Pages 1–5 2j49 Evolutionary trace report by report maker September 27, 2008

4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2j49): Title: Crystal structure of yeast taf5 n-terminal domain Compound: Mol id: 1; molecule: transcription initiation factor tfiid subunit 5; chain: a; fragment: n-terminal domain, residues 147-290; synonym: tbp-associated factor 5,tbp-associated factor 90 kda,tafii- 90; engineered: yes Organism, scientific name: Saccharomyces Cerevisiae; 2j49 contains a single unique chain 2j49A (134 residues long).

2 CHAIN 2J49A CONTENTS 2.1 P38129 overview

1 Introduction 1 From SwissProt, id P38129, 91% identical to 2j49A: Description: Transcription initiation factor TFIID subunit 5 (TBP- 2 Chain 2j49A 1 associated factor 5) (TBP-associated factor 90 kDa) (TAFII-90). 2.1 P38129 overview 1 Organism, scientific name: Saccharomyces cerevisiae (Baker’s 2.2 Multiple sequence alignment for 2j49A 1 yeast). 2.3 Residue ranking in 2j49A 2 Taxonomy: Eukaryota; Fungi; Ascomycota; Saccharomycotina; 2.4 Top ranking residues in 2j49A and their position on Saccharomycetes; Saccharomycetales; Saccharomycetaceae; Sac- the structure 2 charomyces. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Functions as a component of the DNA-binding general 2.4.2 Possible novel functional surfaces at 25% transcription factor complex TFIID and the regulatory transcription coverage. 2 complex SAGA. Binding of TFIID to a promoter (with or without TATA element) is the initial step in pre-initiation complex (PIC) for- 3 Notes on using trace results 3 mation. TFIID plays a key role in the regulation of gene expression 3.1 Coverage 3 by RNA polymerase II through different activities such as trans- 3.2 Known substitutions 3 cription activator interaction, core promoter recognition and selec- 3.3 Surface 4 tivity, TFIIA and TFIIB interaction, chromatin modification (histone 3.4 Number of contacts 4 acetylation by TAF1), facilitation of DNA opening and initiation 3.5 Annotation 4 of transcription. SAGA influences RNA polymerase II transcrip- 3.6 Mutation suggestions 4 tional activity through different activities such as TBP interaction (SPT3 and SPT8) and promoter selectivity, interaction with transcrip- 4 Appendix 4 tion activators (GCN5, ADA2, ADA3, and TRA1), and chromatin 4.1 File formats 4 modification through histone acetylation (GCN5). 4.2 Color schemes used 4 Subunit: The 1.2 MDa TFIID complex is composed of TATA bin- 4.3 Credits 4 ding protein (TBP) and the 14 TBP-associated factors. One copy of 4.3.1 Alistat 4 each TAF1, TAF2, TAF3, TAF7, TAF8, TAF11, TAF13, two copies 4.3.2 CE 5 of each TAF4, TAF5, TAF6, TAF9, TAF10, TAF12, and three copies

1 Lichtarge lab 2006 2.4 Top ranking residues in 2j49A and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 2j49A colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 149-282 in 2j49A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) of TAF14. The 1.8 MDa SAGA complex is composed of at least 17 subunits: TRA1, ADA1, SPT7, SPT20, SPT3, SPT8, GCN5, ADA2, ADA3, TAF5, TAF6, TAF9, TAF10, TAF12, SGF29, SGF73, and UBP8. Interaction: Miscellaneous: Present with 14834 (+/-203) molecules/cell. Similarity: Belongs to the WD-repeat TAF5 family. Similarity: Contains 1 LisH domain. Similarity: Contains 6 WD repeats. 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 2j49A For the chain 2j49A, the alignment 2j49A.msf (attached) with 9 sequences was used. The alignment was assembled through combi- nation of BLAST searching on the UniProt database and alignment using Muscle program. It can be found in the attachment to this Fig. 2. Residues in 2j49A, colored by their relative importance. Clockwise: report, under the name of 2j49A.msf. Its statistics, from the alistat front, back, top and bottom views. program are the following:

Format: MSF 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Number of sequences: 9 top 25% of all residues, this time colored according to clusters they Total number of residues: 1198 belong to. The clusters in Fig.3 are composed of the residues listed Smallest: 130 in Table 1. Largest: 134 Table 1. Average length: 133.1 cluster size member Alignment length: 134 color residues Average identity: 36% red 29 152,156,159,162,166,167,170 Most related pair: 70% 173,174,179,180,182,184,187 Most unrelated pair: 27% 189,192,197,200,208,212,242 Most distant seq: 41% 253,254,257,258,265,266,268 277 Furthermore, 5% of residues show as conserved in this alignment. blue 2 227,230 The alignment consists of 88% eukaryotic ( 66% fungi, 11% plan- tae) sequences. (Descriptions of some sequences were not readily Table 1. Clusters of top ranking residues in 2j49A. available.) The file containing the sequence descriptions can be found in the attachment, under the name 2j49A.descr. 2.4.2 Possible novel functional surfaces at 25% coverage. One 2.3 Residue ranking in 2j49A group of residues is conserved on the 2j49A surface, away from (or The 2j49A sequence is shown in Fig. 1, with each residue colored susbtantially larger than) other functional sites and interfaces reco- according to its estimated importance. The full listing of residues gnizable in PDB entry 2j49. It is shown in Fig. 4. The right panel in 2j49A can be found in the file called 2j49A.ranks sorted in the shows (in blue) the rest of the larger cluster this surface belongs to. attachment. The residues belonging to this surface ”patch” are listed in Table

2 Table 3. res type disruptive mutations 162 W (KE)(TQD)(SNCRG)(M) 167 L (YR)(TH)(SKECG)(FQWD) 170 Y (K)(QM)(NEVLAPIR)(D) 253 L (Y)(R)(TH)(SECG) 173 E (FW)(YH)(VCAG)(T) 257 F (K)(E)(Q)(D) 166 S (R)(K)(FWH)(M)

Table 3. Disruptive mutations for the surface patch in 2j49A.

Another group of surface residues is shown in Fig.5. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 3. Residues in 2j49A, 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 corresponding Pymol script is attached.

Fig. 5. Another possible active surface on the chain 2j49A. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type substitutions(%) cvg 212 H H(88)F(11) 0.13 Fig. 4. A possible active surface on the chain 2j49A. The larger cluster it 184 Y H(55)Y(44) 0.15 belongs to is shown in blue. 208 F H(55)F(44) 0.16 179 Y F(11)Y(66)W(22) 0.18 187 L L(77)I(11)M(11) 0.19 2, while Table 3 suggests possible disruptive replacements for these 242 Y Y(77)W(11)V(11) 0.19 residues (see Section 3.6). 243 R Q(11)R(66)N(22) 0.25 Table 2. res type substitutions(%) cvg Table 4. Residues forming surface ”patch” in 2j49A. 162 W W(100) 0.06 167 L L(100) 0.06 170 Y Y(100) 0.06 Table 5. 253 L L(77)N(22) 0.07 res type disruptive 173 E E(88)K(11) 0.11 mutations 257 F F(88)Y(11) 0.13 212 H (E)(TQD)(KM)(SNCG) 166 S S(66)T(11)N(22) 0.18 184 Y (K)(QM)(E)(NVLAPI) 208 F (E)(K)(TQD)(SNCG) Table 2. Residues forming surface ”patch” in 2j49A. 179 Y (K)(Q)(E)(M) continued in next column

3 Table 5. continued mutation presumably won’t have strong impact). Two heavy atoms res type disruptive are considered to be “in contact” if their centers are closer than 5A˚ . mutations 187 L (Y)(R)(TH)(CG) 3.5 Annotation 242 Y (K)(Q)(E)(R) If the residue annotation is available (either from the pdb file or 243 R (T)(Y)(D)(S) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 5. Disruptive mutations for the surface patch in 2j49A. (indicating existence of related site record in PDB ), S-S (disulfide 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 GST C], medium [LP NQDEMIK], large in a chain], according to trace. The ET results are presented in the [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [W F Y H], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDET Y ], 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, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment 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 • residue# residue number in the PDB file guide - due to rounding errors these percentages often do not add up • type to 100%. amino acid type • rank rank of the position according to older version of ET 3.3 Surface • variability has two subfields: To detect candidates for novel functional interfaces, first we look for 1. number of different amino acids appearing in in this column residues that are solvent accessible (according to DSSP program) by of the alignment A˚ 2 at least 10 , which is roughly the area needed for one water mole- 2. their type cule to come in the contact with the residue. Furthermore, we require • rho that these residues form a “cluster” of residues which have neighbor 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- • gaps percentage of gaps in this column sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4.2 Color schemes used The following color scheme is used in figures with residues colored 3.4 Number of contacts by cluster size: black is a single-residue cluster; clusters composed of Another column worth noting is denoted “noc/bb”; it tells the num- more than one residue colored according to this hierarchy (ordered ber of contacts heavy atoms of the residue in question make across by descending size): red, blue, yellow, green, purple, azure, tur- the interface, as well as how many of them are realized through the quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, backbone atoms (if all or most contacts are through the backbone, bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,

4 Daruvar, 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/

COVERAGE 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- V Wesley,” Reading, Mass. (1986). 50% 30% 5% 100% 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. http://www.drive5.com/muscle/ V

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- Fig. 6. Coloring scheme used to color residues by their relative importance. tific LLC (2005). For more information about Pymol see http://pymol.sourceforge.net/. (Note for Windows 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. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 6. Dan Morgan from the Lichtarge lab has developed a visualization 4.3 Credits tool specifically for viewing trace results. If you are interested, please visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ 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- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ 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 alistat numbers (hence, the most outlying sequence). 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 4.3.2 CE To map ligand binding sites from different of service for comparative analysis of proteins.” Bioinformatics source structures, report maker uses the CE program: 22:1656-7. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , Houston. which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 4.7 Attachments Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following files should accompany this report: by [email protected] November 18,2002, • 2j49A.complex.pdb - coordinates of 2j49A with all of its inter- http://www.cmbi.kun.nl/gv/dssp/descrip.html. acting partners • 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 2j49A.etvx - ET viewer input file for 2j49A gnment as a starting point for the analysis (sequences shorter than • 2j49A.cluster report.summary - Cluster report summary for 75% of the query are taken out, however); R. Schneider, A. de 2j49A

5 • 2j49A.ranks - Ranks file in sequence order for 2j49A • 2j49A.descr - description of sequences used in 2j49A msf • 2j49A.clusters - Cluster descriptions for 2j49A • 2j49A.ranks sorted - full listing of residues and their ranking for • 2j49A.msf - the multiple sequence alignment used for the chain 2j49A 2j49A

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