Pages 1–7 3bxj Evolutionary trace report by report maker May 19, 2009

4.3.1 Alistat 6 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 7 4.4 Note about ET Viewer 7 4.5 Citing this work 7 4.6 About report maker 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3bxj): Title: Crystal structure of the c2-gap fragment of syngap Compound: Mol id: 1; molecule: ras gtpase-activating protein syn- gap; chain: a, b; synonym: synaptic ras gtpase-activating protein 1, synaptic ras-gap 1, neuronal rasgap, p135 syngap; engineered: yes Organism, scientific name: Rattus Norvegicus; 3bxj contains a single unique chain 3bxjB (358 residues long) and its homologue 3bxjA. CONTENTS

1 Introduction 1 2 CHAIN 3BXJB 2.1 Q9QUH6 overview 2 Chain 3bxjB 1 2.1 Q9QUH6 overview 1 From SwissProt, id Q9QUH6, 96% identical to 3bxjB: 2.2 Multiple sequence alignment for 3bxjB 1 Description: Ras GTPase-activating protein SynGAP (Synaptic 2.3 Residue ranking in 3bxjB 2 Ras-GTPase-activating protein 1) (Synaptic Ras-GAP 1) (Neuronal 2.4 Top ranking residues in 3bxjB and their position on RasGAP) (p135 SynGAP). the structure 2 Organism, scientific name: Rattus norvegicus (Rat). 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Verte- 2.4.2 Overlap with known functional surfaces at brata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; 25% coverage. 2 Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Rattus. 2.4.3 Possible novel functional surfaces at 25% Function: Inhibitory regulator of the Ras-cAMP pathway. Member coverage. 3 of the NMDAR signaling complex in excitatory synapses it may play a role in NMDAR-dependent control of AMPAR potentiation and 3 Notes on using trace results 5 synaptic plasticity. 3.1 Coverage 5 Subunit: Isoforms containing the PDZ-binding domain associate 3.2 Known substitutions 5 with DLG4 and DLG3 to form the PSD protein complex colocali- 3.3 Surface 6 zed with GRIN2B at synapses. Interacts with MPDZ, CAMK2A and 3.4 Number of contacts 6 CAMK2B. 3.5 Annotation 6 Subcellular location: Membrane-associated, mostly in excitatory 3.6 Mutation suggestions 6 glutamatergic synapses. Alternative products: 4 Appendix 6 Event=Alternative splicing; Named isoforms=5; Com- 4.1 File formats 6 ment=Additional isoforms seem to exist; Name=1; 4.2 Color schemes used 6 IsoId=Q9QUH6-1; Sequence=Displayed; Name=2; 4.3 Credits 6 Synonyms=SynGAP-a; IsoId=Q9QUH6-2; Sequence=VSP

1 Lichtarge lab 2006 007979; Name=3; Synonyms=SynGAP-b; IsoId=Q9QUH6- 3; Sequence=VSP 007974; Name=4; Synonyms=SynGAP-c; IsoId=Q9QUH6-4; Sequence=VSP 007976, VSP 007980; Name=5; Synonyms=SynGAP-d, SynGAP-beta; IsoId=Q9QUH6-5; Sequence=VSP 007975, VSP 007977, VSP 007978; Tissue specificity: Highly expressed in brain; predominantly in the cortex, hippocampus and olfactory bulb. Domain: The PDZ-binding domain interacts with all three PDZ domains of DGL4. Ptm: Phosphorylated by CaM-kinase II. Dephosphorylated upon NMDA receptor activation or SYNGAP1/MPDZ complex disrup- tion. Fig. 1. Residues 237-538 in 3bxjB colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Similarity: Contains 1 C2 domain. Similarity: Contains 1 PH domain. Similarity: Contains 1 Ras-GAP domain. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 2.2 Multiple sequence alignment for 3bxjB For the chain 3bxjB, the alignment 3bxjB.msf (attached) with 62 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as Fig. 2. Residues 539-719 in 3bxjB colored by their relative importance. (See duplicate sequences were removed. It can be found in the attachment Appendix, Fig.7, for the coloring scheme.) to this report, under the name of 3bxjB.msf. Its statistics, from the alistat program are the following: importance: bright red and yellow indicate more conserved/important Format: MSF residues (see Appendix for the coloring scheme). A Pymol script for Number of sequences: 62 producing this figure can be found in the attachment. Total number of residues: 18083 Smallest: 252 Largest: 358 Average length: 291.7 Alignment length: 358 Average identity: 38% Most related pair: 99% Most unrelated pair: 13% Most distant seq: 33%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 35% eukaryotic ( 24% vertebrata, 1% arthropoda, 8% fungi) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descrip- tions can be found in the attachment, under the name 3bxjB.descr. 2.3 Residue ranking in 3bxjB The 3bxjB sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 3bxjB can be found in the file called 3bxjB.ranks sorted in the attachment. 2.4 Top ranking residues in 3bxjB and their position on Fig. 3. Residues in 3bxjB, colored by their relative importance. Clockwise: the structure front, back, top and bottom views. In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 3bxjB colored by their

2 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Interface with 3bxjA.Table 2 lists the top 25% of residues at the top 25% of all residues, this time colored according to clusters they interface with 3bxjA. The following table (Table 3) suggests possible belong to. The clusters in Fig.4 are composed of the residues listed disruptive replacements for these residues (see Section 3.6).

Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 470 R R(98) 0.01 2/0 4.55 .(1) 467 L I(1) 0.09 1/0 4.53 L(64) T(32) .(1) 452 D S(3) 0.25 3/0 4.27 D(41) A(38) N(3) T(4) I(1) Y(3) C(1) .(1) 564 R R(74) 0.25 63/20 3.09 L(11) H(4) N(1) Q(4) Y(3) Fig. 4. Residues in 3bxjB, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The Table 2. The top 25% of residues in 3bxjB at the interface with 3bxjA. corresponding Pymol script is attached. (Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage 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 the bracket; dist: distance in Table 1. of closest apporach to the ligand. ) Table 1. cluster size member color residues Table 3. red 83 429,433,436,437,439,457,467 res type disruptive 468,469,470,471,472,473,474 mutations 476,477,480,483,484,485,487 470 R (TD)(SVCLAPIG)(YE)(FMW) 490,491,494,495,502,509,510 467 L (R)(Y)(H)(TK) 511,512,513,528,539,542,546 452 D (R)(H)(FW)(K) 547,550,554,558,561,573,574 564 R (T)(D)(E)(SYVCAG) 575,577,578,579,580,581,582 583,584,585,586,587,588,589 Table 3. List of disruptive mutations for the top 25% of residues in 3bxjB, 590,591,592,593,595,600,604 that are at the interface with 3bxjA. 606,607,608,609,610,611,612 613,616,618,619,620,627,628 Figure 5 shows residues in 3bxjB colored by their importance, at the 631,634,648,649,652,653 interface with 3bxjA. blue 4 396,397,402,405 2.4.3 Possible novel functional surfaces at 25% coverage. One Table 1. Clusters of top ranking residues in 3bxjB. group of residues is conserved on the 3bxjB surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 3bxj. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. 2.4.2 Overlap with known functional surfaces at 25% coverage. The residues belonging to this surface ”patch” are listed in Table The name of the ligand is composed of the source PDB identifier 4, while Table 5 suggests possible disruptive replacements for these and the heteroatom name used in that file. residues (see Section 3.6).

3 Table 4. res type substitutions(%) cvg 469 F F(98).(1) 0.01 470 R R(98).(1) 0.01 547 P P(100) 0.01 581 R R(98)S(1) 0.02 606 R R(96)P(1)K(1) 0.03 613 K K(96)P(1)R(1) 0.03 616 Q Q(96)A(1)L(1) 0.03 472 N N(96)Q(1).(1) 0.04 510 E E(98).(1) 0.04 512 D D(98).(1) 0.04 580 L L(95)P(1)M(1) 0.05 T(1) 648 F F(95)L(1)S(1) 0.05 Y(1) 539 V I(96)V(3) 0.06 582 F F(91)L(6)G(1) 0.06 593 F F(95)L(1)N(1) 0.06 R(1) 490 Y Y(95).(3)F(1) 0.07 575 S S(93)C(4)I(1) 0.07 Fig. 5. Residues in 3bxjB, at the interface with 3bxjA, colored by their rela- 585 P P(93)V(3)A(1) 0.07 tive importance. 3bxjA is shown in backbone representation (See Appendix S(1) for the coloring scheme for the protein chain 3bxjB.) 509 C C(93)F(1)I(1) 0.08 .(1)L(1) 627 K K(93)A(3)P(1) 0.08 S(1) 628 E E(93)S(4)A(1) 0.08 467 L I(1)L(64)T(32) 0.09 .(1) 584 C C(66)A(30)L(1) 0.09 S(1) 513 P P(95)D(1)Q(1) 0.10 .(1) 619 A A(66)G(29)R(1) 0.10 S(3) 476 T T(74)S(24).(1) 0.11 Fig. 6. A possible active surface on the chain 3bxjB. The larger cluster it 542 S S(93)T(3)V(1) 0.11 belongs to is shown in blue. N(1) 592 L L(90)T(3)S(3) 0.11 A(1)M(1) 429 K R(3)K(72)L(22) 0.12 I(1) 474 L L(93)F(1)V(1) 0.12 I(1).(1) 607 T S(1)T(80)N(14) 0.12 E(1)D(1) 609 T I(1)T(90)G(1) 0.13 S(1)V(1)L(1) F(1) 471 E G(67)E(27)S(3) 0.14 continued in next column

4 Table 4. continued Table 5. res type substitutions(%) cvg res type disruptive .(1) mutations 574 I V(56)I(40)L(3) 0.14 469 F (KE)(TQD)(SNCG)(R) 595 L L(83)I(9)M(4) 0.14 470 R (TD)(SVCLAPIG)(YE)(FMW) P(1) 547 P (YR)(TH)(SKECG)(FQWD) 457 E E(82)D(14)H(1) 0.15 581 R (D)(TYELPI)(FVMCAWG)(S) .(1) 606 R (T)(Y)(D)(S) 477 K K(87)R(3)Q(8) 0.15 613 K (Y)(T)(FW)(S) .(1) 616 Q (Y)(H)(T)(FW) 484 R K(66)R(30).(1) 0.15 472 N (Y)(FTWH)(SVCAG)(E) Q(1) 510 E (FWH)(VCAG)(YR)(T) 558 R Q(3)R(88)A(1) 0.16 512 D (R)(FWH)(VCAG)(KY) K(4)H(1) 580 L (R)(Y)(H)(T) 620 N N(77)S(20)P(1) 0.16 648 F (K)(E)(Q)(R) 480 E D(53)E(43)H(1) 0.17 539 V (YR)(KE)(H)(QD) .(1) 582 F (KE)(QDR)(T)(SN) 502 L I(58)L(33)V(6) 0.18 593 F (E)(T)(KD)(SCG) .(1) 490 Y (K)(Q)(M)(E) 588 M L(66)M(25)V(3) 0.18 575 S (R)(K)(H)(FW) P(1)I(1)Q(1) 585 P (R)(Y)(H)(K) 485 L L(64)R(20)I(4) 0.19 509 C (R)(KE)(Q)(D) H(1)M(1)F(3) 627 K (Y)(FW)(T)(H) V(1).(1) 628 E (H)(FW)(R)(Y) 587 I I(83)V(6)L(6) 0.19 467 L (R)(Y)(H)(TK) H(1)F(1) 584 C (R)(K)(E)(H) 495 I I(24)L(70)V(3) 0.20 513 P (Y)(R)(H)(T) .(1) 619 A (E)(KY)(R)(D) 546 F C(48)F(40)L(8) 0.20 476 T (KR)(FMWH)(Q)(LPI) V(1)I(1) 542 S (R)(K)(H)(FW) 591 S K(38)S(40)H(3) 0.20 592 L (R)(Y)(H)(K) N(12)F(1)R(1) 429 K (Y)(T)(FW)(SCG) M(1) 474 L (R)(Y)(T)(H) 494 A T(70)A(19)V(4) 0.21 607 T (R)(K)(FWH)(M) S(1).(1)L(1) 609 T (R)(K)(QH)(E) 573 L A(20)L(41)S(24) 0.21 471 E (FWH)(R)(Y)(VA) V(9)I(1)G(1) 574 I (YR)(H)(T)(KE) 468 I I(48)L(35)T(9) 0.22 595 L (Y)(R)(TH)(SCG) V(1)M(3).(1) 457 E (FW)(VCAHG)(R)(Y) 600 P P(82)L(4)V(3) 0.22 477 K (Y)(T)(FW)(SVCAG) A(1)G(1)D(1) 484 R (T)(YD)(SVCAG)(FLWPI) T(4) 558 R (T)(D)(Y)(E) 589 S A(4)S(54)N(29) 0.24 620 N (Y)(H)(FW)(R) G(8)Q(1)T(1) 480 E (FW)(VCAHG)(R)(Y) 604 T T(50)A(30)V(4) 0.24 502 L (YR)(H)(T)(KE) S(3)I(1)P(4) 588 M (Y)(H)(T)(R) L(1)N(3) 485 L (Y)(TR)(E)(H) 649 L I(4)L(72)V(14) 0.25 587 I (R)(Y)(T)(E) A(1)H(1)P(1) 495 I (YR)(H)(T)(KE) M(1)F(1) 546 F (KE)(R)(Q)(D) 591 S (KR)(Y)(FEW)(MH) Table 4. Residues forming surface ”patch” in 3bxjB. 494 A (R)(K)(Y)(E) continued in next column

5 Table 5. continued the interface, as well as how many of them are realized through the res type disruptive backbone atoms (if all or most contacts are through the backbone, mutations mutation presumably won’t have strong impact). Two heavy atoms 573 L (R)(Y)(H)(K) are considered to be “in contact” if their centers are closer than 5A˚ . 468 I (R)(Y)(H)(T) 600 P (R)(Y)(H)(K) 3.5 Annotation 589 S (R)(K)(H)(FW) If the residue annotation is available (either from the pdb file or 604 T (R)(K)(H)(FQW) from other sources), another column, with the header “annotation” 649 L (YR)(T)(EH)(K) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 5. Disruptive mutations for the surface patch in 3bxjB. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- 3.1 Coverage mentarity with the substitutions found in the alignment. Note that Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino couple of residues should affect the protein somehow, with the exact acid, and they are grouped in round brackets if they appear equally effects to be determined experimentally. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 4 APPENDIX an R at that position, it is advisable to try anything, but RVK. Conver- 4.1 File formats sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file: is given in the immediately following bracket. No percentage is given • alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up • residue# residue number in the PDB file to 100%. • type amino acid type 3.3 Surface • rank rank of the position according to older version of ET • To detect candidates for novel functional interfaces, first we look for variability has two subfields: residues that are solvent accessible (according to DSSP program) by 1. number of different amino acids appearing in in this column 2 at least 10A˚ , which is roughly the area needed for one water mole- of the alignment cule to come in the contact with the residue. Furthermore, we require 2. their type that these residues form a “cluster” of residues which have neighbor • rho ET score - the smaller this value, the lesser variability of within 5A˚ from any of their heavy atoms. this position across the branches of the tree (and, presumably, Note, however, that, if our picture of protein evolution is correct, the greater the importance for the protein) the neighboring residues which are not surface accessible might be • cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with • gaps percentage of gaps in this column the surface residues.) 4.2 Color schemes used 3.4 Number of contacts The following color scheme is used in figures with residues colored Another column worth noting is denoted “noc/bb”; it tells the num- by cluster size: black is a single-residue cluster; clusters composed of ber of contacts heavy atoms of the residue in question make across more than one residue colored according to this hierarchy (ordered

6 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- COVERAGE sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/ V A 100% 50% 30% 5% 4.3.5 LaTex The text for this report was processed using LTEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004),

V ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. RELATIVE IMPORTANCE http://www.drive5.com/muscle/

Fig. 7. Coloring scheme used to color residues by their relative importance. 4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. 4.4 Note about ET Viewer The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 7. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- 4.5 Citing this work ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report ˇ sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res, O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics 4.3.2 CE To map ligand binding sites from different 22:1656-7. source structures, report maker uses the CE program: 4.6 About report maker http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension report maker was written in 2006 by Ivana Mihalek. The 1D ran- (CE) of the optimal path . Protein Engineering 11(9) 739-747. king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.3 DSSP In this work a residue is considered solvent accessi- Houston. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in 4.7 Attachments the contact with the residue. DSSP is copyrighted by W. Kabsch, C. The following files should accompany this report: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version • 3bxjB.complex.pdb - coordinates of 3bxjB with all of its inter- by [email protected] November 18,2002, acting partners http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 3bxjB.etvx - ET viewer input file for 3bxjB

7 • 3bxjB.cluster report.summary - Cluster report summary for • 3bxjB.descr - description of sequences used in 3bxjB msf 3bxjB • 3bxjB.ranks sorted - full listing of residues and their ranking for • 3bxjB.ranks - Ranks file in sequence order for 3bxjB 3bxjB • 3bxjB.clusters - Cluster descriptions for 3bxjB • 3bxjB.3bxjA.if.pml - Pymol script for Figure 5 • 3bxjB.msf - the multiple sequence alignment used for the chain • 3bxjB.cbcvg - used by other 3bxjB – related pymol scripts 3bxjB

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