evolutionary trace report by report maker october 10,...

Pages 1–9

2ckrEvolutionary trace report by report maker

October 10, 2010

CONTENTS

1 Introduction 1

2 Chain 2ckrA 12.1 Q01786 overview 12.2 Multiple sequence alignment for 2ckrA 12.3 Residue ranking in 2ckrA 12.4 Top ranking residues in 2ckrA 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. 2

3 Notes on using trace results 73.1 Coverage 73.2 Known substitutions 73.3 Surface 73.4 Number of contacts 83.5 Annotation 83.6 Mutation suggestions 8

4 Appendix 84.1 File formats 84.2 Color schemes used 84.3 Credits 8

4.3.1 Alistat 84.3.2 CE 8

4.3.3 DSSP 94.3.4 HSSP 94.3.5 LaTex 94.3.6 Muscle 94.3.7 Pymol 9

4.4 Note about ET Viewer 94.5 Citing this work 94.6 About reportmaker 94.7 Attachments 9

1 INTRODUCTIONFrom the original Protein Data Bank entry (PDB id 2ckr):Title: X-ray crystal structure of the catalytic domain of thermobifidafusca endoglucanase cel5a (e5) e355q in complex with cellotetraoseCompound: Mol id: 1; molecule: endoglucanase e-5; chain: a, b;fragment: catalytic domain, residues 161-466; synonym: endogluca-nase cel5a, endo-1,4-beta-glucanase e-4, cellulase e-5, cellulase e5;ec: 3.2.1.4; engineered: yes; mutation: yes; other details: mutationglu 355 gln in coordinatesOrganism, scientific name:Thermobifida Fusca;

2ckr contains a single unique chain 2ckrA (305 residues long) andits homologue 2ckrB.

2 CHAIN 2CKRA

2.1 Q01786 overviewFrom SwissProt, id Q01786, 99% identical to 2ckrA:Description: Endoglucanase E-5 precursor (EC 3.2.1.4) (Endo-1,4-beta-glucanase E-4) (Cellulase E-5) (Cellulase E5).Organism, scientific name:Thermomonospora fusca.Taxonomy: Bacteria; Actinobacteria; Actinobacteridae; Actinomy-cetales; Streptosporangineae; Nocardiopsaceae; Thermobifida.Catalytic activity: Endohydrolysis of 1,4-beta-D-glucosidic linka-ges in cellulose, lichenin and cereal beta-D-glucans.Pathway: Cellulose degradation.Similarity: Belongs to the glycosyl hydrolase 5 (cellulase A) family.Similarity: Contains 1 CBM2 (carbohydrate binding type-2)domain.About: This Swiss-Prot entry is copyright. It is produced through acollaboration between the Swiss Institute of Bioinformatics and theEMBL outstation - the European Bioinformatics Institute. There areno restrictions on its use as long as its content is in no way modifiedand this statement is not removed.

2.2 Multiple sequence alignment for 2ckrAFor the chain 2ckrA, the alignment 2ckrA.msf (attached) with 144sequences was used. The alignment was downloaded from the HSSPdatabase, and fragments shorter than 75% of the query as well asduplicate sequences were removed. It can be found in the attachment

1

Lichtarge lab 2006

Fig. 1. Residues 126-277 in 2ckrA colored by their relative importance. (SeeAppendix, Fig.12, for the coloring scheme.)

Fig. 2. Residues 278-430 in 2ckrA colored by their relative importance. (SeeAppendix, Fig.12, for the coloring scheme.)

to this report, under the name of 2ckrA.msf. Its statistics, from thealistat program are the following:

Format: MSFNumber of sequences: 144Total number of residues: 40496Smallest: 115Largest: 305Average length: 281.2Alignment length: 305Average identity: 41%Most related pair: 99%Most unrelated pair: 0%Most distant seq: 39%

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

The alignment consists of 18% eukaryotic ( 1% arthropoda,<1% fungi), and 31% prokaryotic sequences. (Descriptions ofsome sequences were not readily available.) The file containing thesequence descriptions can be found in the attachment, under the name2ckrA.descr.

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

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

In the following we consider residues ranking among top 25% of resi-dues in the protein . Figure 3 shows residues in 2ckrA colored by theirimportance: bright red and yellow indicate more conserved/importantresidues (see Appendix for the coloring scheme). A Pymol script forproducing this figure can be found in the attachment.

Fig. 3. Residues in 2ckrA, colored by their relative importance. Clockwise:front, back, top and bottom views.

2.4.1 Clustering of residues at 25% coverage.Fig. 4 shows thetop 25% of all residues, this time colored according to clusters theybelong to. The clusters in Fig.4 are composed of the residues listedin Table 1.

Table 1.cluster size membercolor residuesred 72 133,135,152,154,156,159,185

187,189,194,213,217,219,220221,222,223,224,225,227,239242,243,247,250,256,257,258259,262,263,264,269,273,275276,283,284,287,292,294,295296,297,299,300,301,322,325328,329,330,331,333,334,352354,355,356,357,358,361,363375,384,385,388,389,394,396420,421

blue 3 175,179,417

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

2

Fig. 4. Residues in 2ckrA, 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.

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.

Tetraethylene glycol binding site. Table 2 lists the top 25%of residues at the interface with 2ckrAPG41431 (tetraethylene gly-col). The following table (Table 3) suggests possible disruptivereplacements for these residues (see Section 3.6).

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

(%) bb (A)389 W W(93) 0.07 9/0 3.17 site

.(4)FY363 G G(90) 0.12 2/2 4.31

.(4)A(3)K(1)

189 Y Y(46) 0.15 3/0 4.15G(39)S(2).(6)TA(4)

394 D K(79) 0.20 1/0 4.96D(4)G(2)A(3).(4)M(2)

continued in next column

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

(%) bb (A)I(2)STQ

396 R E(84)N 0.20 23/0 3.62R(4)G.(4)QAILDK

Table 2. The top 25% of residues in 2ckrA at the interface with tetraethy-lene glycol.(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. )

Table 3.res type disruptive

mutations389 W (K)(E)(Q)(D)363 G (E)(R)(FKWHD)(Y)189 Y (K)(Q)(M)(R)394 D (R)(H)(FW)(Y)396 R (TY)(D)(CG)(S)

Table 3. List of disruptive mutations for the top 25% of residues in 2ckrA,that are at the interface with tetraethylene glycol.

Fig. 5. Residues in 2ckrA, at the interface with tetraethylene glycol, colo-red by their relative importance. The ligand (tetraethyleneglycol) is coloredgreen. Atoms further than 30A away from the geometric center of the ligand,as well as on the line of sight to the ligand were removed. (See Appendix forthe coloring scheme for the protein chain 2ckrA.)

3

Figure 5 shows residues in 2ckrA colored by their importance, at theinterface with 2ckrAPG41431.

Beta-d-glucose binding site. By analogy with 2ckrB –2ckrBBGC1432 interface. Table 4 lists the top 25% of residues atthe interface with 2ckrBBGC1432 (beta-d-glucose). The followingtable (Table 5) suggests possible disruptive replacements for theseresidues (see Section 3.6).

Table 4.res type subst’s cvg noc/ dist

(%) bb (A)330 Y Y(99). 0.01 5/0 3.77263 E E(98) 0.02 5/0 3.62

.(1)334 H H(95) 0.03 5/0 3.17

W(1)P(1)N.S

299 W W(94) 0.09 55/0 3.73Y(2)SF(1).

227 L L(39) 0.15 1/0 4.76H(44).(2)E(1)F(6)G(2)TMSY

361 Y A(81) 0.24 35/10 3.81Y(4)S(5).(4)C(1)E(1)Q

Table 4. The top 25% of residues in 2ckrA at the interface with beta-d-glucose.(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. )


mutations330 Y (K)(QM)(NVLAPI)(ER)263 E (FWH)(VCAG)(YR)(T)334 H (E)(T)(QD)(M)299 W (K)(E)(Q)(D)227 L (R)(Y)(KH)(T)361 Y (K)(M)(QR)(ELPI)

Table 5. List of disruptive mutations for the top 25% of residues in 2ckrA,that are at the interface with beta-d-glucose.

Figure 6 shows residues in 2ckrA colored by their importance, at theinterface with 2ckrBBGC1432.

Zinc ion binding site. Table 6 lists the top 25% of residues at theinterface with 2ckrAZN1434 (zinc ion). The following table (Table

Fig. 6. Residues in 2ckrA, at the interface with beta-d-glucose, colored bytheir relative importance. The ligand (beta-d-glucose) is colored green. Atomsfurther than 30A away from the geometric center of the ligand, as well as onthe line of sight to the ligand were removed. (See Appendix forthe coloringscheme for the protein chain 2ckrA.)

7) suggests possible disruptive replacements for these residues (seeSection 3.6).


(%) bb (A)287 D D(73)H 0.21 6/2 2.23 site

G(10)A(6).(1)EY(1)S(3)N(1)

Table 6. The top 25% of residues in 2ckrA at the interface with zincion.(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. )


mutations287 D (R)(FWH)(K)(Y)

Table 7. List of disruptive mutations for the top 25% of residues in 2ckrA,that are at the interface with zinc ion.

4

Fig. 7. Residues in 2ckrA, at the interface with zinc ion, colored bytheirrelative importance. The ligand (zinc ion) is colored green.Atoms furtherthan 30A away from the geometric center of the ligand, as well as on the lineof sight to the ligand were removed. (See Appendix for the coloring schemefor the protein chain 2ckrA.)

Figure 7 shows residues in 2ckrA colored by their importance, at theinterface with 2ckrAZN1434.



(%) bb (A)334 H H(95) 0.03 13/0 3.04

W(1)P(1)N.S

299 W W(94) 0.09 15/0 3.79Y(2)SF(1).

361 Y A(81) 0.24 9/0 4.04Y(4)S(5).(4)C(1)E(1)Q

Table 8. The top 25% of residues in 2ckrA at the interface with beta-d-glucose.(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. )


mutations334 H (E)(T)(QD)(M)299 W (K)(E)(Q)(D)361 Y (K)(M)(QR)(ELPI)

Table 9. List of disruptive mutations for the top 25% of residues in 2ckrA,that are at the interface with beta-d-glucose.


Figure 8 shows residues in 2ckrA colored by their importance, at theinterface with 2ckrBBGC1431.

Benzamidine binding site.Table 10 lists the top 25% of residuesat the interface with 2ckrABEN1432 (benzamidine). The followingtable (Table 11) suggests possible disruptive replacements for theseresidues (see Section 3.6).


(%) bb (A)284 R R(97)KH 0.04 6/0 3.56 site

.322 N N(94)K 0.07 8/4 4.25

H(4).

Table 10. The top 25% of residues in 2ckrA at the interface with benza-midine.(Field names: res: residue number in the PDB entry; type: amino acidtype; substs: substitutions seen in the alignment; with the percentage of each

5

type 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. )


mutations284 R (T)(D)(SVCAG)(YELPI)322 N (Y)(T)(FW)(SVCAG)

Table 11. List of disruptive mutations for the top 25% of residues in2ckrA, that are at the interface with benzamidine.

Fig. 9. Residues in 2ckrA, at the interface with benzamidine, colored by theirrelative importance. The ligand (benzamidine) is colored green. Atoms furtherthan 30A away from the geometric center of the ligand, as well as on the lineof sight to the ligand were removed. (See Appendix for the coloring schemefor the protein chain 2ckrA.)

Figure 9 shows residues in 2ckrA colored by their importance, at theinterface with 2ckrABEN1432.



(%) bb (A)328 H H(99). 0.01 1/0 4.82330 Y Y(99). 0.01 16/0 3.00


Table 12.continuedres type subst’s cvg noc/ dist antn

(%) bb (A)263 E E(98) 0.02 18/0 2.52

.(1)355 Q E(98)Q. 0.02 10/0 3.08262 N N(97) 0.05 1/0 4.88

.(1)D225 H H(92). 0.06 2/0 4.12

Q(6)A389 W W(93) 0.07 7/0 4.00 site

.(4)FY299 W W(94) 0.09 1/0 4.94

Y(2)SF(1).

189 Y Y(46) 0.15 3/0 4.41G(39)S(2).(6)TA(4)

227 L L(39) 0.15 9/0 4.21H(44).(2)E(1)F(6)G(2)TMSY

361 Y A(81) 0.24 8/8 3.75Y(4)S(5).(4)C(1)E(1)Q

Table 12. The top 25% of residues in 2ckrA at the interface with beta-d-glucose.(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. )


mutations328 H (E)(TQMD)(SNVCLAPIG)(K)330 Y (K)(QM)(NVLAPI)(ER)263 E (FWH)(VCAG)(YR)(T)355 Q (Y)(FWH)(T)(VCAG)262 N (Y)(FWH)(T)(VCARG)225 H (E)(TD)(QM)(SCG)389 W (K)(E)(Q)(D)299 W (K)(E)(Q)(D)189 Y (K)(Q)(M)(R)227 L (R)(Y)(KH)(T)361 Y (K)(M)(QR)(ELPI)


6

Table 13.continuedres type disruptive

mutations

Table 13. List of disruptive mutations for the top 25% of residues in2ckrA, that are at the interface with beta-d-glucose.


Figure 10 shows residues in 2ckrA colored by their importance, atthe interface with 2ckrBBGC1433.



(%) bb (A)394 D K(79) 0.20 8/0 2.92

D(4)G(2)A(3).(4)M(2)I(2)STQ

396 R E(84)N 0.20 13/0 3.29continued in next column

Table 14.continuedres type subst’s cvg noc/ dist

(%) bb (A)R(4)G.(4)QAILDK

Table 14. The top 25% of residues in 2ckrA at the interface with beta-d-glucose.(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. )


mutations394 D (R)(H)(FW)(Y)396 R (TY)(D)(CG)(S)

Table 15. List of disruptive mutations for the top 25% of residues in2ckrA, that are at the interface with beta-d-glucose.


Figure 11 shows residues in 2ckrA colored by their importance, atthe interface with 2ckrBBGC1435.

7

3 NOTES ON USING TRACE RESULTS

3.1 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 strongerthepressure 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.

3.2 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%.

3.3 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.)

3.4 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.

3.5 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).

3.6 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 listedaccording 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.

4 APPENDIX

4.1 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

4.2 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,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. 12.

4.3 Credits4.3.1 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 the

8

5%30%50%100%

COVERAGE

V

VRELATIVE IMPORTANCE

Fig. 12. Coloring scheme used to color residues by their relative importance.

alignment 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.

4.3.2 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.

4.3.3 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.

4.3.4 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/

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

4.3.6 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/

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

4.4 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.

4.5 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.

4.6 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.

4.7 AttachmentsThe following files should accompany this report:

• 2ckrA.complex.pdb - coordinates of 2ckrA with all of its inter-acting partners

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

• 2ckrA.clusterreport.summary - Cluster report summary for2ckrA

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

• 2ckrA.clusters - Cluster descriptions for 2ckrA

• 2ckrA.msf - the multiple sequence alignment used for the chain2ckrA

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

• 2ckrA.rankssorted - full listing of residues and their ranking for2ckrA

• 2ckrA.2ckrAPG41431.if.pml - Pymol script for Figure 5

9

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

• 2ckrA.2ckrBBGC1432.if.pml - Pymol script for Figure 6

• 2ckrA.2ckrAZN1434.if.pml - Pymol script for Figure 7


• 2ckrA.2ckrABEN1432.if.pml - Pymol script for Figure 9



10

evolutionary trace report by report maker october 10,...

Documents