In the PAM matrices this substitution is quite okay. In BLOSUM62 it is disfavored. SIFT predicts the mutation to be damaging, but the score is in the twilight-zone. Polyphen predicts it to be damaging. SNAP predicts the mutation to be neutral, but with low reliability and average accuracy.

Since the residue is probably at the border of a disordered region. It's impact on the structure is perhaps not strong.

R158Q

The mutation from amino acid arginine to amino acid glutamine on position 158 leads to the effect that this residue is no longer positive charged which might have an influence on its neighbors in close proximity. In fact, we observed that this mutation leads probably to a loss of polar contacts to E280, Y154 and E141 which certainly influences the overall protein stability. In addition this mutation is located within a alpha-helix. Although, the mutant glutamine is not know to be a helix breaker as proline or glycine as well as it is not known to be one of the 5 most common alpha-helix amino acids (known as MALEK = methionine, alanine, leucine, uncharged glutamate, and lysine). Therefore, it is hard for us to tell whether this mutant has a good or bad influence on the structure of the alpha-helix. Another interesting fact is that this mutation is not close to the catalytic which has been identified to be the residues HIS 285 and SER 349. Hence, the possibility of a direct influence of this mutation to the catalytic reaction can be excluded and other reasons as overall protein stability which is violated by the loss of three polar interactions is more likely to harm the proper function.

In a next step we analyzed the scores and conservation of this mutation to see how established scoring matrices like BLOSUM and PAM judge this mutation and how conserved this residue is in all mammalian homologous. In BLOSUM62 we got a score of 1 which is the second best score. The worst score for a mutation from arginine to another amino acid is produces by either cysteine, isoleucine, phenylalanine or tryptophan with a score of -3. The best mutation score is produced by a mutation to lysine with a score of 2. Hence, we assume that our mutation from arginine to glutamine is quite accepted by BLOSUM62.

We got similar results for the PAM1 and PAM250 scoring matrices. For PAM1 we have got an score of 9 which is quite good with respect to a worst score of 0 and a best mutation score of 37. For PAM250 it is similar but not the same. Here we got a score of 5 with a worst score of 1 and the best mutation score of 9.

However, when we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation is not quite accepted in evolution. To be more precise: The mutant glutamine has been observed in 6% of all cases for the position 158 whereas the WT arginine has a conservation of 50% in our PSSM. The conservation of arginine is even more present in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 62/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this conservation is driven by some positive selection pressure which is suppressing a more liberal incorporation of other amino acids for this position.

Finally, we run the in silico effect predictors SNAP, SIFT and Polyphen2. All three programs predicted with high confidence to have an bad influence on the proteins function.

In summary we got the following pro and contra arguments whether this mutation should be treated as disease causing or not:

Pro:

• loss of positive charge
• loss of three polar interactions
• strong conservation of this position in our PSSM and MSA
• strong confidence of all three in silico predictors to have negative effect on protein function

Contra:

• mutant is not a helix breaker
• not close to catalytic site
• neutral score in BLOSUM, PAM1 and PAM250

After thinking about the pro and contra arguments for a while we decided to associate this mutation as disease causing.The reason for this decision is that we think that our pro arguments can be considered as "strong" arguments and the contra arguments are more weak and general which apply to a lot of amino acids.

Real annotation: disease causing. (-> our prediction was correct)

R261Q

The mutated residue Q differs to R in the loss of the positive charge. Both residues are structurally very similar. The residue is part of a PFAM domain. Pymol was able to find a rotamer for the shorter residue Q, which fits almost perfectly the native rotamer of R. This change seems to be supported by the general substitution matrices. DSSP predicts the residue to be part of a turn. Therefore the positive charge of R is probably essential to establish the bonds, which are necessary for the turn. Directly after the residue starts a sheet. That means small changes can influence the sheet and therefore the protein structure and its function.

SNAP predicts the mutation to be harmful with high accuracy and reliability. SIFT and Polyphen2 agree to this prediction.

T266A

The mutation from amino acid threonine to amino acid alanine on position 266 leads to the minor effect that this residue is no longer polar but the rest of the physicochemical properties stay pretty much the same. In fact, we observed that this mutation leads probably to a loss of a polar contact to E286 which maybe has some influence. In addition this mutation is located within a loop which are known to be more liberal to mutations than secondary structure elements like alpha-helices and beta-strands. Another interesting fact is that this mutation is close to the catalytic site which has been identified to be the residues HIS 285 and SER 349. Hence, the possibility of a indirect or direct influence of this mutation to the catalytic reaction can be not completely excluded.

In a next step we analyzed the scores and conservation of this mutation to see how established scoring matrices like BLOSUM and PAM judge this mutation and how conserved this residue is in all mammalian homologous. In BLOSUM62 we got a score of 0 which is the second best score. The worst score for a mutation from threonine to another amino acid is produced by either glycine, histidine, phenylalanine, tryptophan or tyrosine with a score of -2. The best mutation score is produced by a mutation to serine with a score of 1. Hence, we assume that our mutation from threonine to alanine is quite accepted by BLOSUM62.

We got similar results for the PAM1 and PAM250 scoring matrices. For PAM1 we have got an score of 32 which is quite good with respect to a worst score of 0 and a best mutation score of 38. For PAM250 it is similar but not the same. Here we got a score of 11 with a worst score of 0 and the best mutation score of 11.

When we looked for the conservation of this mutation in our PSSM and MSA we noticed that this mutation is can be told to be tolerated from a evolutionary point of view. To be more precise: The mutant alanine has been observed in 40% of all cases for the position 266 whereas the WT arginine has a conservation of 40% in our PSSM. The conservation of threonine is more present in our MSA which is constructed from homologous mammalian sequences only. Here we have a conservation of 48/74 for the WT and a conservation of 0/74 for the mutant. So we assume that this position is not so much under positive selection pressure since we observed that both alleles can exists at this position (at least in our PSSM).

Finally, we run the in silico effect predictors SNAP, SIFT and Polyphen2. Two of three programms predicted with high confidence to have an bad influence on the proteins function (SIFT and Polyphen2). The predictor SNAP is not that certain about the influence of this mutation since it predicted it only with a 70% accuracy.

In summary we got the following pro and contra arguments whether this mutation should be treated as disease causing or not:

Pro:

• probably loss of one polar interactions
• close to catalytic site
• strong confidence of two in silico predictors to have negative effect on protein function (SIFT and PolyPhen2)

Contra:

• not so much change in the physicochemical properties
• neutral and slight good scores in BLOSUM, PAM1 and PAM250
• mutant and WT are both accepted with 40% in our PSSM
• MSA conservation not that high
• not so much confidence of the predictor SNAP about harmful effect of mutation (only 70%)

After thinking about the pro and contra arguments for a while we decided to associate this mutation as not disease causing.

Real annotation: not disease causing. (-> our prediction was correct)

G312D

This mutation changes the physico-chemical properties of the acid in a quite drastic way. G is mutated to the polar, negatively charged and acidic residue D. At the wrong position in the structure this can cause quite some trouble. In the structure superposition both residues seem to occupy quite the same volume. The general substitution matrices differ in the scoring for this substitution. In BLOSUM62 this exchange seems to be slightly unfavored. In both PAM matrices the substitution is okay. DSSP predicts the residue to be part of a turn. Compared to G D does not loose any properties, which could be essential for the stabilization of the turn. The structure is perhaps not influenced.

SNAP predicts the mutation to be non-neutral, but the program is as unsure as we are with respect to the reliability score and the accuracy. SIFT and Polyphen2 predict it to be harmful without doubts.

If there is an influence of G312D then we would predict that its influence on the protein function is not that strong. Perhaps it does not even cause a symptom.

R408W

The physico-chemical properties of the wildtype and mutated residue are quite different. The mutation is aromatic and not positively charged. The position 408 is part of a PFAM domain, which means that this region is quite compact. Therefore even little changes can cause quite some trouble. In the superposition of the two residues, one can see that they differ quite strongly in their structures. Pymol was unable to find a more overlapping rotamer for the mutated W with the wildtype R rotamer. The scores in the substitution matrices support the assumption, that such a change is not very likely. DSSP predicts the residue to be unstructured. But there is a helix to the left and a sheet to the right. Small changes might influence these two structures and cause trouble in the protein function.

SNAP, SIFT and Polyphen2 agree with our observations. The mutation is very likely harmful.