Pages 1–5 1ydl Evolutionary trace report by report maker December 13, 2009

4.3.3 DSSP 4 4.3.4 HSSP 4 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 Data Bank entry (PDB id 1ydl): Title: Crystal structure of the human tfiih, northeast structural genomics target hr2045. Compound: Mol id: 1; molecule: general transcription factor iih, polypeptide 5; chain: a; synonym: tfiih; engineered: yes Organism, scientific name: Homo Sapiens; 1ydl contains a single unique chain 1ydlA (71 residues long).

2 CHAIN 1YDLA 2.1 Q6ZYL4 overview CONTENTS From SwissProt, id Q6ZYL4, 95% identical to 1ydlA: 1 Introduction 1 Description: TFIIH basal transcription factor complex TTD-A subu- nit (General transcription factor IIH polypeptide 5) (TFB5 ortholog). 2 Chain 1ydlA 1 Organism, scientific name: Homo sapiens (Human). 2.1 Q6ZYL4 overview 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.2 Multiple sequence alignment for 1ydlA 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.3 Residue ranking in 1ydlA 1 Catarrhini; Hominidae; Homo. 2.4 Top ranking residues in 1ydlA and their position on Function: Component of the TFIIH basal transcription factor invol- the structure 2 ved in nucleotide excision repair (NER) of DNA and, when comple- 2.4.1 Clustering of residues at 25% coverage. 2 xed to CAK, in RNA transcription by RNA polymerase II. Necessary 2.4.2 Possible novel functional surfaces at 25% for the stability of the TFIIH complex and for the presence of normal coverage. 2 levels of TFIIH in the cell. Subunit: Subunit of the TFIIH basal transcription factor com- 3 Notes on using trace results 3 plex that contains ERCC2, ERCC3, GTF2H1, GTF2H2, GTF2H3, 3.1 Coverage 3 GTF2H4, GTF2H5, MNAT1, CDK7 and CCNH. 3.2 Known substitutions 3 Subcellular location: Nuclear. 3.3 Surface 3 Disease: Defects in GTF2H5 are a cause of 3.4 Number of contacts 3 (TTD) [MIM:601675]. TTD is an autosomal recessive disease cha- 3.5 Annotation 4 racterized by sulfur-deficient brittle hair and nails, mental retardation, 3.6 Mutation suggestions 4 impaired sexual development, ichthyotic skin, abnormal facies and in some but not all instances photosensitivity. There are no reports of 4 Appendix 4 skin cancer associated with TTD. Photosensitive patients have a defi- 4.1 File formats 4 ciency in excision- repair which in most cases is indistinguishable 4.2 Color schemes used 4 from that in XP patients. 4.3 Credits 4 Similarity: Belongs to the TFB5 family. 4.3.1 Alistat 4 About: This Swiss-Prot entry is copyright. It is produced through a 4.3.2 CE 4 collaboration between the Swiss Institute of Bioinformatics and the

1 Lichtarge lab 2006 Fig. 1. Residues 1-71 in 1ydlA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.)

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 1ydlA For the chain 1ydlA, the alignment 1ydlA.msf (attached) with 28 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1ydlA.msf. Its statistics, from the alistat program are the following:

Format: MSF Fig. 2. Residues in 1ydlA, colored by their relative importance. Clockwise: Number of sequences: 28 front, back, top and bottom views. Total number of residues: 1801 Smallest: 57 Largest: 71 Average length: 64.3 Alignment length: 71 Average identity: 43% Most related pair: 98% Most unrelated pair: 14% Most distant seq: 38%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 35% eukaryotic ( 21% vertebrata, 3% fungi, 3% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1ydlA.descr. 2.3 Residue ranking in 1ydlA The 1ydlA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1ydlA can be found in the file called 1ydlA.ranks sorted in the attachment.

2.4 Top ranking residues in 1ydlA and their position on Fig. 3. Residues in 1ydlA, colored according to the cluster they belong to: the structure red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The In the following we consider residues ranking among top 25% of resi- corresponding Pymol script is attached. dues in the protein . Figure 2 shows residues in 1ydlA 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. 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed in Table 1.

2 Table 1. Table 2. continued cluster size member res type substitutions(%) cvg color residues 24 D D(71)A(3)N(17) 0.14 red 11 9,12,13,14,15,17,18,37,38,39 M(3)S(3) 42 37 D D(71)E(10)M(10) 0.15 blue 6 7,24,32,33,34,45 R(3)V(3) 32 K D(14)K(75)N(7) 0.17 Table 1. Clusters of top ranking residues in 1ydlA. H(3) 18 Q A(10)Q(78)E(7) 0.18 S(3) 2.4.2 Possible novel functional surfaces at 25% coverage. One 12 C C(85)F(3)N(3) 0.20 group of residues is conserved on the 1ydlA surface, away from (or S(7) susbtantially larger than) other functional sites and interfaces reco- 34 I I(78)V(17)E(3) 0.21 gnizable in PDB entry 1ydl. It is shown in Fig. 4. The residues 9 L L(82).(3)F(14) 0.23 38 I L(78)V(3)I(17) 0.24

Table 2. Residues forming surface ”patch” in 1ydlA.

Table 3. res type disruptive mutations 13 D (R)(FWH)(YVCAG)(K) 39 D (R)(FWH)(K)(Y) 42 H (E)(MD)(TQ)(VLAPI) 33 F (K)(E)(Q)(D) 7 G (KR)(E)(FMWH)(Q) 17 K (Y)(T)(FW)(SCG) 15 A (R)(K)(YE)(H) 14 P (R)(Y)(H)(K) 24 D (R)(H)(FYW)(K) 37 D (R)(H)(FYW)(CG) 32 K (Y)(T)(FW)(VCAG) 18 Q (Y)(H)(FW)(T) 12 C (KER)(QMHD)(FW)(Y) 34 I (YR)(H)(T)(K) 9 L (R)(Y)(T)(H) 38 I (YR)(H)(T)(KE) Fig. 4. A possible active surface on the chain 1ydlA.

Table 3. Disruptive mutations for the surface patch in 1ydlA. belonging to this surface ”patch” are listed in Table 2, while Table 3 suggests possible disruptive replacements for these residues (see Section 3.6). 3 NOTES ON USING TRACE RESULTS Table 2. 3.1 Coverage res type substitutions(%) cvg Trace results are commonly expressed in terms of coverage: the resi- 13 D D(96)E(3) 0.01 due is important if its “coverage” is small - that is if it belongs to 39 D D(96)G(3) 0.03 some small top percentage of residues [100% is all of the residues 42 H R(14)H(82)Y(3) 0.04 in a chain], according to trace. The ET results are presented in the 33 F Y(10)F(85)A(3) 0.06 form of a table, usually limited to top 25% percent of residues (or 7 G G(92).(3)S(3) 0.07 to some nearby percentage), sorted by the strength of the presumed 17 K K(75)R(14)A(10) 0.09 evolutionary pressure. (I.e., the smaller the coverage, the stronger the 15 A S(10)A(71)T(3) 0.10 pressure on the residue.) Starting from the top of that list, mutating a P(14) couple of residues should affect the protein somehow, with the exact 14 P P(85)S(3)V(7) 0.13 effects to be determined experimentally. I(3) continued in next column 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types

3 may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, COVERAGE one may try replacing, R with K, or (perhaps more surprisingly), with

V. The percentage of times the substitution appears in the alignment V is given in the immediately following bracket. No percentage is given 100% 50% 30% 5% 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 to 100%.

3.3 Surface V To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by RELATIVE IMPORTANCE 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor Fig. 5. Coloring scheme used to color residues by their relative importance. within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be choose, however, the straightforward alanine mutations, especially in equally important in maintaining the interaction specificity - they the beginning stages of their investigation. should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4 APPENDIX 3.4 Number of contacts 4.1 File formats Another column worth noting is denoted “noc/bb”; it tells the num- Files with extension “ranks sorted” are the actual trace results. The ber of contacts heavy atoms of the residue in question make across fields in the table in this file: the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • alignment# number of the position in the alignment mutation presumably won’t have strong impact). Two heavy atoms • residue# residue number in the PDB file ˚ are considered to be “in contact” if their centers are closer than 5A. • type amino acid type • 3.5 Annotation rank rank of the position according to older version of ET • variability If the residue annotation is available (either from the pdb file or has two subfields: from other sources), another column, with the header “annotation” 1. number of different amino acids appearing in in this column appears. Annotations carried over from PDB are the following: site of the alignment (indicating existence of related site record in PDB ), S-S (disulfide 2. their type bond forming residue), hb (hydrogen bond forming residue, jb (james • rho ET score - the smaller this value, the lesser variability of bond forming residue), and sb (for salt bridge forming residue). this position across the branches of the tree (and, presumably, the greater the importance for the protein) 3.6 Mutation suggestions • cvg coverage - percentage of the residues on the structure which Mutation suggestions are completely heuristic and based on comple- have this rho or smaller mentarity with the substitutions found in the alignment. Note that • gaps percentage of gaps in this column they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following properties: small [AV GSTC], medium [LPNQDEMIK], large 4.2 Color schemes used [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- The following color scheme is used in figures with residues colored tively [KHR], or negatively [DE] charged, aromatic [WFYH], by cluster size: black is a single-residue cluster; clusters composed of long aliphatic chain [EKRQM], OH-group possession [SDETY ], more than one residue colored according to this hierarchy (ordered and NH2 group possession [NQRK]. The suggestions are listed by descending size): red, blue, yellow, green, purple, azure, tur- according to how different they appear to be from the original amino quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, acid, and they are grouped in round brackets if they appear equally bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, disruptive. From left to right, each bracketed group of amino acid DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, types resembles more strongly the original (i.e. is, presumably, less tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive) These suggestions are tentative - they might prove disrup- The colors used to distinguish the residues by the estimated tive to the fold rather than to the interaction. Many researcher will evolutionary pressure they experience can be seen in Fig. 5.

4 4.3 Credits is an open-source application copyrighted by DeLano Scien- 4.3.1 Alistat alistat reads a multiple sequence alignment from the tific LLC (2005). For more information about Pymol see file and shows a number of simple statistics about it. These stati- http://pymol.sourceforge.net/. (Note for Windows stics include the format, the number of sequences, the total number users: the attached package needs to be unzipped for Pymol to read of residues, the average and range of the sequence lengths, and the the scripts and launch the viewer.) alignment length (e.g. including gap characters). Also shown are 4.4 Note about ET Viewer some percent identities. A percent pairwise alignment identity is defi- Dan Morgan from the Lichtarge lab has developed a visualization ned as (idents / MIN(len1, len2)) where idents is the number of tool specifically for viewing trace results. If you are interested, please exact identities and len1, len2 are the unaligned lengths of the two visit: sequences. The ”average percent identity”, ”most related pair”, and http://mammoth.bcm.tmc.edu/traceview/ ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant The viewer is self-unpacking and self-installing. Input files to be used seq” is calculated by finding the maximum pairwise identity (best with ETV (extension .etvx) can be found in the attachment to the relative) for all N sequences, then finding the minimum of these N main report. numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, 4.5 Citing this work and freely distributed under the GNU General Public License. The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 4.3.2 CE To map ligand binding sites from different Evolution-Entropy Hybrid Methods for Ranking of Protein Residues source structures, report maker uses the CE program: by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://cl.sdsc.edu/ . Shindyalov IN, Bourne PE (1998) of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- ”Protein structure alignment by incremental combinatorial extension tionary Trace Method Defines Binding Surfaces Common to Protein (CE) of the optimal path . Protein Engineering 11(9) 739-747. Families” J. Mol. Bio. 257: 342-358. 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker itself is described in Mihalek I., I. Res and O. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type which is roughly the area needed for one water molecule to come in of service for comparative analysis of .” Bioinformatics the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 22:1656-7. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 4.6 About report maker by [email protected] November 18,2002, report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://www.cmbi.kun.nl/gv/dssp/descrip.html. king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.4 HSSP Whenever available, report maker uses HSSP ali- Houston. gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de 4.7 Attachments Daruvar, and C. Sander. ”The HSSP database of protein structure- The following files should accompany this report: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 1ydlA.complex.pdb - coordinates of 1ydlA with all of its inter- http://swift.cmbi.kun.nl/swift/hssp/ acting partners • 1ydlA.etvx - ET viewer input file for 1ydlA 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 1ydlA.cluster report.summary - Cluster report summary for Wesley,” Reading, Mass. (1986). 1ydlA • 1ydlA.ranks - Ranks file in sequence order for 1ydlA 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), • 1ydlA.clusters - Cluster descriptions for 1ydlA ”MUSCLE: multiple sequence alignment with high accuracy and • 1ydlA.msf - the multiple sequence alignment used for the chain high throughput.” Nucleic Acids Research 32(5), 1792-97. 1ydlA • http://www.drive5.com/muscle/ 1ydlA.descr - description of sequences used in 1ydlA msf • 1ydlA.ranks sorted - full listing of residues and their ranking for 4.3.7 Pymol The figures in this report were produced using 1ydlA Pymol. The scripts can be found in the attachment. Pymol

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