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2Kht Lichtarge Lab 2006

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2Kht Lichtarge Lab 2006 Pages 1–4 2kht Evolutionary trace report by report maker March 31, 2010 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 2kht): Title: Nmr structure of human alpha defensin hnp-1 Compound: Mol id: 1; molecule: neutrophil defensin 1; chain: a; fragment: residues 65-94; synonym: hnp-1, hp-1, hp1, defensin, alpha 1, hp 1-56, neutrophil defensin 2, hnp-2, hp-2, hp2; engineered: yes Organism, scientific name: Homo Sapiens; CONTENTS 2kht contains a single unique chain 2khtA (30 residues long). This is an NMR-determined structure – in this report the first model in the 1 Introduction 1 file was used. 2 Chain 2khtA 1 2.1 Q6EZF6 overview 1 2.2 Multiple sequence alignment for 2khtA 1 2.3 Residue ranking in 2khtA 1 2.4 Top ranking residues in 2khtA and their position on 2 CHAIN 2KHTA the structure 1 2.1 Q6EZF6 overview 2.4.1 Clustering of residues at 80% coverage. 1 From SwissProt, id Q6EZF6, 100% identical to 2khtA: Description: Novel protein, similar to DEFA1 (Defensin, alpha 1, 3 Notes on using trace results 2 myeloid-related sequence) (Defensin, alpha 1, preproprotein). 3.1 Coverage 2 Organism, scientific name: Homo sapiens (Human). 3.2 Known substitutions 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.3 Surface 2 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 3.4 Number of contacts 2 Catarrhini; Hominidae; Homo. 3.5 Annotation 3 3.6 Mutation suggestions 3 4 Appendix 3 4.1 File formats 3 2.2 Multiple sequence alignment for 2khtA 4.2 Color schemes used 3 For the chain 2khtA, the alignment 2khtA.msf (attached) with 5 4.3 Credits 3 sequences was used. The alignment was assembled through combi- 4.3.1 Alistat 3 nation of BLAST searching on the UniProt database and alignment 4.3.2 CE 3 using Muscle program. It can be found in the attachment to this 4.3.3 DSSP 3 report, under the name of 2khtA.msf. Its statistics, from the alistat 4.3.4 HSSP 3 program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 1-30 in 2khtA colored by their relative importance. (See Appendix, Fig.4, for the coloring scheme.) Format: MSF Number of sequences: 5 Total number of residues: 150 Smallest: 30 Largest: 30 Average length: 30.0 Alignment length: 30 Average identity: 88% Most related pair: 97% Most unrelated pair: 80% Most distant seq: 93% Furthermore, 80% of residues show as conserved in this alignment. The alignment consists of 80% eukaryotic ( 80% vertebrata) Fig. 2. Residues in 2khtA, colored by their relative importance. Clockwise: sequences. (Descriptions of some sequences were not readily availa- front, back, top and bottom views. ble.) The file containing the sequence descriptions can be found in the attachment, under the name 2khtA.descr. 2.3 Residue ranking in 2khtA The 2khtA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2khtA can be found in the file called 2khtA.ranks sorted in the attachment. 2.4 Top ranking residues in 2khtA and their position on the structure In the following we consider residues ranking among top 80% of resi- dues in the protein (the closest this analysis allows us to get to 25%). Figure 2 shows residues in 2khtA 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 80% coverage. Fig. 3 shows the top 80% 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. Fig. 3. Residues in 2khtA, colored according to the cluster they belong to: Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for cluster size member the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. color residues red 24 2,3,4,5,6,7,8,9,11,12,13,14 15,16,17,18,19,21,24,26,27 28,29,30 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Table 1. Clusters of top ranking residues in 2khtA. Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to some small top percentage of residues [100% is all of the residues 2 in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a COVERAGE couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. V 3.2 Known substitutions 100% 50% 30% 5% One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types 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 V an R at that position, it is advisable to try anything, but RVK. Conver- RELATIVE IMPORTANCE sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment Fig. 4. Coloring scheme used to color residues by their relative importance. is given in the immediately following bracket. No percentage is given 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 [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to 100%. tively [KHR], or negatively [DE] charged, aromatic [WFYH], long aliphatic chain [EKRQM], OH-group possession [SDETY ], 3.3 Surface and NH2 group possession [NQRK]. The suggestions are listed To detect candidates for novel functional interfaces, first we look for according to how different they appear to be from the original amino residues that are solvent accessible (according to DSSP program) by acid, and they are grouped in round brackets if they appear equally 2 at least 10A˚ , which is roughly the area needed for one water mole- disruptive. From left to right, each bracketed group of amino acid cule to come in the contact with the residue. Furthermore, we require types resembles more strongly the original (i.e. is, presumably, less that these residues form a “cluster” of residues which have neighbor disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4.1 File formats Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • ber of contacts heavy atoms of the residue in question make across alignment# number of the position in the alignment the interface, as well as how many of them are realized through the • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 3.5 Annotation 1. number of different amino acids appearing in in this column If the residue annotation is available (either from the pdb file or of the alignment from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • gaps percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of 3 more than one residue colored according to this hierarchy (ordered http://www.drive5.com/muscle/ by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.7 Pymol The figures in this report were produced using quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, Pymol.
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