Pages 1–4 1zkh Evolutionary trace report by report maker September 19, 2008

4.3.5 LaTex 4 4.3.6 Muscle 4 4.3.7 Pymol 4 4.4 Note about ET Viewer 4 4.5 Citing this work 4 4.6 About report maker 4 4.7 Attachments 4

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1zkh): Title: Solution structure of a human ubiquitin-like domain in sf3a1 Compound: Mol id: 1; molecule: splicing factor 3 subunit 1; chain: a; fragment: ubiquitin-like domain; synonym: spliceosome associated protein 114, sap 114, sf3a120, sf3a1; engineered: yes Organism, scientific name: Homo Sapiens; 1zkh contains a single unique chain 1zkhA (86 residues long). This is an NMR-determined structure – in this report the first model in the file was used.

2 CHAIN 1ZKHA 2.1 Q15459 overview CONTENTS From SwissProt, id Q15459, 100% identical to 1zkhA: Description: 1 Introduction 1 Splicing factor 3 subunit 1 (Spliceosome associated protein 114) (SAP 114) (SF3a120). 2 Chain 1zkhA 1 Organism, scientific name: Homo sapiens (Human). 2.1 Q15459 overview 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.2 Multiple sequence alignment for 1zkhA 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.3 Residue ranking in 1zkhA 1 Catarrhini; Hominidae; Homo. 2.4 Top ranking residues in 1zkhA and their position on Function: Subunit of the splicing factor SF3A required for ’A’ com- the structure 1 plex assembly formed by the stable binding of U2 snRNP to the 2.4.1 Clustering of residues at 44% coverage. 2 branchpoint sequence (BPS) in pre-mRNA. Sequence independent binding of SF3A/SF3B complex upstream of the branch site is essen- 3 Notes on using trace results 2 tial, it may anchor U2 snRNP to the pre-mRNA. May also be involved 3.1 Coverage 2 in the assembly of the ’E’ complex. 3.2 Known substitutions 2 Subunit: Component of splicing factor SF3A which is composed 3.3 Surface 2 of three subunits; SF3A3/SAP61, SF3A2/SAP62, SF3A1/SAP114. 3.4 Number of contacts 3 SF3A associates with the splicing factor SF3B and a 12S RNA unit to 3.5 Annotation 3 form the U2 small nuclear ribonucleoproteins complex (U2 snRNP). 3.6 Mutation suggestions 3 Interacts with SF3A3. Subcellular location: Nuclear (By similarity). 4 Appendix 3 Tissue specificity: Ubiquitously expressed. 4.1 File formats 3 Similarity: Contains 2 SURP motif repeats. 4.2 Color schemes used 3 Similarity: Contains 1 ubiquitin-like domain. 4.3 Credits 3 About: This Swiss-Prot entry is copyright. It is produced through a 4.3.1 Alistat 3 collaboration between the Swiss Institute of Bioinformatics and the 4.3.2 CE 4 EMBL outstation - the European Bioinformatics Institute. There are 4.3.3 DSSP 4 no restrictions on its use as long as its content is in no way modified 4.3.4 HSSP 4 and this statement is not removed.

1 Lichtarge lab 2006 Fig. 1. Residues 1-86 in 1zkhA colored by their relative importance. (See Appendix, Fig.4, for the coloring scheme.)

2.2 Multiple sequence alignment for 1zkhA For the chain 1zkhA, the alignment 1zkhA.msf (attached) with 3 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 report, under the name of 1zkhA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 3 Total number of residues: 257 Smallest: 85 Largest: 86 Fig. 2. Residues in 1zkhA, colored by their relative importance. Clockwise: Average length: 85.7 front, back, top and bottom views. Alignment length: 86 Average identity: 57% Most related pair: 62% Most unrelated pair: 53% Most distant seq: 54%

Furthermore, 44% of residues show as conserved in this alignment. The alignment consists of 66% eukaryotic ( 66% arthropoda) sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 1zkhA.descr. 2.3 Residue ranking in 1zkhA The 1zkhA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1zkhA can be found in the file called 1zkhA.ranks sorted in the attachment. 2.4 Top ranking residues in 1zkhA and their position on the structure In the following we consider residues ranking among top 44% of resi- dues in the protein (the closest this analysis allows us to get to 25%). Figure 2 shows residues in 1zkhA colored by their importance: bright red and yellow indicate more conserved/important residues (see Fig. 3. Residues in 1zkhA, colored according to the cluster they belong to: Appendix for the coloring scheme). A Pymol script for producing red, followed by blue and yellow are the largest clusters (see Appendix for this figure can be found in the attachment. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. 2.4.1 Clustering of residues at 44% coverage. Fig. 3 shows the top 44% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed Table 1. in Table 1. cluster size member color residues continued in next column

2 Table 1. continued backbone atoms (if all or most contacts are through the backbone, cluster size member mutation presumably won’t have strong impact). Two heavy atoms color residues are considered to be “in contact” if their centers are closer than 5A˚ . red 33 6,9,10,17,18,19,20,21,22,30 38,40,45,46,47,48,51,52,53 3.5 Annotation 56,58,60,62,63,65,68,70,71 If the residue annotation is available (either from the pdb file or 80,83,84,85,86 from other sources), another column, with the header “annotation” blue 4 1,27,74,75 appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 1. Clusters of top ranking residues in 1zkhA. 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 they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GST C], medium [LP NQDEMIK], large some small top percentage of residues [100% is all of the residues [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [W F Y H], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDET Y ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. 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 4 APPENDIX 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- 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 amino acid type to 100%. • 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 2 at least 10A˚ , 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 ET score - the smaller this value, the lesser variability of that these residues form a “cluster” of residues which have neighbor this position across the branches of the tree (and, presumably, within 5A˚ from any of their heavy atoms. the greater the importance for the protein) Note, however, that, if our picture of protein evolution is correct, • 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 4.2 Color schemes used the surface residues.) 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,

3 75% of the query are taken out, however); R. Schneider, A. de 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; V Leslie Lamport, “LaTeX: A Document Preparation System Addison- 100% 50% 30% 5% Wesley,” Reading, Mass. (1986).

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. V http://www.drive5.com/muscle/ RELATIVE IMPORTANCE 4.3.7 Pymol The figures in this report were produced using Fig. 4. Coloring scheme used to color residues by their relative importance. Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- tific LLC (2005). For more information about Pymol see bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, http://pymol.sourceforge.net/. (Note for Windows DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, users: the attached package needs to be unzipped for Pymol to read tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. the scripts and launch the viewer.) 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. 4. Dan Morgan from the Lichtarge lab has developed a visualization 4.3 Credits tool specifically for viewing trace results. If you are interested, please 4.3.1 Alistat alistat reads a multiple sequence alignment from the visit: 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 of residues, the average and range of the sequence lengths, and the The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two 4.5 Citing this work sequences. The ”average percent identity”, ”most related pair”, and The method used to rank residues and make predictions in this report ”most unrelated pair” of the alignment are the average, maximum, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant Evolution-Entropy Hybrid Methods for Ranking of Protein Residues seq” is calculated by finding the maximum pairwise identity (best by Importance” J. Mol. Bio. 336: 1265-82. For the original version relative) for all N sequences, then finding the minimum of these N of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- numbers (hence, the most outlying sequence). alistat is copyrighted tionary Trace Method Defines Binding Surfaces Common to Protein by HHMI/Washington University School of Medicine, 1992-2001, Families” J. Mol. Bio. 257: 342-358. and freely distributed under the GNU General Public License. report maker itself is described in Mihalek I., I. Res and O. 4.3.2 CE To map ligand binding sites from different Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type source structures, report maker uses the CE program: of service for comparative analysis of proteins.” Bioinformatics http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 22:1656-7. ”Protein structure alignment by incremental combinatorial extension 4.6 About report maker (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker 2 king visualization program was written by Ivica Res.ˇ ble if the DSSP program finds it exposed to water by at least 10A˚ , is copyrighted by Lichtarge Lab, Baylor College of Medicine, which is roughly the area needed for one water molecule to come in Houston. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 4.7 Attachments by [email protected] November 18,2002, The following files should accompany this report: http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1zkhA.complex.pdb - coordinates of 1zkhA with all of its 4.3.4 HSSP Whenever available, report maker uses HSSP ali- interacting partners gnment as a starting point for the analysis (sequences shorter than • 1zkhA.etvx - ET viewer input file for 1zkhA

4 • 1zkhA.cluster report.summary - Cluster report summary for • 1zkhA.descr - description of sequences used in 1zkhA msf 1zkhA • 1zkhA.ranks sorted - full listing of residues and their ranking • 1zkhA.ranks - Ranks file in sequence order for 1zkhA for 1zkhA • 1zkhA.clusters - Cluster descriptions for 1zkhA • 1zkhA.msf - the multiple sequence alignment used for the chain 1zkhA

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