Evolutionary trace report by report maker February 10, 2010mammoth.bcm.tmc.edu/report_maker/reports/pdb... · February 10, 2010 CONTENTS 1 Introduction 1 2 Chain 2vpwB 1 2.1 Q72LA5

Pages 1–25

2vpwEvolutionary trace report by report maker

February 10, 2010

CONTENTS

1 Introduction 1

2 Chain 2vpwB 12.1 Q72LA5 overview 12.2 Multiple sequence alignment for 2vpwB 12.3 Residue ranking in 2vpwB 12.4 Top ranking residues in 2vpwB and their position on

the structure 12.4.1 Clustering of residues at 25% coverage. 22.4.2 Overlap with known functional surfaces at

25% coverage. 22.4.3 Possible novel functional surfaces at 25%

coverage. 7

3 Chain 2vpwA 93.1 Q72LA4 overview 93.2 Multiple sequence alignment for 2vpwA 93.3 Residue ranking in 2vpwA 93.4 Top ranking residues in 2vpwA and their position on

the structure 93.4.1 Clustering of residues at 25% coverage. 93.4.2 Overlap with known functional surfaces at

25% coverage. 103.4.3 Possible novel functional surfaces at 25%

coverage. 22

4 Notes on using trace results 224.1 Coverage 224.2 Known substitutions 234.3 Surface 234.4 Number of contacts 234.5 Annotation 234.6 Mutation suggestions 23

5 Appendix 235.1 File formats 235.2 Color schemes used 235.3 Credits 24

5.3.1 Alistat 245.3.2 CE 245.3.3 DSSP 245.3.4 HSSP 245.3.5 LaTex 245.3.6 Muscle 245.3.7 Pymol 24

5.4 Note about ET Viewer 245.5 Citing this work 245.6 About reportmaker 245.7 Attachments 24

1 INTRODUCTIONFrom the original Protein Data Bank entry (PDB id 2vpw):Title: Polysulfide reductase with bound menaquinoneCompound: Mol id: 1; molecule: thiosulfate reductase; chain: a, e;synonym: polysulfide reductase; mol id: 2; molecule: nrfc protein;chain: b, f; mol id: 3; molecule: hypothetical membrane spanningprotein; chain: c, gOrganism, scientific name:Thermus Thermophilus;

2vpw contains unique chains 2vpwB (194 residues) and 2vpwA(735 residues) 2vpwF is a homologue of chain 2vpwB. 2vpwE isa homologue of chain 2vpwA. Not enough homologous sequencescould be found to permit analysis for chain 2vpwC.

2 CHAIN 2VPWB

2.1 Q72LA5 overviewFrom SwissProt, id Q72LA5, 88% identical to 2vpwB:Description: NrfC protein.Organism, scientific name:Thermus thermophilus (strain HB27 /ATCC BAA-163 / DSM 7039).Taxonomy: Bacteria; Deinococcus-Thermus; Deinococci; Therma-les; Thermaceae; Thermus.Function: Ferredoxins are iron-sulfur proteins that transfer electronsin a wide variety of metabolic reactions (By similarity).

1

Lichtarge lab 2006

Fig. 1. Residues 1-194 in 2vpwB colored by their relative importance. (SeeAppendix, Fig.24, for the coloring scheme.)

Cofactor: Binds 1 4Fe-4S cluster and 1 3Fe-4S cluster (By simila-rity).

2.2 Multiple sequence alignment for 2vpwBFor the chain 2vpwB, the alignment 2vpwB.msf (attached) with 24sequences was used. The alignment was assembled through combi-nation of BLAST searching on the UniProt database and alignmentusing Muscle program. It can be found in the attachment to thisreport, under the name of 2vpwB.msf. Its statistics, from thealistatprogram are the following:

Format: MSFNumber of sequences: 24Total number of residues: 4462Smallest: 175Largest: 194Average length: 185.9Alignment length: 194Average identity: 42%Most related pair: 99%Most unrelated pair: 28%Most distant seq: 48%

Furthermore, 10% of residues show as conserved in this alignment.The alignment consists of 79% prokaryotic, and 33% archaean

sequences. (Descriptions of some sequences were not readily availa-ble.) The file containing the sequence descriptions can be found inthe attachment, under the name 2vpwB.descr.

2.3 Residue ranking in 2vpwBThe 2vpwB sequence is shown in Fig. 1, with each residue coloredaccording to its estimated importance. The full listing of residuesin 2vpwB can be found in the file called 2vpwB.rankssorted in theattachment.

2.4 Top ranking residues in 2vpwB and their position onthe structure

In the following we consider residues ranking among top 25% ofresidues in the protein . Figure 2 shows residues in 2vpwB coloredby their importance: bright red and yellow indicate more conser-ved/important residues (see Appendix for the coloring scheme). APymol script for producing this figure can be found in the attachment.

Fig. 2. Residues in 2vpwB, colored by their relative importance. Clockwise:front, back, top and bottom views.

2.4.1 Clustering of residues at 25% coverage.Fig. 3 shows thetop 25% of all residues, this time colored according to clusters theybelong to. The clusters in Fig.3 are composed of the residues listed

Fig. 3. Residues in 2vpwB, colored according to the cluster they belong to:red, followed by blue and yellow are the largest clusters (see Appendix forthe coloring scheme). Clockwise: front, back, top and bottomviews. Thecorresponding Pymol script is attached.

in Table 1.

2

Table 1.cluster size membercolor residuesred 46 4,6,9,13,15,16,19,23,27,55

58,60,61,65,66,70,71,72,7481,83,90,92,93,96,99,100,101102,105,113,115,116,118,119129,130,131,132,135,140,142143,171,173,177

blue 2 147,150

Table 1. Clusters of top ranking residues in 2vpwB.

2.4.2 Overlap with known functional surfaces at 25% coverage.The name of the ligand is composed of the source PDB identifierand the heteroatom name used in that file.

Iron/sulfur cluster binding site. Table 2 lists the top 25% of resi-dues at the interface with 2vpwBSF41195 (iron/sulfur cluster). Thefollowing table (Table 3) suggests possible disruptive replacementsfor these residues (see Section 4.6).

Table 2.res type subst’s cvg noc/ dist antn

(%) bb (A)19 C C(100) 0.10 1/1 4.9323 C C(100) 0.10 18/7 2.27 site27 N N(100) 0.10 8/0 3.14 site

116 C C(100) 0.10 17/6 2.30 site118 F F(100) 0.10 18/13 3.68119 C C(100) 0.10 15/6 2.48 site131 C C(100) 0.10 17/6 2.26129 P P(95) 0.16 9/4 3.30 site

T(4)140 R R(62) 0.19 1/0 4.74 site

L(37)130 A A(83) 0.21 8/6 3.51 site

H(4)I(12)

6 M M(83) 0.23 1/0 4.18L(8)F(8)

Table 2. The top 25% of residues in 2vpwB at the interface withiron/sulfur cluster.(Field names: res: residue number in thePDB entry; type:amino acid type; substs: substitutions seen in the alignment;with the percen-tage of each type in the bracket; noc/bb: number of contacts with the ligand,with the number of contacts realized through backbone atoms given in thebracket; dist: distance of closest apporach to the ligand. )

Table 3.res type disruptive

mutations19 C (KER)(FQMWHD)(NYLPI)(SVA)23 C (KER)(FQMWHD)(NYLPI)(SVA)27 N (Y)(FTWH)(SEVCARG)(MD)

continued in next column

3

Table 3.continuedres type disruptive

mutations116 C (KER)(FQMWHD)(NYLPI)(SVA)118 F (KE)(TQD)(SNCRG)(M)119 C (KER)(FQMWHD)(NYLPI)(SVA)131 C (KER)(FQMWHD)(NYLPI)(SVA)129 P (R)(YH)(K)(E)140 R (T)(YD)(SECG)(VA)130 A (E)(KR)(Y)(QD)6 M (Y)(T)(HR)(CG)

Table 3. List of disruptive mutations for the top 25% of residues in2vpwB, that are at the interface with iron/sulfur cluster.

Fig. 4. Residues in 2vpwB, at the interface with iron/sulfur cluster, coloredby their relative importance. The ligand (iron/sulfur cluster) is colored green.Atoms further than 30A away from the geometric center of the ligand, as wellas on the line of sight to the ligand were removed. (See Appendix for thecoloring scheme for the protein chain 2vpwB.)

Figure 4 shows residues in 2vpwB colored by their importance, at theinterface with 2vpwBSF41195.

Interface with 2vpwC.Table 4 lists the top 25% of residues atthe interface with 2vpwC. The following table (Table 5) suggestspossible disruptive replacements for these residues (see Section 4.6).


(%) bb (A)90 C C(100) 0.10 22/20 2.93 site

101 P P(100) 0.10 32/20 3.38102 Y Y(100) 0.10 5/5 3.75


Table 4.continuedres type subst’s cvg noc/ dist antn

(%) bb (A)93 C C(91) 0.12 11/10 2.74 site

Y(8)99 A A(91) 0.12 24/22 3.28

G(8)71 P P(95) 0.13 70/46 2.90 site

L(4)100 C C(95) 0.14 19/19 3.01 site

D(4)105 R R(87) 0.17 16/16 3.38

I(4)V(8)

70 C C(95) 0.21 17/17 3.48 siteL(4)

72 T T(70) 0.23 47/30 3.21V(20)R(8)

15 G G(87) 0.24 7/7 3.14N(4)Q(4)A(4)

92 A A(12) 0.25 22/12 3.50 siteG(83)M(4)

Table 4. The top 25% of residues in 2vpwB at the interface with 2vpwC.(Field names: res: residue number in the PDB entry; type: amino acid type;substs: substitutions seen in the alignment; with the percentage of each typein the bracket; noc/bb: number of contacts with the ligand, with the number ofcontacts realized through backbone atoms given in the bracket; dist: distanceof closest apporach to the ligand. )


mutations90 C (KER)(FQMWHD)(NYLPI)(SVA)

101 P (YR)(TH)(SKECG)(FQWD)102 Y (K)(QM)(NEVLAPIR)(D)93 C (K)(ER)(QM)(D)99 A (KER)(Y)(QHD)(N)71 P (YR)(TH)(SKECG)(FQWD)

100 C (R)(K)(FWH)(EQM)105 R (TYD)(E)(SCG)(FVLAWPI)70 C (R)(KE)(H)(FYQWD)72 T (K)(R)(FWH)(EQM)15 G (ER)(H)(FKYW)(D)92 A (Y)(R)(KE)(H)

Table 5. List of disruptive mutations for the top 25% of residues in2vpwB, that are at the interface with 2vpwC.

Figure 5 shows residues in 2vpwB colored by their importance, at theinterface with 2vpwC.

Iron/sulfur cluster binding site. Table 6 lists the top 25% of resi-dues at the interface with 2vpwBSF41196 (iron/sulfur cluster). The

4

Fig. 5. Residues in 2vpwB, at the interface with 2vpwC, colored by their rela-tive importance. 2vpwC is shown in backbone representation (See Appendixfor the coloring scheme for the protein chain 2vpwB.)

following table (Table 7) suggests possible disruptive replacementsfor these residues (see Section 4.6).


(%) bb (A)58 C C(100) 0.10 19/8 2.29 site61 C C(100) 0.10 21/9 2.2166 C C(100) 0.10 15/4 2.24 site

101 P P(100) 0.10 3/1 3.85102 Y Y(100) 0.10 10/0 4.0065 P P(91) 0.12 5/1 4.07

S(8)99 A A(91) 0.12 1/1 4.88

G(8)83 V V(95) 0.14 11/0 3.61

T(4)100 C C(95) 0.14 14/4 2.52 site

D(4)115 K K(91) 0.15 6/0 3.31 site

N(8)105 R R(87) 0.17 9/0 3.58

I(4)V(8)

60 H H(83) 0.20 13/11 3.33 siteQ(16)

171 T T(79) 0.24 1/0 4.84G(12)



(%) bb (A)V(8)



mutations58 C (KER)(FQMWHD)(NYLPI)(SVA)61 C (KER)(FQMWHD)(NYLPI)(SVA)66 C (KER)(FQMWHD)(NYLPI)(SVA)

101 P (YR)(TH)(SKECG)(FQWD)102 Y (K)(QM)(NEVLAPIR)(D)65 P (R)(Y)(H)(K)99 A (KER)(Y)(QHD)(N)83 V (KR)(E)(YQH)(D)

100 C (R)(K)(FWH)(EQM)115 K (Y)(FTW)(SVCAG)(H)105 R (TYD)(E)(SCG)(FVLAWPI)60 H (TE)(D)(SVMCAG)(QLPI)

171 T (KR)(QH)(FMW)(E)



Menaquinone-7 binding site.Table 8 lists the top 25% of resi-dues at the interface with 2vpwCMQ71252 (menaquinone-7). Thefollowing table (Table 9) suggests possible disruptive replacementsfor these residues (see Section 4.6).


(%) bb (A)93 C C(91) 0.12 18/12 3.75 site

Y(8)

Table 8. The top 25% of residues in 2vpwB at the interface withmenaquinone-7.(Field names: res: residue number in the PDB entry; type:amino acid type; substs: substitutions seen in the alignment;with the percen-tage of each type in the bracket; noc/bb: number of contacts with the ligand,with the number of contacts realized through backbone atoms given in thebracket; dist: distance of closest apporach to the ligand. )

5



mutations93 C (K)(ER)(QM)(D)

Table 9. List of disruptive mutations for the top 25% of residues in2vpwB, that are at the interface with menaquinone-7.

Figure 7 shows residues in 2vpwB colored by their importance, at theinterface with 2vpwCMQ71252.

Interface with 2vpwA.Table 10 lists the top 25% of residues atthe interface with 2vpwA. The following table (Table 11) suggestspossible disruptive replacements for these residues (see Section 4.6).


(%) bb (A)13 C C(100) 0.10 8/8 2.71 site16 C C(100) 0.10 33/20 3.51 site27 N N(100) 0.10 4/1 4.42 site

135 C C(100) 0.10 19/19 3.07 site30 P P(91) 0.12 6/6 4.50

E(8)130 A A(83) 0.21 3/1 4.44 site

H(4)I(12)

9 D D(91) 0.22 1/0 4.51continued in next column

Fig. 7. Residues in 2vpwB, at the interface with menaquinone-7, coloredby their relative importance. The ligand (menaquinone-7) is colored green.Atoms further than 30A away from the geometric center of the ligand, as wellas on the line of sight to the ligand were removed. (See Appendix for thecoloring scheme for the protein chain 2vpwB.)


(%) bb (A)K(4)R(4)

15 G G(87) 0.24 4/4 3.84N(4)Q(4)A(4)

Table 10. The top 25% of residues in 2vpwB at the interface with 2vpwA.(Field names: res: residue number in the PDB entry; type: amino acid type;substs: substitutions seen in the alignment; with the percentage of each typein the bracket; noc/bb: number of contacts with the ligand, with the number ofcontacts realized through backbone atoms given in the bracket; dist: distanceof closest apporach to the ligand. )


mutations13 C (KER)(FQMWHD)(NYLPI)(SVA)16 C (KER)(FQMWHD)(NYLPI)(SVA)27 N (Y)(FTWH)(SEVCARG)(MD)

135 C (KER)(FQMWHD)(NYLPI)(SVA)30 P (YR)(H)(T)(CG)

130 A (E)(KR)(Y)(QD)9 D (FW)(Y)(VCAHG)(R)


6


mutations15 G (ER)(H)(FKYW)(D)

Table 11. List of disruptive mutations for the top 25% of residues in2vpwB, that are at the interface with 2vpwA.

Fig. 8. Residues in 2vpwB, at the interface with 2vpwA, colored by their rela-tive importance. 2vpwA is shown in backbone representation (See Appendixfor the coloring scheme for the protein chain 2vpwB.)

Figure 8 shows residues in 2vpwB colored by their importance, at theinterface with 2vpwA.



(%) bb (A)74 A A(100) 0.10 9/5 4.0390 C C(100) 0.10 17/7 2.23 site96 C C(100) 0.10 15/5 2.19 site93 C C(91) 0.12 18/8 2.30 site

Y(8)71 P P(95) 0.13 9/2 3.68 site

L(4)105 R R(87) 0.17 2/0 4.11

I(4)V(8)



(%) bb (A)113 V V(50) 0.20 11/1 3.63

A(41)S(8)

70 C C(95) 0.21 15/5 2.32 siteL(4)

72 T T(70) 0.23 5/0 4.03V(20)R(8)

92 A A(12) 0.25 12/11 3.46 siteG(83)M(4)



mutations74 A (KYER)(QHD)(N)(FTMW)90 C (KER)(FQMWHD)(NYLPI)(SVA)96 C (KER)(FQMWHD)(NYLPI)(SVA)93 C (K)(ER)(QM)(D)71 P (YR)(TH)(SKECG)(FQWD)

105 R (TYD)(E)(SCG)(FVLAWPI)113 V (KR)(YE)(H)(Q)70 C (R)(KE)(H)(FYQWD)72 T (K)(R)(FWH)(EQM)92 A (Y)(R)(KE)(H)





(%) bb (A)13 C C(100) 0.10 14/4 2.30 site16 C C(100) 0.10 23/15 2.42 site19 C C(100) 0.10 18/6 2.25

135 C C(100) 0.10 11/4 2.77 site55 P P(66) 0.15 9/0 3.91

R(33)continued in next column

7



(%) bb (A)140 R R(62) 0.19 9/0 3.65 site

L(37)15 G G(87) 0.24 11/11 3.93

N(4)Q(4)A(4)



mutations13 C (KER)(FQMWHD)(NYLPI)(SVA)16 C (KER)(FQMWHD)(NYLPI)(SVA)19 C (KER)(FQMWHD)(NYLPI)(SVA)

135 C (KER)(FQMWHD)(NYLPI)(SVA)55 P (Y)(T)(SECHRG)(D)

140 R (T)(YD)(SECG)(VA)15 G (ER)(H)(FKYW)(D)



mutations



Figure 10 shows residues in 2vpwB colored by their importance, atthe interface with 2vpwBSF41194.

2.4.3 Possible novel functional surfaces at 25% coverage.Onegroup of residues is conserved on the 2vpwB surface, away from (orsusbtantially larger than) other functional sites and interfaces reco-gnizable in PDB entry 2vpw. It is shown in Fig. 11. The right panelshows (in blue) the rest of the larger cluster this surface belongs to.The residues belonging to this surface ”patch” are listed in Table 16,while Table 17 suggests possible disruptive replacements for theseresidues (see Section 4.6).

Table 16.res type substitutions(%) cvg antn13 C C(100) 0.10 site16 C C(100) 0.10 site19 C C(100) 0.1023 C C(100) 0.10 site27 N N(100) 0.10 site58 C C(100) 0.10 site


8

Fig. 11. A possible active surface on the chain 2vpwB. The larger cluster itbelongs to is shown in blue.

Table 16.continuedres type substitutions(%) cvg antn61 C C(100) 0.1066 C C(100) 0.10 site74 A A(100) 0.1081 G G(100) 0.1090 C C(100) 0.10 site96 C C(100) 0.10 site

101 P P(100) 0.10102 Y Y(100) 0.10116 C C(100) 0.10 site118 F F(100) 0.10119 C C(100) 0.10 site131 C C(100) 0.10135 C C(100) 0.10 site143 G G(100) 0.1065 P P(91)S(8) 0.1293 C C(91)Y(8) 0.12 site99 A A(91)G(8) 0.1271 P P(95)L(4) 0.13 site

100 C C(95)D(4) 0.14 site173 P P(95)A(4) 0.14115 K K(91)N(8) 0.15 site105 R R(87)I(4)V(8) 0.17132 V V(87)Q(4)A(8) 0.17142 F F(83)I(8)V(8) 0.17147 D D(87)S(12) 0.18140 R R(62)L(37) 0.19 site150 S S(79).(12)G(8) 0.19113 V V(50)A(41)S(8) 0.2070 C C(95)L(4) 0.21 site

130 A A(83)H(4)I(12) 0.21 site9 D D(91)K(4)R(4) 0.22

72 T T(70)V(20)R(8) 0.2315 G G(87)N(4)Q(4) 0.24

A(4)171 T T(79)G(12)V(8) 0.24

4 Y Y(75)W(16)V(8) 0.2592 A A(12)G(83)M(4) 0.25 site


Table 16.continuedres type substitutions(%) cvg antn

Table 16. Residues forming surface ”patch” in 2vpwB.


mutations13 C (KER)(FQMWHD)(NYLPI)(SVA)16 C (KER)(FQMWHD)(NYLPI)(SVA)19 C (KER)(FQMWHD)(NYLPI)(SVA)23 C (KER)(FQMWHD)(NYLPI)(SVA)27 N (Y)(FTWH)(SEVCARG)(MD)58 C (KER)(FQMWHD)(NYLPI)(SVA)61 C (KER)(FQMWHD)(NYLPI)(SVA)66 C (KER)(FQMWHD)(NYLPI)(SVA)74 A (KYER)(QHD)(N)(FTMW)81 G (KER)(FQMWHD)(NYLPI)(SVA)90 C (KER)(FQMWHD)(NYLPI)(SVA)96 C (KER)(FQMWHD)(NYLPI)(SVA)

101 P (YR)(TH)(SKECG)(FQWD)102 Y (K)(QM)(NEVLAPIR)(D)116 C (KER)(FQMWHD)(NYLPI)(SVA)118 F (KE)(TQD)(SNCRG)(M)119 C (KER)(FQMWHD)(NYLPI)(SVA)131 C (KER)(FQMWHD)(NYLPI)(SVA)135 C (KER)(FQMWHD)(NYLPI)(SVA)143 G (KER)(FQMWHD)(NYLPI)(SVA)65 P (R)(Y)(H)(K)93 C (K)(ER)(QM)(D)99 A (KER)(Y)(QHD)(N)71 P (YR)(TH)(SKECG)(FQWD)

100 C (R)(K)(FWH)(EQM)173 P (YR)(H)(TKE)(SQCDG)115 K (Y)(FTW)(SVCAG)(H)105 R (TYD)(E)(SCG)(FVLAWPI)132 V (Y)(ER)(H)(K)142 F (KE)(TQDR)(SNCG)(Y)147 D (R)(FWH)(K)(Y)140 R (T)(YD)(SECG)(VA)150 S (KR)(FWH)(QM)(NELPI)113 V (KR)(YE)(H)(Q)70 C (R)(KE)(H)(FYQWD)

130 A (E)(KR)(Y)(QD)9 D (FW)(Y)(VCAHG)(R)72 T (K)(R)(FWH)(EQM)15 G (ER)(H)(FKYW)(D)

171 T (KR)(QH)(FMW)(E)4 Y (K)(Q)(E)(R)92 A (Y)(R)(KE)(H)

Table 17. Disruptive mutations for the surface patch in 2vpwB.

9

Fig. 12. Residues 30-274 in 2vpwA colored by their relative importance. (SeeAppendix, Fig.24, for the coloring scheme.)

3 CHAIN 2VPWA

3.1 Q72LA4 overviewFrom SwissProt, id Q72LA4, 95% identical to 2vpwA:Description: Thiosulfate reductase (EC 1.-.-.-).Organism, scientific name:Thermus thermophilus (strain HB27 /ATCC BAA-163 / DSM 7039).Taxonomy: Bacteria; Deinococcus-Thermus; Deinococci; Therma-les; Thermaceae; Thermus.

3.2 Multiple sequence alignment for 2vpwAFor the chain 2vpwA, the alignment 2vpwA.msf (attached) with 95sequences was used. The alignment was assembled through combi-nation of BLAST searching on the UniProt database and alignmentusing Muscle program. It can be found in the attachment to thisreport, under the name of 2vpwA.msf. Its statistics, from thealistatprogram are the following:

Format: MSFNumber of sequences: 95Total number of residues: 62704Smallest: 583Largest: 735Average length: 660.0Alignment length: 735Average identity: 27%Most related pair: 99%Most unrelated pair: 15%Most distant seq: 30%

Furthermore,<1% of residues show as conserved in this ali-gnment.

The alignment consists of 2% eukaryotic ( 2% fungi), 83%prokaryotic, and 18% archaean sequences. (Descriptions of somesequences were not readily available.) The file containing thesequence descriptions can be found in the attachment, under the name2vpwA.descr.

3.3 Residue ranking in 2vpwAThe 2vpwA sequence is shown in Figs. 12–14, with each residuecolored according to its estimated importance. The full listing of

Fig. 13. Residues 275-519 in 2vpwA colored by their relative importance.(See Appendix, Fig.24, for the coloring scheme.)

Fig. 14. Residues 520-764 in 2vpwA colored by their relative importance.(See Appendix, Fig.24, for the coloring scheme.)

residues in 2vpwA can be found in the file called 2vpwA.rankssortedin the attachment.

3.4 Top ranking residues in 2vpwA and their position onthe structure

In the following we consider residues ranking among top 25% ofresidues in the protein . Figure 15 shows residues in 2vpwA coloredby their importance: bright red and yellow indicate more conser-ved/important residues (see Appendix for the coloring scheme). APymol script for producing this figure can be found in the attachment.

3.4.1 Clustering of residues at 25% coverage.Fig. 16 shows thetop 25% of all residues, this time colored according to clusters theybelong to. The clusters in Fig.16 are composed of the residues listedin Table 18.

Table 18.cluster size membercolor residuesred 177 44,46,47,48,50,51,67,74,76

79,81,82,94,95,98,101,112115,117,120,124,143,145,146147,150,154,155,157,161,162


10

Fig. 15. Residues in 2vpwA, colored by their relative importance. Clockwise:front, back, top and bottom views.

Fig. 16. Residues in 2vpwA, colored according to the cluster they belong to:red, followed by blue and yellow are the largest clusters (see Appendix forthe coloring scheme). Clockwise: front, back, top and bottomviews. Thecorresponding Pymol script is attached.

Table 18.continuedcluster size membercolor residues

163,164,166,170,171,172,173174,176,180,186,191,194,196202,206,207,208,209,212,213


Table 18.continuedcluster size membercolor residues

214,215,216,233,236,237,238240,243,246,249,254,255,256257,259,260,263,275,278,279285,289,298,305,306,312,315316,320,327,330,331,332,334335,340,341,345,349,350,351353,354,355,356,357,358,361362,414,436,439,440,441,442445,447,456,463,464,465,466470,472,473,476,477,478,480481,482,487,488,490,511,513514,518,519,521,583,585,588589,590,592,619,621,623,624625,627,628,629,630,631,632633,634,640,652,653,655,656661,667,671,674,691,692,694697,698,699,717,719,720,726729,731,732,735,738,739

blue 3 225,229,230yellow 2 526,530

Table 18. Clusters of top ranking residues in 2vpwA.

3.4.2 Overlap with known functional surfaces at 25% coverage.The name of the ligand is composed of the source PDB identifierand the heteroatom name used in that file.

Iron/sulfur cluster binding site. Table 19 lists the top 25% of resi-dues at the interface with 2vpwASF41764 (iron/sulfur cluster). Thefollowing table (Table 20) suggests possible disruptive replacementsfor these residues (see Section 4.6).


(%) bb (A)51 C C(91) 0.04 14/4 2.14 site

G(4).(3)D(1)

81 R K(72) 0.04 28/10 3.27 siteR(22)N(3).(2)

44 C C(91) 0.05 18/7 2.07 siteE(3)T(1)N(1).(2)S(1)

47 C C(91) 0.05 21/11 2.39 siteT(3)F(1)S(1)


11


(%) bb (A).(2)L(1)

79 C C(89) 0.05 16/7 2.48 siteP(2)M(3)G(2)Y(1).(2)

82 G G(75) 0.07 10/10 3.41 siteM(4)V(5)A(5)T(4).(2)D(1)P(1)L(1)

48 F G(61) 0.08 1/1 4.85 siteS(8)Y(7)P(11)N(1)E(4)A(1).(2)H(1)F(1)T(1)

214 T H(44) 0.14 3/3 4.12 siteI(5)D(1)Q(6)C(2)S(5)N(16)R(12)A(2)F(1)V(1)M(1)T(1)

50 R G(45) 0.15 6/6 3.66 siteR(16)Q(4)W(3)T(11)N(1)S(2)A(6).(3)I(4)K(1)



(%) bb (A)F(1)

216 N V(33) 0.15 11/4 3.25 siteI(15)M(1)T(4)D(3)H(21)S(16)G(1)L(2)N(1)

215 H P(51) 0.16 7/3 3.77 siteN(1)M(18)L(4)I(3)E(5)G(5)T(2)S(3)V(4)H(1)

630 T T(44) 0.16 1/0 4.63 siteG(5)V(7)L(21).(2)I(3)F(1)A(2)C(2)N(4)S(6)E(1)

46 G Y(38) 0.25 13/13 3.69 siteM(5)F(8)I(3)R(7)L(13)V(12)S(2)H(3)W(1).(2)A(1)G(1)

Table 19. The top 25% of residues in 2vpwA at the interface withiron/sulfur cluster.(Field names: res: residue number in thePDB entry; type:amino acid type; substs: substitutions seen in the alignment;with the percen-tage of each type in the bracket; noc/bb: number of contacts with the ligand,with the number of contacts realized through backbone atoms given in thebracket; dist: distance of closest apporach to the ligand. )

12


mutations51 C (R)(K)(FEWH)(QM)81 R (T)(YD)(SVCAG)(FELWPI)44 C (R)(K)(FWH)(E)47 C (KR)(E)(Q)(H)79 C (R)(K)(E)(H)82 G (R)(K)(EH)(Y)48 F (K)(E)(Q)(R)

214 T (R)(K)(H)(FW)50 R (D)(T)(YE)(SCG)

216 N (Y)(H)(R)(FW)215 H (E)(TQ)(KD)(M)630 T (R)(K)(H)(Q)46 G (E)(K)(R)(D)

Table 20. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with iron/sulfur cluster.

Fig. 17. Residues in 2vpwA, at the interface with iron/sulfur cluster, coloredby their relative importance. The ligand (iron/sulfur cluster) is colored green.Atoms further than 30A away from the geometric center of the ligand, as wellas on the line of sight to the ligand were removed. (See Appendix for thecoloring scheme for the protein chain 2vpwA.)

Figure 17 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwASF41764.

Interface with 2vpwB.Table 21 lists the top 25% of residues atthe interface with 2vpwB. The following table (Table 22) suggestspossible disruptive replacements for these residues (see Section 4.6).

13


(%) bb (A)79 C C(89) 0.05 13/12 3.78 site

P(2)M(3)G(2)Y(1).(2)

76 G G(68) 0.11 9/9 3.69D(2)V(12)M(1)P(4)I(2)F(1)T(3).(2)N(3)


216 N V(33) 0.15 4/1 4.08 siteI(15)M(1)T(4)D(3)H(21)S(16)G(1)L(2)N(1)

627 P Q(22) 0.15 17/3 3.51A(4)H(36)D(4)R(6)V(5)K(13)P(4)L(1)N(2)



(%) bb (A)249 W F(15) 0.19 5/0 3.88

H(44)W(24)Y(7)V(2)Q(3)P(1)K(1)R(1)

640 L I(8) 0.19 3/0 4.03L(69)V(5)M(4)H(2)G(2)Y(3)F(1)E(1)A(2)T(1)

67 G P(20) 0.20 1/1 4.80G(61).(9)S(1)A(3)Y(2)E(2)Q(1)

653 K P(80) 0.20 4/0 4.33I(1)E(3)T(2)D(1)M(1)Q(1)R(2)A(2)V(2)K(3)S(1)

652 H N(32) 0.24 11/0 3.56H(47)A(2)S(15)C(2)

628 V Y(23) 0.25 5/0 3.82W(18)V(7)F(15)N(4)M(2)S(2)


14


(%) bb (A)A(8)R(7)G(2)H(5)L(1)K(1)Q(1)

Table 21. The top 25% of residues in 2vpwA at the interface with 2vpwB.(Field names: res: residue number in the PDB entry; type: amino acid type;substs: substitutions seen in the alignment; with the percentage of each typein the bracket; noc/bb: number of contacts with the ligand, with the number ofcontacts realized through backbone atoms given in the bracket; dist: distanceof closest apporach to the ligand. )


mutations79 C (R)(K)(E)(H)76 G (R)(K)(E)(H)214 T (R)(K)(H)(FW)216 N (Y)(H)(R)(FW)627 P (Y)(R)(T)(H)249 W (E)(T)(K)(D)640 L (R)(Y)(H)(K)67 G (R)(K)(EH)(FW)653 K (Y)(FW)(T)(H)652 H (E)(Q)(D)(M)628 V (E)(Y)(KR)(D)

Table 22. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with 2vpwB.

Figure 18 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwB.

MGD binding site. Table 23 lists the top 25% of residues at theinterface with 2vpwAMGD1765 (mgd). The following table (Table24) suggests possible disruptive replacements for these residues (seeSection 4.6).


(%) bb (A)173 C C(76) 0.01 12/3 3.55 site

S(18)D(4)

441 N N(88) 0.02 42/19 2.84 siteD(10)S(1)

625 R R(54) 0.03 22/0 2.82 siteK(6)H(23)


Fig. 18. Residues in 2vpwA, at the interface with 2vpwB, colored bytheir relative importance. 2vpwB is shown in backbone representation (SeeAppendix for the coloring scheme for the protein chain 2vpwA.)


(%) bb (A)P(12)T(2)A(1)

472 H T(80) 0.04 4/0 3.48 siteA(1)S(16)H(2)

717 N N(92) 0.04 11/2 2.98 siteK(1)D(1)Q(3)G(2)

470 Q T(71) 0.05 16/0 3.41 siteS(8)N(15)E(1)D(1)Q(2)

466 D D(83) 0.06 54/14 2.51 siteG(1)E(13)T(1)N(1)

445 S S(49) 0.07 48/23 2.86 siteT(11)R(3)Q(17)


15


(%) bb (A)V(13)H(3)C(1)


623 Y T(56) 0.09 13/0 3.29 siteQ(5)S(11)P(2)G(17)N(3)F(1)I(1)Y(1)

473 V A(69) 0.10 1/0 4.87C(6)V(9)S(3)T(7)M(1)N(1)I(1)G(1)

482 E A(56) 0.12 23/1 2.46E(5)G(8)S(5)V(7)D(14)T(1)P(1)

488 R K(43) 0.12 11/0 2.21 siteR(16)D(2)E(5)N(6)M(1)Q(11)H(12)S(1)

631 F T(3) 0.12 72/9 3.19 siteG(27)N(22)



(%) bb (A)H(23)A(1)Q(2)R(8)M(7)L(1).(1)D(1)F(1)I(1)

145 H S(52) 0.13 26/0 2.86 site.(7)Q(2)Y(2)A(12)T(14)G(6)D(1)H(1)

720 Q T(75) 0.13 7/0 2.93 siteP(5)I(1)A(6)V(6)S(2)Q(1)M(1)N(1)

439 G G(45) 0.14 39/39 3.06 siteC(12)R(5)S(1)N(12)A(21)H(1)T(1)

172 L L(35) 0.15 25/15 3.19 siteT(7)V(12)Y(4)P(4).(25)I(7)S(2)F(1)

630 T T(44) 0.16 3/2 4.52 siteG(5)V(7)L(21).(2)I(3)F(1)


16


(%) bb (A)A(2)C(2)N(4)S(6)E(1)

697 H F(28) 0.16 48/0 3.36 siteM(16)E(16)G(10)K(3)N(1)Y(2)Q(6)H(5)S(2)W(2)L(2)A(1)I(2)

632 A T(25) 0.19 14/10 2.94 siteS(51)G(5)V(1)E(4)A(7)N(3)Q(1).(1)

633 R M(23) 0.21 57/10 2.47 siteR(11)A(4)Q(20)L(3)T(15)V(2)S(9)W(4)K(3)I(2)H(1)

465 I Q(34) 0.23 9/7 2.90C(5)V(3)I(23)A(6)H(14)T(2)L(5)S(3)M(2)

440 I E(28) 0.24 30/26 3.46T(24)



(%) bb (A)S(18)K(4).(1)G(10)N(4)Q(1)A(1)C(1)H(3)Y(1)I(1)

Table 23. The top 25% of residues in 2vpwA at the interface withMGD.(Field names: res: residue number in the PDB entry; type: amino acidtype; substs: substitutions seen in the alignment; with the percentage of eachtype in the bracket; noc/bb: number of contacts with the ligand, with the num-ber of contacts realized through backbone atoms given in the bracket; dist:distance of closest apporach to the ligand. )


mutations173 C (R)(K)(FWH)(EQM)441 N (Y)(FWH)(R)(T)625 R (D)(T)(E)(Y)472 H (E)(Q)(KM)(D)717 N (Y)(FW)(H)(T)470 Q (Y)(FWH)(T)(VA)466 D (R)(FWH)(K)(Y)445 S (K)(R)(FW)(M)48 F (K)(E)(Q)(R)623 Y (K)(R)(Q)(EM)473 V (R)(KY)(E)(H)482 E (H)(R)(FW)(Y)488 R (T)(Y)(CG)(VAD)631 F (E)(K)(T)(D)145 H (E)(Q)(M)(K)720 Q (Y)(H)(FW)(T)439 G (E)(K)(R)(MD)172 L (R)(Y)(K)(H)630 T (R)(K)(H)(Q)697 H (E)(T)(D)(Q)632 A (R)(Y)(K)(H)633 R (TD)(Y)(E)(CG)465 I (R)(Y)(H)(K)440 I (R)(Y)(H)(T)

Table 24. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with MGD.

Figure 19 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwAMGD1765.

MGD binding site. Table 25 lists the top 25% of residues at theinterface with 2vpwAMGD1766 (mgd). The following table (Table

17

Fig. 19. Residues in 2vpwA, at the interface with MGD, colored by theirrelative importance. The ligand (MGD) is colored green. Atomsfurther than30A away from the geometric center of the ligand, as well as on the line ofsight to the ligand were removed. (See Appendix for the coloring scheme forthe protein chain 2vpwA.)

26) suggests possible disruptive replacements for these residues (seeSection 4.6).


(%) bb (A)173 C C(76) 0.01 4/0 3.17 site

S(18)D(4)

207 G G(95) 0.02 41/41 1.89A(2)S(2)

209 H N(92) 0.02 64/12 2.74 siteD(5)S(1)H(1)

236 D D(95) 0.02 52/11 2.40 siteE(3)N(1)

257 D N(12) 0.02 19/0 2.78D(87)

332 R G(86) 0.02 36/12 3.21 siteR(6)C(5)A(1)Y(1)

237 P P(87) 0.03 30/5 3.37continued in next column


(%) bb (A)V(8)T(2)I(2)

254 P P(92) 0.03 9/6 3.62 siteN(2)L(1)H(1)G(1)S(2)

255 G G(88) 0.03 6/6 3.00 siteQ(5)D(3)H(1)T(1)R(1)

625 R R(54) 0.03 75/1 3.35 siteK(6)H(23)P(12)T(2)A(1)

81 R K(72) 0.04 35/0 3.00 siteR(22)N(3).(2)

238 R R(74) 0.07 24/3 2.59 siteV(7)L(4)K(1)H(2)I(2)Y(7)E(1)

735 R K(49) 0.07 49/0 2.82H(31)F(2)Y(8)N(5)R(2)A(1)


623 Y T(56) 0.09 4/4 4.19 sitecontinued in next column

18


(%) bb (A)Q(5)S(11)P(2)G(17)N(3)F(1)I(1)Y(1)

624 G G(51) 0.10 1/1 4.92V(7)P(14)D(1)F(11)A(2)H(2)C(2)R(4)T(1)Y(1)S(1)

629 H H(41) 0.10 12/12 2.83 siteN(9)L(2)R(18)G(5)F(9)T(4)S(6)A(1)Y(1).(1)

256 T T(70) 0.11 1/1 4.65S(17)G(6)Y(2)N(1)H(1)Q(1)

331 T M(34) 0.11 19/3 2.49 siteQ(8)H(5)L(6)R(2)W(18)Y(14)N(3)A(1)S(1)G(1)E(1)F(1)T(1)



(%) bb (A)631 F T(3) 0.12 31/5 2.71 site

G(27)N(22)H(23)A(1)Q(2)R(8)M(7)L(1).(1)D(1)F(1)I(1)

694 Y F(42) 0.12 7/0 4.22Y(9)R(5)V(17)S(7)I(2)A(13)D(1)G(1)


627 P Q(22) 0.15 5/5 3.76A(4)H(36)D(4)R(6)V(5)K(13)P(4)L(1)N(2)

215 H P(51) 0.16 36/4 3.48 siteN(1)M(18)L(4)I(3)E(5)G(5)


19


(%) bb (A)T(2)S(3)V(4)H(1)

630 T T(44) 0.16 20/11 3.37 siteG(5)V(7)L(21).(2)I(3)F(1)A(2)C(2)N(4)S(6)E(1)

206 I T(2) 0.20 10/7 3.67 siteA(7)F(21)I(22)V(9)W(28)L(5)M(2)S(2)

212 E E(45) 0.22 8/8 3.25 siteY(5)D(6)W(14)A(11)S(9)V(5)T(1)N(1)

213 D N(7) 0.24 50/15 2.85A(13)G(7)C(12)W(2)T(36)S(12)Q(5)L(1)D(1)

240 S I(8) 0.24 2/0 4.30T(49)S(23)N(11)H(3)V(1)Q(2)P(1)



(%) bb (A)208 H S(22) 0.25 18/14 3.41 site

A(15)H(11)T(11)I(10)N(14)Y(5)G(5)L(1)R(1)E(1)

628 V Y(23) 0.25 1/1 4.39W(18)V(7)F(15)N(4)M(2)S(2)A(8)R(7)G(2)H(5)L(1)K(1)Q(1)

Table 25. The top 25% of residues in 2vpwA at the interface withMGD.(Field names: res: residue number in the PDB entry; type: amino acidtype; substs: substitutions seen in the alignment; with the percentage of eachtype in the bracket; noc/bb: number of contacts with the ligand, with the num-ber of contacts realized through backbone atoms given in the bracket; dist:distance of closest apporach to the ligand. )


mutations173 C (R)(K)(FWH)(EQM)207 G (KR)(E)(QH)(FMW)209 H (E)(M)(TQ)(K)236 D (R)(FWH)(Y)(VCAG)257 D (R)(FWH)(Y)(VCAG)332 R (D)(E)(LPI)(TY)237 P (R)(Y)(H)(K)254 P (R)(Y)(H)(TE)255 G (E)(KR)(FW)(M)625 R (D)(T)(E)(Y)81 R (T)(YD)(SVCAG)(FELWPI)

238 R (T)(D)(Y)(ECG)735 R (TD)(E)(SCG)(Y)48 F (K)(E)(Q)(R)

623 Y (K)(R)(Q)(EM)continued in next column

20


mutations624 G (K)(ER)(Q)(M)629 H (E)(D)(Q)(M)256 T (KR)(M)(FW)(QH)331 T (KR)(H)(Q)(FMW)631 F (E)(K)(T)(D)694 Y (K)(Q)(R)(M)214 T (R)(K)(H)(FW)627 P (Y)(R)(T)(H)215 H (E)(TQ)(KD)(M)630 T (R)(K)(H)(Q)206 I (R)(Y)(K)(H)212 E (R)(H)(FW)(Y)213 D (R)(H)(FW)(K)240 S (R)(K)(H)(FYW)208 H (E)(Q)(MD)(K)628 V (E)(Y)(KR)(D)

Table 26. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with MGD.

Fig. 20. Residues in 2vpwA, at the interface with MGD, colored by theirrelative importance. The ligand (MGD) is colored green. Atomsfurther than30A away from the geometric center of the ligand, as well as on the line ofsight to the ligand were removed. (See Appendix for the coloring scheme forthe protein chain 2vpwA.)

Figure 20 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwAMGD1766.

Molybdenum atom binding site. Table 27 lists the top 25% ofresidues at the interface with 2vpwAMO1767 (molybdenum atom).The following table (Table 28) suggests possible disruptive replace-ments for these residues (see Section 4.6).

21


(%) bb (A)173 C C(76) 0.01 3/1 2.60 site

S(18)D(4)

332 R G(86) 0.02 3/0 4.21 siteR(6)C(5)A(1)Y(1)

625 R R(54) 0.03 1/0 4.52 siteK(6)H(23)P(12)T(2)A(1)

631 F T(3) 0.12 2/0 4.48 siteG(27)N(22)H(23)A(1)Q(2)R(8)M(7)L(1).(1)D(1)F(1)I(1)

Table 27. The top 25% of residues in 2vpwA at the interface with molyb-denum atom.(Field names: res: residue number in the PDB entry; type: aminoacid type; substs: substitutions seen in the alignment; withthe percentage ofeach type in the bracket; noc/bb: number of contacts with the ligand, withthe number of contacts realized through backbone atoms given in the bracket;dist: distance of closest apporach to the ligand. )


mutations173 C (R)(K)(FWH)(EQM)332 R (D)(E)(LPI)(TY)625 R (D)(T)(E)(Y)631 F (E)(K)(T)(D)

Table 28. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with molybdenum atom.

Figure 21 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwAMO1767.

Interface with 2vpwC.Table 29 lists the top 25% of residues atthe interface with 2vpwC. The following table (Table 30) suggestspossible disruptive replacements for these residues (see Section 4.6).

Fig. 21. Residues in 2vpwA, at the interface with molybdenum atom, coloredby their relative importance. The ligand (molybdenum atom) is colored green.Atoms further than 30A away from the geometric center of the ligand, as wellas on the line of sight to the ligand were removed. (See Appendix for thecoloring scheme for the protein chain 2vpwA.)

Table 29.res type subst’s cvg noc/ dist

(%) bb (A)627 P Q(22) 0.15 13/1 3.02

A(4)H(36)D(4)R(6)V(5)K(13)P(4)L(1)N(2)

628 V Y(23) 0.25 5/0 4.40W(18)V(7)F(15)N(4)M(2)S(2)A(8)R(7)G(2)H(5)L(1)


22

Table 29.continuedres type subst’s cvg noc/ dist

(%) bb (A)K(1)Q(1)

Table 29. The top 25% of residues in 2vpwA at the interface with 2vpwC.(Field names: res: residue number in the PDB entry; type: amino acid type;substs: substitutions seen in the alignment; with the percentage of each typein the bracket; noc/bb: number of contacts with the ligand, with the number ofcontacts realized through backbone atoms given in the bracket; dist: distanceof closest apporach to the ligand. )


mutations627 P (Y)(R)(T)(H)628 V (E)(Y)(KR)(D)

Table 30. List of disruptive mutations for the top 25% of residues in2vpwA, that are at the interface with 2vpwC.

Fig. 22. Residues in 2vpwA, at the interface with 2vpwC, colored bytheir relative importance. 2vpwC is shown in backbone representation (SeeAppendix for the coloring scheme for the protein chain 2vpwA.)

Figure 22 shows residues in 2vpwA colored by their importance, atthe interface with 2vpwC.

3.4.3 Possible novel functional surfaces at 25% coverage.Onegroup of residues is conserved on the 2vpwA surface, away from (orsusbtantially larger than) other functional sites and interfaces reco-gnizable in PDB entry 2vpw. It is shown in Fig. 23. The right panelshows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 23. A possible active surface on the chain 2vpwA. The larger cluster itbelongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 31,while Table 32 suggests possible disruptive replacements for theseresidues (see Section 4.6).

Table 31.res type substitutions(%) cvg186 G G(97)N(1)S(1) 0.01588 G G(92)K(2)Q(3) 0.07

A(1)R(1)583 F C(1)F(56)L(26) 0.10

W(4)A(5)Y(5)S(1)

589 K K(61)R(25)L(10) 0.12T(1)Q(1)P(1)

585 T R(13)S(7)T(68) 0.17N(3)H(1)F(2)E(2)K(1)G(1)

Table 31. Residues forming surface ”patch” in 2vpwA.


mutations186 G (R)(KE)(FWH)(M)588 G (E)(D)(FYW)(H)583 F (K)(E)(Q)(R)589 K (Y)(FTW)(S)(CG)585 T (KR)(FW)(M)(H)

Table 32. Disruptive mutations for the surface patch in 2vpwA.

4 NOTES ON USING TRACE RESULTS

4.1 CoverageTrace results are commonly expressed in terms of coverage: the resi-due is important if its “coverage” is small - that is if it belongs tosome small top percentage of residues [100% is all of the residuesin a chain], according to trace. The ET results are presented in theform of a table, usually limited to top 25% percent of residues (orto some nearby percentage), sorted by the strength of the presumedevolutionary pressure. (I.e., the smaller the coverage, the strongerthe

23

pressure on the residue.) Starting from the top of that list, mutating acouple of residues should affect the protein somehow, with the exacteffects to be determined experimentally.

4.2 Known substitutionsOne of the table columns is “substitutions” - other amino acid typesseen at the same position in the alignment. These amino acid typesmay be interchangeable at that position in the protein, so if one wantsto affect the protein by a point mutation, they should be avoided. Forexample if the substitutions are “RVK” and the original protein hasan R at that position, it is advisable to try anything, but RVK. Conver-sely, when looking for substitutions which willnot affect the protein,one may try replacing, R with K, or (perhaps more surprisingly), withV. The percentage of times the substitution appears in the alignmentis given in the immediately following bracket. No percentage is givenin the cases when it is smaller than 1%. This is meant to be a roughguide - due to rounding errors these percentages often do not add upto 100%.

4.3 SurfaceTo detect candidates for novel functional interfaces, first we look forresidues that are solvent accessible (according to DSSP program) byat least10A2, which is roughly the area needed for one water mole-cule to come in the contact with the residue. Furthermore, we requirethat these residues form a “cluster” of residues which have neighborwithin 5A from any of their heavy atoms.

Note, however, that, if our picture of protein evolution is correct,the neighboring residues whichare notsurface accessible might beequally important in maintaining the interaction specificity - theyshould not be automatically dropped from consideration when choo-sing the set for mutagenesis. (Especially if they form a cluster withthe surface residues.)

4.4 Number of contactsAnother column worth noting is denoted “noc/bb”; it tells the num-ber of contacts heavy atoms of the residue in question make acrossthe interface, as well as how many of them are realized through thebackbone atoms (if all or most contacts are through the backbone,mutation presumably won’t have strong impact). Two heavy atomsare considered to be “in contact” if their centers are closer than5A.

4.5 AnnotationIf the residue annotation is available (either from the pdb file orfrom other sources), another column, with the header “annotation”appears. Annotations carried over from PDB are the following: site(indicating existence of related site record in PDB ), S-S (disulfidebond forming residue), hb (hydrogen bond forming residue, jb (jamesbond forming residue), and sb (for salt bridge forming residue).

4.6 Mutation suggestionsMutation suggestions are completely heuristic and based on comple-mentarity with the substitutions found in the alignment. Note thatthey are meant to bedisruptive to the interaction of the proteinwith its ligand. The attempt is made to complement the followingproperties: small[AV GSTC], medium[LPNQDEMIK], large[WFY HR], hydrophobic[LPV AMWFI], polar [GTCY ]; posi-tively [KHR], or negatively[DE] charged, aromatic[WFY H],long aliphatic chain[EKRQM ], OH-group possession[SDETY ],and NH2 group possession[NQRK]. The suggestions are listed

5%30%50%100%

COVERAGE

V

V

RELATIVE IMPORTANCE

Fig. 24. Coloring scheme used to color residues by their relative importance.

according to how different they appear to be from the original aminoacid, and they are grouped in round brackets if they appear equallydisruptive. From left to right, each bracketed group of amino acidtypes resembles more strongly the original (i.e. is, presumably, lessdisruptive) These suggestions are tentative - they might prove disrup-tive to the fold rather than to the interaction. Many researcher willchoose, however, the straightforward alanine mutations, especially inthe beginning stages of their investigation.

5 APPENDIX

5.1 File formatsFiles with extension “rankssorted” are the actual trace results. Thefields in the table in this file:

• alignment# number of the position in the alignment

• residue# residue number in the PDB file

• type amino acid type

• rank rank of the position according to older version of ET

• variability has two subfields:1. number of different amino acids appearing in in this column

of the alignment

2. their type

• rho ET score - the smaller this value, the lesser variability ofthis position across the branches of the tree (and, presumably,the greater the importance for the protein)

• cvg coverage - percentage of the residues on the structure whichhave this rho or smaller

• gaps percentage of gaps in this column

5.2 Color schemes usedThe following color scheme is used in figures with residues coloredby cluster size: black is a single-residue cluster; clusters composed ofmore than one residue colored according to this hierarchy (orderedby descending size): red, blue, yellow, green, purple, azure, tur-quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold,bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,

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DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen,tan, DarkOrange, DeepPink, maroon, BlanchedAlmond.

The colors used to distinguish the residues by the estimatedevolutionary pressure they experience can be seen in Fig. 24.

5.3 Credits5.3.1 Alistat alistat reads a multiple sequence alignment from thefile and shows a number of simple statistics about it. These stati-stics include the format, the number of sequences, the total numberof residues, the average and range of the sequence lengths, and thealignment length (e.g. including gap characters). Also shown aresome percent identities. A percent pairwise alignment identity is defi-ned as (idents / MIN(len1, len2)) where idents is the number ofexact identities and len1, len2 are the unaligned lengths of the twosequences. The ”average percent identity”, ”most related pair”, and”most unrelated pair” of the alignment are the average, maximum,and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distantseq” is calculated by finding the maximum pairwise identity (bestrelative) for all N sequences, then finding the minimum of these Nnumbers (hence, the most outlying sequence).alistat is copyrightedby HHMI/Washington University School of Medicine, 1992-2001,and freely distributed under the GNU General Public License.

5.3.2 CE To map ligand binding sites from differentsource structures, reportmaker uses the CE program:http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998)”Protein structure alignment by incremental combinatorial extension(CE) of the optimal path. Protein Engineering 11(9) 739-747.

5.3.3 DSSP In this work a residue is considered solvent accessi-ble if the DSSP program finds it exposed to water by at least 10A2,which is roughly the area needed for one water molecule to come inthe contact with the residue. DSSP is copyrighted by W. Kabsch, C.Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI versionby [email protected] November 18,2002,

http://www.cmbi.kun.nl/gv/dssp/descrip.html.

5.3.4 HSSP Whenever available, reportmaker uses HSSP ali-gnment as a starting point for the analysis (sequences shorter than75% of the query are taken out, however); R. Schneider, A. deDaruvar, and C. Sander.”The HSSP database of protein structure-sequence alignments.”Nucleic Acids Res., 25:226–230, 1997.

http://swift.cmbi.kun.nl/swift/hssp/

5.3.5 LaTex The text for this report was processed using LATEX;Leslie Lamport, “LaTeX: A Document Preparation System Addison-Wesley,” Reading, Mass. (1986).

5.3.6 Muscle When making alignments “from scratch”, reportmaker uses Muscle alignment program: Edgar, Robert C. (2004),”MUSCLE: multiple sequence alignment with high accuracy andhigh throughput.”Nucleic Acids Research 32(5), 1792-97.

http://www.drive5.com/muscle/

5.3.7 Pymol The figures in this report were produced usingPymol. The scripts can be found in the attachment. Pymolis an open-source application copyrighted by DeLano Scien-tific LLC (2005). For more information about Pymol seehttp://pymol.sourceforge.net/. (Note for Windows

users: the attached package needs to be unzipped for Pymol to readthe scripts and launch the viewer.)

5.4 Note about ET ViewerDan Morgan from the Lichtarge lab has developed a visualizationtool specifically for viewing trace results. If you are interested, pleasevisit:

http://mammoth.bcm.tmc.edu/traceview/

The viewer is self-unpacking and self-installing. Input files to be usedwith ETV (extension .etvx) can be found in the attachment to themain report.

5.5 Citing this workThe method used to rank residues and make predictions in this reportcan be found in Mihalek, I., I. Res, O. Lichtarge. (2004).”A Family ofEvolution-Entropy Hybrid Methods for Ranking of Protein Residuesby Importance” J. Mol. Bio.336: 1265-82. For the original versionof ET see O. Lichtarge, H.Bourne and F. Cohen (1996).”An Evolu-tionary Trace Method Defines Binding Surfaces Common to ProteinFamilies” J. Mol. Bio.257: 342-358.

report maker itself is described in Mihalek I., I. Res and O.Lichtarge (2006).”Evolutionary Trace Report Maker: a new typeof service for comparative analysis of proteins.”Bioinformatics22:1656-7.

5.6 About report makerreport maker was written in 2006 by Ivana Mihalek. The 1D ran-king visualization program was written by Ivica Res. report makeris copyrighted by Lichtarge Lab, Baylor College of Medicine,Houston.

5.7 AttachmentsThe following files should accompany this report:

• 2vpwB.complex.pdb - coordinates of 2vpwB with all of itsinteracting partners

• 2vpwB.etvx - ET viewer input file for 2vpwB

• 2vpwB.clusterreport.summary - Cluster report summary for2vpwB

• 2vpwB.ranks - Ranks file in sequence order for 2vpwB

• 2vpwB.clusters - Cluster descriptions for 2vpwB

• 2vpwB.msf - the multiple sequence alignment used for the chain2vpwB

• 2vpwB.descr - description of sequences used in 2vpwB msf

• 2vpwB.rankssorted - full listing of residues and their rankingfor 2vpwB

• 2vpwB.2vpwBSF41195.if.pml - Pymol script for Figure 4

• 2vpwB.cbcvg - used by other 2vpwB – related pymol scripts

• 2vpwB.2vpwC.if.pml - Pymol script for Figure 5


• 2vpwB.2vpwCMQ71252.if.pml - Pymol script for Figure 7

• 2vpwB.2vpwA.if.pml - Pymol script for Figure 8



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• 2vpwA.complex.pdb - coordinates of 2vpwA with all of itsinteracting partners

• 2vpwA.etvx - ET viewer input file for 2vpwA

• 2vpwA.clusterreport.summary - Cluster report summary for2vpwA

• 2vpwA.ranks - Ranks file in sequence order for 2vpwA

• 2vpwA.clusters - Cluster descriptions for 2vpwA

• 2vpwA.msf - the multiple sequence alignment used for the chain2vpwA

• 2vpwA.descr - description of sequences used in 2vpwA msf

• 2vpwA.rankssorted - full listing of residues and their rankingfor 2vpwA

• 2vpwA.2vpwASF41764.if.pml - Pymol script for Figure 17

• 2vpwA.cbcvg - used by other 2vpwA – related pymol scripts

• 2vpwA.2vpwB.if.pml - Pymol script for Figure 18

• 2vpwA.2vpwAMGD1765.if.pml - Pymol script for Figure 19

• 2vpwA.2vpwAMGD1766.if.pml - Pymol script for Figure 20

• 2vpwA.2vpwAMO1767.if.pml - Pymol script for Figure 21

• 2vpwA.2vpwC.if.pml - Pymol script for Figure 22

• 2vpwC.complex.pdb - coordinates of 2vpwC with all of itsinteracting partners

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Documents

Evolutionary trace report by report maker February 10, 2010mammoth.bcm.tmc.edu/report_maker/reports/pdb... · February 10, 2010 CONTENTS 1 Introduction 1 2 Chain 2vpwB 1 2.1 Q72LA5