Pages 1–7 1y4j Evolutionary trace report by report maker September 20, 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 Data Bank entry (PDB id 1y4j): Title: Crystal structure of the paralogue of the human formylglycine generating Compound: Mol id: 1; molecule: sulfatase modifying factor 2; chain: a, b; synonym: c-alpha-formyglycine- generating enzyme 2, unq1968/pro4500; engineered: yes Organism, scientific name: Homo Sapiens; CONTENTS 1y4j contains a single unique chain 1y4jB (268 residues long) and 1 Introduction 1 its homologue 1y4jA.

2 Chain 1y4jB 1 2.1 Q5RCR5 overview 1 2 CHAIN 1Y4JB 2.2 Multiple sequence alignment for 1y4jB 1 2.1 Q5RCR5 overview 2.3 Residue ranking in 1y4jB 1 2.4 Top ranking residues in 1y4jB and their position on From SwissProt, id Q5RCR5, 100% identical to 1y4jB: the structure 1 Description: Sulfatase modifying factor 2 precursor (C-alpha- 2.4.1 Clustering of residues at 25% coverage. 2 formyglycine- generating enzyme 2). 2.4.2 Overlap with known functional surfaces at Organism, scientific name: Pongo pygmaeus (Orangutan). 25% coverage. 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.4.3 Possible novel functional surfaces at 25% Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; coverage. 3 Catarrhini; Hominidae; Pongo. Function: Seems to lack formyl-glycine generating activity and to be 3 Notes on using trace results 5 unable to convert newly synthesized inactive sulfatases to their active 3.1 Coverage 5 form by modifying an residue to 3-oxoalanine. 3.2 Known substitutions 5 Overexpression of SUMF2 inhibits the activation of sulfatases by 3.3 Surface 6 SUMF1 (By similarity). 3.4 Number of contacts 6 Subcellular location: Endoplasmic reticulum lumenal protein (By 3.5 Annotation 6 similarity). 3.6 Mutation suggestions 6 Similarity: Belongs to the sulfatase modifying factor family. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 6 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 6 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 6 no restrictions on its use as long as its content is in no way modified 4.3 Credits 6 and this statement is not removed.

1 Lichtarge lab 2006 2.4 Top ranking residues in 1y4jB and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1y4jB 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 27-160 in 1y4jB colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 161-294 in 1y4jB colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

2.2 Multiple sequence alignment for 1y4jB For the chain 1y4jB, the alignment 1y4jB.msf (attached) with 739 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 1y4jB.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1y4jB, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 739 Total number of residues: 164649 Smallest: 102 Largest: 268 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Average length: 222.8 top 25% of all residues, this time colored according to clusters they Alignment length: 268 belong to. The clusters in Fig.4 are composed of the residues listed Average identity: 29% in Table 1. Most related pair: 99% Most unrelated pair: 4% Table 1. Most distant seq: 30% cluster size member color residues red 65 30,36,38,40,41,51,53,62,65 Furthermore, <1% of residues show as conserved in this ali- 68,69,70,71,74,77,143,144 gnment. 145,147,148,149,151,152,155 The alignment consists of 1% eukaryotic ( 1% vertebrata, <1% 156,158,160,162,163,164,165 arthropoda), 11% prokaryotic, and <1% archaean sequences. (Des- 166,167,168,169,170,171,172 criptions of some sequences were not readily available.) The file 173,174,175,176,223,226,227 containing the sequence descriptions can be found in the attachment, 229,230,232,233,234,235,236 under the name 1y4jB.descr. 237,238,240,253,254,255,256 270,274,287,288,289,290 2.3 Residue ranking in 1y4jB blue 2 184,185 The 1y4jB sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues Table 1. Clusters of top ranking residues in 1y4jB. in 1y4jB can be found in the file called 1y4jB.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) D(7) H(1) L(2) G(1)Y S(2)E Q(1) A(1)IKT R.VC 229 D D(72) 0.07 3/3 2.59 Q(10) N(8)S E(1)CA .(2) G(2)T H(1) 234 V V(82) 0.12 5/4 2.56 I(3) L(4)P A(6)C Fig. 4. Residues in 1y4jB, colored according to the cluster they belong to: T(1)EGS red, followed by blue and yellow are the largest clusters (see Appendix for .MF the coloring scheme). Clockwise: front, back, top and bottom views. The 255 A G(78) 0.13 1/1 4.11 corresponding Pymol script is attached. N(1) A(7) S(7)DEI 2.4.2 Overlap with known functional surfaces at 25% coverage. .LTCRPH The name of the ligand is composed of the source PDB identifier VKQ and the heteroatom name used in that file. 253 R R(69) 0.15 1/1 4.99 Calcium ion binding site. Table 2 lists the top 25% of residues K(20) at the interface with 1y4jCA1003 (calcium ion). The following table G(2)QTP (Table 3) suggests possible disruptive replacements for these residues DA.(1)C (see Section 3.6). SHEL Table 2. 235 W E(1) 0.16 2/2 4.76 res type subst’s cvg noc/ dist W(69) (%) bb (A˚ ) G(2) 169 W W(94)L 0.01 1/0 4.95 M(1) R(2)Y A(5) F(2)HV. Y(5) T D(1) 289 R R(97) 0.01 1/0 4.73 S(4)IH .(1)SHT R(2) N F(2) 232 G G(97)AI 0.03 4/4 2.68 L(1)NQP S.DHQ T.KC 236 E E(89) 0.03 5/0 2.77 230 L M(68) 0.17 4/4 2.53 Q(9)HTS T(4) VG.D L(12) 254 G G(94) 0.05 3/3 4.48 I(2) S(1)NT A(4)G .(1)EQA V(2) DKIRF .(2)QWC 233 N N(77) 0.06 3/2 4.86 FS continued in next column continued in next column

3 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ )

Table 2. The top 25% of residues in 1y4jB at the interface with calcium ion.(Field names: res: residue number in the PDB entry; type: 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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations 169 W (E)(K)(Q)(D) 289 R (D)(T)(E)(LPI) 232 G (R)(K)(E)(H) 236 E (FWH)(R)(Y)(VA) 254 G (R)(KE)(H)(FW) 233 N (Y)(H)(FW)(T) 229 D (R)(FWH)(KY)(VA) 234 V (R)(K)(Y)(E) Fig. 5. Residues in 1y4jB, at the interface with calcium ion, colored by their 255 A (Y)(R)(E)(K) relative importance. The ligand (calcium ion) is colored green. Atoms further 253 R (Y)(TD)(E)(FW) than 30A˚ away from the geometric center of the ligand, as well as on the line 235 W (KE)(T)(D)(Q) of sight to the ligand were removed. (See Appendix for the coloring scheme 230 L (R)(Y)(H)(K) for the protein chain 1y4jB.)

Table 3. List of disruptive mutations for the top 25% of residues in 1y4jB, that are at the interface with calcium ion.

Figure 5 shows residues in 1y4jB colored by their importance, at the interface with 1y4jCA1003. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1y4jB surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1y4j. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these Fig. 6. A possible active surface on the chain 1y4jB. The larger cluster it residues (see Section 3.6). belongs to is shown in blue. Table 4. res type substitutions(%) cvg Table 4. continued 163 L L(96)M(1)I.NVS 0.01 res type substitutions(%) cvg 169 W W(94)LR(2)YF(2) 0.01 .(3)S(1)ELDQITV HV.T Y 168 E E(86)Q(13)LV.T 0.04 158 W W(73)S(1)K(6) 0.08 170 E E(95)SRLQMY.ADG 0.04 R(11)A(1)F(1)IQ NPHI .GTDMCLVNY 166 E E(94)A(1)GDTSI. 0.05 162 R R(77)E(2)H(3) 0.08 NRMPWQ D(4)T(2)S(2)K 226 G G(85).(6)D(1) 0.06 Q(2)A(1)YV.FING N(1)HSA(1)ERMKC WC TV 36 G A(1)IG(87)TD(1) 0.10 41 G G(90)APR(1)N 0.07 P.(4)Q(1)KRNMES continued in next column HL continued in next column

4 Table 4. continued Table 4. continued res type substitutions(%) cvg res type substitutions(%) cvg 77 F F(64)V(11)WC(5) 0.10 A(5)QW(2)H(2)CM Y(5).(3)QI(6)LA TSI(1)NE. DS 184 W W(48)KF(14)T(3) 0.21 274 R R(87)TMGH.(1) 0.10 P(1).(14)D(1) S(1)Q(1)IPNY E(1)LA(1)Y(3)R L(1)FKAECVD S(1)G(5)INHV 155 Y Y(60)F(34)CHM 0.11 C(1)MQ W(2)Q.DGLEAV 270 R R(72)LA(1)E(1) 0.22 223 N S(12)N(65).(11) 0.12 K(2)PT(1)I.(5) G(2)T(2)QAEHPLI Q(3)G(1)H(1)D DYVRF S(2)Y(1)MCVNWF 185 G G(67)LKM.(13) 0.15 40 M L(8)AM(59)R(2) 0.23 S(2)A(4)D(4)H F(3)I(9)KWGV(4) P(2)E(1)NQTRIVW SY(1).(3)PQ(2)C 149 W PW(57)Q(3)Y(20) 0.16 DETNH L(3)F(6)H(1) 51 E E(70)IT(1)VN(2) 0.25 A(1)STRGKV.CEI GLSD(4)A(2)RY 235 W E(1)W(69)G(2) 0.16 .(7)K(2)P(2) M(1)A(5)Y(5) Q(1)FCH D(1)S(4)IHR(2) 154 A A(66)R(1)K(1) 0.25 F(2)L(1)NQPT.KC T(1)S(2)E(14) 165 T T(66)S(26)GLEF 0.17 Q(2)N(1)V(1) A(1)RKDN(1)V.YI D(2)YG(1)H.LFMI H 176 G G(63)D(6)K(1) 0.25 175 G G(44)A(36)YW(3) 0.17 R(2)Q(3).(2) T(2)S(3)D(1)LV V(1)MT(2)E(2) H(1)NQ(1)EMK P(2)N(3)A(2) .(1)FRI S(2)L(1)H(1)ICF 160 G G(50)S(23)N(4) 0.18 W T(6)D(2)A(2) E(1)P(1)K(1) Table 4. Residues forming surface ”patch” in 1y4jB. R(2)IH.CVYQL 174 R G(2)R(72)A(5)W 0.18 S(2)T(2)L(1)YVP Table 5. Q(2)K(4)H(1)DCN res type disruptive EM. mutations 38 F V(7)F(69)Y(7)T 0.19 163 L (Y)(R)(H)(T) A(2)PH(2)I(1) 169 W (E)(K)(Q)(D) .(3)W(1)QMS(1)G 168 E (H)(FW)(Y)(R) DLRC 170 E (H)(FW)(Y)(R) 240 S D(55)S(30)T(8)K 0.19 166 E (H)(FW)(Y)(R) N(2)IGAER.QV 226 G (R)(KE)(FWH)(YD) 53 P P(76)R(1)S(1) 0.20 41 G (R)(K)(E)(H) Q(1)T(2)VH.(6) 158 W (E)(K)(D)(T) A(2)NG(2)ME(1) 162 R (D)(T)(E)(Y) W(1)LKY 36 G (R)(E)(KH)(FW) 167 E A(74)PF(2)Q(1) 0.20 77 F (K)(E)(QR)(D) V(2)GYS(3)WT(1) 274 R (T)(D)(Y)(E) E(3)RD(2)NM(2) 155 Y (K)(Q)(R)(M) I(1)KL.H 223 N (Y)(H)(FTWR)(E) 171 F Y(52)R(2)L(3) 0.21 185 G (R)(E)(K)(H) F(9)K(9)V(5) 149 W (E)(K)(D)(Q) continued in next column 235 W (KE)(T)(D)(Q) continued in next column

5 Table 5. continued Note, however, that, if our picture of protein evolution is correct, res type disruptive the neighboring residues which are not surface accessible might be mutations equally important in maintaining the interaction specificity - they 165 T (R)(K)(H)(FQW) should not be automatically dropped from consideration when choo- 175 G (R)(KE)(H)(D) sing the set for mutagenesis. (Especially if they form a cluster with 160 G (R)(KE)(H)(FW) the surface residues.) 174 R (TD)(Y)(E)(CG) 38 F (K)(E)(Q)(D) 3.4 Number of contacts 240 S (R)(H)(FW)(K) Another column worth noting is denoted “noc/bb”; it tells the num- 53 P (Y)(R)(H)(T) ber of contacts heavy atoms of the residue in question make across 167 E (H)(FW)(YR)(CG) the interface, as well as how many of them are realized through the 171 F (E)(K)(TD)(Q) backbone atoms (if all or most contacts are through the backbone, 184 W (K)(E)(T)(D) mutation presumably won’t have strong impact). Two heavy atoms 270 R (T)(D)(Y)(E) are considered to be “in contact” if their centers are closer than 5A˚ . 40 M (Y)(H)(T)(R) 51 E (H)(FW)(R)(Y) 3.5 Annotation 154 A (Y)(R)(KE)(H) 176 G (R)(E)(K)(H) If the residue annotation is available (either from the pdb file or 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 1y4jB. (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 NOTES ON USING TRACE RESULTS 3.6 Mutation suggestions 3.1 Coverage Mutation suggestions are completely heuristic and based on comple- Trace results are commonly expressed in terms of coverage: the resi- mentarity with the substitutions found in the alignment. Note that due is important if its “coverage” is small - that is if it belongs to they are meant to be disruptive to the interaction of the protein some small top percentage of residues [100% is all of the residues with its ligand. The attempt is made to complement the following in a chain], according to trace. The ET results are presented in the properties: small [AV GSTC], medium [LPNQDEMIK], large form of a table, usually limited to top 25% percent of residues (or [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to some nearby percentage), sorted by the strength of the presumed tively [KHR], or negatively [DE] charged, aromatic [WFYH], evolutionary pressure. (I.e., the smaller the coverage, the stronger the long aliphatic chain [EKRQM], OH-group possession [SDETY ], pressure on the residue.) Starting from the top of that list, mutating a and NH2 group possession [NQRK]. The suggestions are listed couple of residues should affect the protein somehow, with the exact according to how different they appear to be from the original amino effects to be determined experimentally. acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid 3.2 Known substitutions types resembles more strongly the original (i.e. is, presumably, less One of the table columns is “substitutions” - other amino acid types disruptive) These suggestions are tentative - they might prove disrup- seen at the same position in the alignment. These amino acid types tive to the fold rather than to the interaction. Many researcher will may be interchangeable at that position in the protein, so if one wants choose, however, the straightforward alanine mutations, especially in to affect the protein by a point mutation, they should be avoided. For the beginning stages of their investigation. 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, 4 APPENDIX one may try replacing, R with K, or (perhaps more surprisingly), with 4.1 File formats V. The percentage of times the substitution appears in the alignment Files with extension “ranks sorted” are the actual trace results. The is given in the immediately following bracket. No percentage is given fields in the table in this file: 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 • alignment# number of the position in the alignment to 100%. • residue# residue number in the PDB file 3.3 Surface • type amino acid type To detect candidates for novel functional interfaces, first we look for • rank rank of the position according to older version of ET residues that are solvent accessible (according to DSSP program) by 2 • variability has two subfields: at least 10A˚ , which is roughly the area needed for one water mole- 1. number of different amino acids appearing in in this column cule to come in the contact with the residue. Furthermore, we require of the alignment that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. 2. their type

6 http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.3.3 DSSP In this work a residue is considered solvent accessi- COVERAGE 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 V the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 100% 50% 30% 5% Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, http://www.cmbi.kun.nl/gv/dssp/descrip.html.

4.3.4 HSSP Whenever available, report maker uses HSSP ali-

V gnment as a starting point for the analysis (sequences shorter than RELATIVE IMPORTANCE 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. Fig. 7. Coloring scheme used to color residues by their relative importance. http://swift.cmbi.kun.nl/swift/hssp/

4.3.5 LaTex The text for this report was processed using LATEX; • rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered 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. 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. 7. 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 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 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two 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. 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 .” Bioinformatics source structures, report maker uses the CE program: 22:1656-7.

7 4.6 About report maker • 1y4jB.ranks - Ranks file in sequence order for 1y4jB report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 1y4jB.clusters - Cluster descriptions for 1y4jB king visualization program was written by Ivica Res.ˇ report maker • 1y4jB.msf - the multiple sequence alignment used for the chain is copyrighted by Lichtarge Lab, Baylor College of Medicine, 1y4jB Houston. • 1y4jB.descr - description of sequences used in 1y4jB msf 4.7 Attachments • 1y4jB.ranks sorted - full listing of residues and their ranking for The following files should accompany this report: 1y4jB • • 1y4jB.complex.pdb - coordinates of 1y4jB with all of its inter- 1y4jB.1y4jCA1003.if.pml - Pymol script for Figure 5 acting partners • 1y4jB.cbcvg - used by other 1y4jB – related pymol scripts • 1y4jB.etvx - ET viewer input file for 1y4jB • 1y4jB.cluster report.summary - Cluster report summary for 1y4jB

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