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FR3D User`s manual

1. To specify that all candidate motifs must have a tVVH basepair between the nucleotides corresponding to the first and second nucleotides in the query motif type VA in the first row second column This means that the nucleotide in the first row must use its vatson Crick edge and the nucleotide in the second column must use its Hoogsteen edge Valid basepair specifications are CAME CAAM cWH cH CAH TAS cio com OAS tov CHA tHH cHS csH ths t5H c55 t55 Note however that the cSS and t55 Interactions are not in fact symmetric because each base can use the sugar edge differently Folhwing Leontis Stombaugh Westhof MAR 2002 type cSs to specify that the first base has priority cso forthe second or cSo for either specitying multiple interactions allows more ways a candidate can satisfy the constraints for example typing ciH cH requires a cis VWatson Crick Hoogsteen basepair but either base can use the WYvatson Crick edge and the other uses the Hoogsteen edge The abbreviation trans gives all trans categories cis for cis Type bif for bifurcated basepairs see L amp W 2002 Type cVV to exclude candidates having a cVVV basepair some pairs of bases are close to say CAWAN but do not meet the strict criteria for membership in the GAA classification Type acy near cvV to get basepairs that are not classified into any category but for which the CAAA category Is the closest match up to a certain fairly gen
2. ger Query PDB 1872 LH M View query la7y Query NTs 2701 2702 2703 2691 2693 2694 l Read Query Generate Interaction Matrix ides Query Chains o MW Po Ar Ar A A 1696 Configuration anti fag anti age anti pga anti fg anti eget anti pga 1qa6 G2701 A2702 A2703 A2691 U2693 A2594 tacu mask BRR e O 1 Tlm tan Distanco E a E O eee seno DD a tau LL JE dp tava sd J J JL JIE irae JL jt 1si2 Search name l Sarcin ricin Motif l Search 4szi Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Display Candidates Guaranteed Cutor 0 5 Relaxed Cutor 1 0 Exclude Overlaps List Candi 14 candidates found 2 When the user is Displaying the Candidates they can press the List Candidates button located on the menu MENU ea eA Figure 1 pispiayoptons File Edit View Insert Tools Desktop Window Help Dn d amp RACH e 08 20 fae MO 1 2 dd FOS 8269 12695 42694 a72 Next candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to sc Qa Write to PDB Sort by centrality Group Candidates Show Alignment Quit display 5 Plot 1 of 14 Discrepancy 1 1213e 15 The output is displayed in the Matlab command window or with the PC executable in two pop up windows The first columns of the output look like this Query Sarcin 5 nucleotide geometric Sarcin
3. Configuration anti f anti kH anti FSA anti Sl anti BSM anti ESA NTI NIZ N13 NI4 NIS NIG Mask NT Distance NIZ Interaction NIg NI4 NI5 NIG Search name Sarcin ricin Motif Search description Guaranteed Cutott 0 5 Relaxed Cutoft 0 5 Exclude Overlaps ca 21 Load previous results 2006 07 12_10_55_17 44 mat KS Generate Interaction Matrix Search Ready to search 7 The user should enter a name the type of search that they will be performing in the text box labeled Search name 1 e Sarcin ricin Motif This will be the name used to recall a previous search so the name should not use or N because these refer to folders The user can add more descriptive information about their search in the text box labeled Search description 1 e Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortui Other comments about the search can be added here as well TT FR3D GUI Load previous results 2006 07 12 10 55 17 44 mat ES SEE RENN Geometric symbolic search f Pure symbolic search 1034 0 1a9n Number of NTs 6 Generate Interaction Matrix nas Configuration anti H anti HH anti HHJ anti HH anti H anti ben 2 3 5 tav6 NTI NI 1b36 1b7f Mask 1br3 Distance bei Interaction 1dul can Search name Sarcin ri E Search 1e7k Search description Symbolic Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula Guaranteed Cutoft 0 5 Relaxed Cutoft 0 5 Exclud
4. Often the slowest part of the search is loading PDB data If a PDB file has not already been analyzed by FR3D it will need to be analyzed which is rather slow Even loading pre computed data may be slow The length of the search itself will vary depending on the number of nucleotides in the Query motif and the guaranteed discrepancy cutoff 80 FR3D_GUI Load previous results Geometric symbolic search Pure symbolic search ee eee z 2006 07 12 10 55 17 44 mat E 1981 Query PDB 1872 ES v View query 1q86 Query NTs 2701 2702 2703 2691 2693 2694 Read Query 1q96 a a a a ieee Query Chains 0 he 0 he 0 HA 0 HH 0 HH 0 he igin Mask tgru Distance Interaction 1sz1 Search name Sarcin ricin Motif 100d 1t0e Search description Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Guaranteed Cutoff 0 5 Relaxed Cutoff 1 0 Exclude Overlaps Fd Generate Interaction Matrix sca GE itty v gt Searching Please wait press Ctrl C to interrupt 14 Once the search is complete the total number of Candidates found will be displayed in the bottom right corner of the GUI e g 400 Candidates found To learn about Displaying or Listing Candidates refer to those sections within this manual 10 FR3D GUI De RO taco 2701 2702 2703 2691 2693 2694 po Mo RM Oo Ao jo CO O O O 11 2006 07 12_10_55_17 44 mat TS Performing Mixed Geometric and Symbolic searches wi
5. uit display The Display Candidates menu has been updated as of April 2009 Most features work the same as described above but there are some new features which are explained below Graphical display of discrepancies between candidates Figure 99 29 66 When the user presses the buttons Sort by centrality Order by similarity or Navigate using Fig 99 FR3D calculates the geometric discrepancies between the candidates and displays them in Figure 99 The candidates are listed in the same order on the vertical and horizontal axes The discrepancy between a candidate and itself is zero so the discrepancies down the diagonal are zero which are displayed as red boxes All pairs of candidates are considered and displayed 40 gt menu ix Display options Next candidate Previous Candidate Figure 99 Navigation window click here then click the Navigate button a ol File Edit View Insert Tools Desktop Window Help E 0 Esg S Elia n Discrepancies between all candidates ordered by by discrepancy from query Add plot Larger Neighborhood Toggle sugar Toggle display MarkUnrmark current Reverse all marks List to screen Write to PDB Sort by centrality Order by Similarity Show Alignment Show Scatterplot Navigate with Fig 99 __ Previous Conte At _bergerNeitoohood o Tee ower o Tedy Mercer cuert _Feverse amas __tsttoscreen topos _sotby
6. Cutoff 0 5 Relaxed Cutoff 1 0 Exclude Overlaps Ha ema I Ready to Search 17 Perform the Search by pressing the Search Button Information about the progress of the search is displayed in the bottom right corner of the GUI 23 ear FR3D_GUI Load previous results Structures to search Geometric symbolic search Pure symbolic search eee ger Query PDB 1872 _ Vi View query 1a7y Query NTS 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix ids Query Chains 0 Hid 0 H 0 HH 0 HH 0 HH 0 He 1696 Configuration anti SM anti BSM anti BSM anti BSM anti BSM anti ge 1906 G2701 A2702 A2703 A2691 U2693 A2594 ce e e se a SS SS ESS taco Mask G2701 R his Distance aro oo ns qea ee 1 EE ee RR RN qa ies si Lake O ie L__ J J J Jig h a a 1ser Search name Sarcin ricin Motif soo 1sz1 Search description Sarcinricin sub motif comprised of 6 NTs from 23S Halarcula marismortul x Guaranteed Cutott 0 5 Relaxed Cutott 1 0 Exclude Overlaps HH Searching Please wait press Ctrl C to interrupt 18 Once the search is complete the total number of Candidate will be displayed in the bottom right corner of the GUI i e 400 Candidates found To learn about Displaying or Listing Candidates refer to those sections within this manual ear FR3D_GUI Load previous results 3 Geometric symbolic search Pure symbolic search Seuctw
7. cetaty __Orcer by smarty __ Stow Agent __ Stew Seaterpt _Nevigete win Fi 98 Sady Quit display Initially FR3D only calculates discrepancies between the first 300 candidates even this takes time To increase the limit go to the first candidate press Previous Candidate then Next Candidate Each time you do this it doubles the number of candidates whose discrepancies will be calculated Be patient as FR3D calculates more discrepancies The candidate currently being displayed is indicated with a white star on the diagonal If multiple plot windows are open to show candidates the other candidates being displayed are indicated by other white symbols If some candidates have been marked this is shown with white dots on the diagonal Navigating with Figure 99 To jump to a candidate of interest click on the diagonal in Figure 99 in the row and column corresponding to the candidate then click Navigate with Fig 99 in the menu FR3D will jump to the specified candidate To mark a group of candidates that are contiguous in Figure 99 click off the diagonal at the upper right or lower left corner of the square containing the discrepancies between the candidates you want to mark then click Navigate with Fig 99 in the menu FR3D will mark the candidates you indicated Sorting by Centrality 4 Pressing the Sort by Centrality button on the menu will re order the candidates The candidate whose average discrepancy
8. marismortul 1e80 Guaranteed Cutott ex Relaxed Cutoff 0 5 Exclude Overlaps 1ec6 v Ready to Search 10 The user must specify the Relaxed Cutoff discrepancy using the text box labeled Relaxed Cutoff e g 1 0 This number must be equal to or greater than the Guaranteed Cutoff Making the relaxed cutoff larger than the guaranteed cutoff will retain some candidates which are similar to the Query motif without greatly increasing the running time The algorithm is not guaranteed to find all candidates whose discrepancy from the Query motif is between the guaranteed cutoff and the relaxed cutoff ear FR3D_GUI Load previous results Geometric symbolic search Pure symbolic search Structures to search y y 2006 07 12 10 55 17 44 mat Hef 157d a 165d Query PDB 2h M View query a ee 0 Query NTs 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix 1aq3 a a tag Query Chains 0 HA 9 HH 0 FH 0 hey 0 HH 0 bed tas tam Configuration HA HHA FH Hid HH pid 1aud ma G2701 A2702 A2703 A2691 U2693 A2694 1b36 E j ib7t Mask G2701 N 1bmv ibra Distance ago iby4 1 tebe moracton et E 1c9s icsl E icw 1cx tet aces O j f rag Search name Sarcin ricin Motit Susa 1e7k Search description Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul 1e80 Guaranteed Cutott 0 5 Relaxed Cutoff q Exclude Overlaps Ready to Search 11 Using
9. of the GUI 1 e 400 Candidates found To learn about Displaying or Listing Candidates refer to those sections within this manual 009 FR3D GUI Geometric symbolic sareh Pwre symbolic search Number of NTS HE MEL Generate Interaction Matrix ant BSM ant BSM ani PSA ani BSM ant SM anti ES o EEE Viewing candidates After performing a Geometric Symbolic or mixed search the user can view the candidate motifs by pressing the Display Candidates button in the bottom right corner of the GUI The green arrows will direct the user to places in the figures where changes may have occurred while the red arrows direct the attention to user actions 30 ear FR3D_GUI gt Load previous results 2006 07 12_10_55_17 44 mat LH pwf 1q2r Query PDB 1872 H V View query Structures to search fe Geometric symbolic search 2 Pure symbolic search bp Query NTs 2701 2702 2703 2691 2693 2694 l Read Query E iri ides Query Chains o MW Po Ar Ar A A 1998 Configuration anti HH anti HH anti a anti bid anti HH anti Hid tn o 2701 A2702 A2703 A2691 U26593 A2694 ice mask lc lt i Tlm iam Distance woz At E J O E O Tau LJ gt el fee RU t o O O RR gt 1si2 search name l Sarcin ricin Motif l Search 4sz1 Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Display Ca Guaranteed Cutor 0 5 Relaxed Cutor 1 0 Exclude Overlaps gt Li
10. shortens the running time of the search algorithm ean FR3D_GUI O To GEE ps 2761 2702 2703 2001 2063 2006 __ReadQuery Generate interact HHK po a a S223 8RRRSeRT SERS ERE RES 7 The user can specify the glycosidic bond conformation anti or syn for each base in their search using the drop down menu labeled Configuration The order of the drop down menus corresponds to the same order as the Query nucleotides To allow both conformations leave the selection s blank 14 Distance Interaction FR3D GUI fe Geometric symbolic search Query PDB 1872 ref Query NTs 2701 2702 2703 2691 2693 2694 Query Chains o Wajo Walo o o o RH Configuration Pure symbolic search Mask Search name Search description Guaranteed Cutott 0 5 Relaxed Cutoff 0 5 Load previous results 2006 07 12 10 55 17 44 mat H v View query Read Query Generate Interaction Matrix Search Exclude Overlaps HA Ready to Search 8 The user can impose a basepair identify constraint nucleotide mask for their search by putting in nucleotide constraints in the text boxes on the diagonal in the Interaction Matrix which has a white background Typing A for instance means that only candidate motifs with an A in the corresponding position will be kept Typing AG allows either A or G etc The program uses these standard abbreviations for other combinations M fo
11. the drop down menu to the right of the Relaxed Cutoff text box the user can specify whether to Exclude Overlaps or Include Overlaps An example of this is when performing a search using nucleotides 10 11 12 13 14 15 from some PDB file The algorithm will certainly return the Query motif but 1t may also return slight variations of the same motif such as nucleotides 9 11 12 13 14 15 These are referring to the same motif just one nucleotide 1s different and so we consider this an overlap or redundant version of the motif The option Include Overlaps would keep this candidate while Exclude Overlaps will remove candidates which have more than half of their nucleotides in common with another candidate having lower discrepancy from the Query motif In this search we are Excluding Overlaps eer FR3D_GUI fe Geometric symboli h Ss mboli h ae noone ere e s c searc ure s c searc Structures to search y y l 2006 07 12 10 55 17 44 mat 157d 165d Query PDB 1872 E V View query a E He 0 Query NTs 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix tag br Query Chains o jo Ralo Mao 1082 Configuration he hed HH 1av6 G2701 A2702 A2703 b36 1b7f Mask G2701 N 1bmv ie Distance aroe M iby4 o toy Interaction A2703 i tesi T HM icw tcx E aoe O idaf 1dah idrz iduh 1dul rn Search name Sarcin ricin Motif Search 1e6t 167k Search description Geometric Sarcinricin sub motif compris
12. 3 2694 To Rao EMO Ballo aro aro M Cam pe anv pe am pep ant ped ant jaf am 54 1 1 1 1 1 1 1 832888 For more information on using the sequence constraints the user can press the Distance buttons located to the left of the Interaction Matrix eeo FR3D GUI lo 6 GEE 2707 2702 2703 2691 2693 2694 DR OR OR TIO TOR TIO Cam pe an pe am bef ant peq ant a4 am pe This is the pop up menu which is displayed after the user presses the Distance button 20 lox Set limits on the diference between nucleotide numbers using the boxes below the diagonal Actually what is used is the difference between the index of nucleotides in the file not NDE nucleotide number To put an upper limit on the difference type something like lt 5 or lt 5 To put a lower limit on the diference type something like gt 5 or 5 To put both limits at once type something like gt 5 lt 12 To insist that the nucleotide in the given row have a lower nucleotide number than the nucleotide in the given column type separated by a space fram other specifications For greater type gt 12 The user should enter a name the type of search that they will be performing in the text box labeled Search name 1 e Sarcin ricin Motif This will be the name used to recall a previous search so the name should not use or V because these refer to folders The user can add more descriptive information about their search in
13. 3 Ranges may be increasing or decreasing The chain may be indicated with the syntax 2701 0 or 2701_0 or it may be specified later see below FR3D_GUI 2006 07 12 10 55 17 44 mat 2701 2702 2703 2001 q 4 The user tells FR3D to read the crystal structure information about the Query motif by pressing the button labeled Read Query If the user would like to view the motif they inputted they may check the check box labeled View query and then press the Read Query button and new figure will pop up to show the user the Query motif The interactions present will be displayed in the console window this will be explained in more detail below 809 mara 1055 1744mat OOOO O O Read Query 1b36 1b7f 1bmv ibys cbs 1 1c0a 1c9s 1cco ics ion The figure below shows the Query motif which is displayed by checking the Read query checkbox It may be rotated in the figure window 8ece Figure 3 File Edit View Insert Tools Desktop Window Help Shs k Aare e E so 5 Some PDB files have multiple RNA chains For instance 1s72 pdb contains a 5S chain and a 23S chain and both chains have some of the same nucleotide numbers If there is any ambiguity in the chain for the Query nucleotides the user must specify the chain which contains the Query motif This may be selected using the drop down menu labeled Query Chains The order of the drop down menus corresponds to the order of the nucleotides supplied by the us
14. 3D Structures To appear in the Journal of Mathematical Biology 46
15. 8 10 Sarcin ricin_ Motif mat ios 2007 01 17 12 00 31 Sarcin ricin_Motif mat 1521 Contguraton anti anti Es anti s anti anti anti s 2007 01 17_12_03_18 Sarcin ricin_Motif mat iz Ke be Fid he H hee 2007 01 18 11 05 09 Sarcin ricin_Motif mat 0074 R 09 40 Sarcin icin_ Moti 1t0d G2701 A2702 A2703 A2691 U2693 A2694 5 Ex E oe E 2007 01 18 11 d2 53 Sarein nen Motf mat 110k 2007 01 23_10_37_56 K_tum_1_23_07 mat 2007 01 23_10_47_13 K_tum_1_23_07 mat 4 2007 01 23 10 51 52 K tum 1 23 07 mat 4u0b Interaction N a 2007 01 23 10 53 38 K tum 1 23 07 mat 0 2007 01 24 10 21 59 K tum 1 23 07 mat yy N Do a 2007 01 24 14 54 04 K tum_1_23_07 mat 2007 01 24 14 55 39 K tum 1 23 07 mat Tull 1un6 tum oo O i ee eee RR tutd lt iwmq Search name Sarcin ricin Motif 1wvd Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Halarcula marismortul Display Candidates 1xbp es Guaranteed Cutott 0 5 Relaxed Cutott 1 Exciude Overiaps Hd List Candidates Loaded saved search results You can examine these results using Display Candidates or List Candidates or you can repeat the analysis by starting trom Head Query User maintained lists of PDB files To facilitate searching a subset of the entire collection of PDB files in the Matlab search path the user may maintain lists of PDB files To do so create a text file with a name ending with _list pdb For example the file Nonredundant_list pdb has thes
16. 81369 Ul37l AlS 2 G2053 A2054 A2055 AGUA GAA 1a72 0 1046 Al369 Ul371 AlS 2 G2053 A2054 A2055 AGUA GAA 1a72 0 1451 A 380 U 382 A 383 G 406 A 407 A 408 AGUA GAA lqvf 0 1518 A 380 U 382 A 383 G 406 A 407 A 408 AGUA GAA ls 72 0 1742 A 77 U 79 A 80 G 102 A 103 A 104 AGUA GAA 1a72 0 1790 A 212 U 214 A 215 G 225 A 226 A 227 AGUA GAA lqvf 0 1804 A3077 U3079 A3080 G amp 3102 A3103 A3104 AGUA GAA 1a72 0 1841 A 174 U 176 A 177 G 159 A 160 A 161 AGUA GAA lavf 0 1861 A 212 U 214 A 215 G 225 A 226 A 227 AGUA GAA lavf 0 1872 A 463 U 465 A 466 G 475 A 476 A ATi AGUA GAA lavt 0 1875 A 174 U 176 A 177 G 159 A 160 A 16l AGUA GAR ls 72 0 1937 A 463 U 465 A 466 G 475 A 476 A 477 AGUA GAA Retrieving the results of previous searches To retrieve previous search results the user can select one of their previous searches using the Load previous search drop down menu located at the top right corner of the GUI 44 ear FR3D GUI Load previous results Geometric symbolic search Pure symbolic search em om Sy 2007 01 18_11_09_40 Sarcin ricin_Motif mat e WR 2006 12 21 13 11 36 Sarein rein Motf mat Al 1576 Query PUB 1872 2006 12 27 14 57 _43 Sarcin ricin_ Motif mat 1877 2007 01 12 21 55 40 mat pas Query NIs 2701 2702 2703 2691 2693 2694 2007 01 17_11_29 57 Sarcin ricin_ Motif mat i 2007 01 17 11 51 18 Sarcin rcin Motif mat Query Chains o Elo Mo io io io 2007 01 17_11_5
17. DB files which you would like to search in for your particular motif 1 e Iqrs Iqvf Irc7 1s72 On a PC by holding down the Control key on your keyboard one can select multiple files which are not consecutive in the list menu To do this on a Mac the user must hold down the Command or Open Apple key If the user would like to select consecutive file in the list menu they should hold down the Shift Key PC Mac and select their files A A Structures to search FR3D GUI Geometric symbolic search Number of NTs 6 f Pure symbolic search Load previous results 2006 07 12 10 55 17 44 mat ES Generate Interaction Matrix Configuration anti f anti HH anti SA anti BSM anti BSM anti BSM NI NIZ NT3 NI4 NI5 NIG Mask l l tSH Distance Ji A thiw Interaction a tHH Em Ea Ea U Es SC Search name Sarcin ricin Motif Search Search description Symbolic Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Guaranteed Cutoff 0 5 Relaxed Cutoff 1 0 Exclude Overlaps he Ready to search 11 Perform the Search by pressing the Search Button Information about the progress of the search is displayed in the bottom right corner of the GUI 29 ee0 FR3D GUI je cz EE ICE CE CE CE Symbolic Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul os o 12 Once the search is complete the total number of Candidate will be displayed in the bottom right corner
18. FR3D User s Manual Table of Contents Tables or Contents asas e Taio Rian bi fi TA l ESEE LEETE a AES E EE E E A E E E A E A E NR TO 2 Runnin FR SD Om M Wla Dee RS O ien 2 Using the compiled version or FR3D Tor the PC aicsiecesinieniinee ai E adia louis 2 Step By Step Tutorial on Performing Motif Searches using FR3D sssssnessssssssseeressssssssceerssssssssseeressssssseeees 3 Performing Purely Geometric Search with FRSD asas eds a a a 3 Performing Mixed Geometric and Symbolic searches with FR3D sssssssssenssssssssscerrssssssssserresssssssees 12 Conducting Symbolic searches with PRSD isso ri ias Elas DIRETORES UNE na ala 24 Viewing CANGIGALES rails ra O E utawneen O ae 30 LAS CC andidAtes quase autres aguas A again qr a QU req E A E Sl E 36 Waung Candidates toa PDB Piles ssrepicascerrase dana eee Dinha Diga s told ni DEL aaa ERR ada a ART aa aa ea 38 New Display Candidates Menu as of April 2009 cc e eeeeeerrrreeer aeee aaa rrrreeeea aaa aa a eerreaanda 40 Graphical display of discrepancies between candidates Figure 99 00 cccccccccccccsssseseeececeeceeaeeeeeeeeeeeeeeeaas 40 INAV le tino With PIS URS DO n E ss taseacecesncnn ao ai a pn Innetedieieaetouzed 4 SONNE CUM AE yea cera siete nae arse a O tian ate dupe E E eden nega 41 Order DV Sia ATE rp E a a RP ST eR 42 Ao nino candidate S aso A E tele daly cuscaentied ua eeadianw temenstale iene 43 Retrievine the results of previous SCALCICG css ssnteaciicknonsdetaanat
19. Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortui Other comments about the search can be added here as well 00 FR3D_GUI gee 2707 2702 2703 2691 2693 2694 o OR CC O VC A CRM EM BM BM MM Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula m EC 9 The user sets the Guaranteed Cutoff discrepancy in the text box labeled Guaranteed Cutoff 1 e 0 5 The search algorithm is guaranteed to find all candidates whose geometric discrepancy with the Query motif is less than this number The discrepancy is roughly comparable to RMS discrepancy Increasing the value of the guaranteed cutoff will rapidly increase the running time of the program Values above 1 0 are often impractical ear FR3D_GUI Load previous results e Geometric symbolic search Pure symbolic search nincakan nica id 2006 07 12 10 55 17 44 mat Hj N 165d Query PDB View que 1a34 0 sro 4 Tapa Query NTS 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix 1aq3 a a bow Query Chains o mao mio Ao o Ralo RW lasy a tasz Configuration He H 4 HH be bed a G2701 A2702 A2691 U26943 A2594 1b36 E E J 1bmv o 1br3 Distance A2702 E 4 E EE 1bys E morcton aero O E EE 1c9s ics icw 1cx tat aee Lee Search name Sarcin ricin Motif cove 107k Search description Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula
20. H 569 00000 cSH ntSH s33 ncSH 635 s33 tWH tH 1s72 0 1784 175 U 176 A A A 160 00000 CSH ntSH s33 ncSH s35 33 tWH tH 1s72 0 1844 464 U 465 A 466 A A A G G 476 00000 CSH ntSH s33 ncSH 535 s33 tWH tH 1s72 0 1976 G 358 U 359 293 00000 cSH ntSH s33 ncSH s35 s33 tWH tH 1s72 0 2284 G 213 U 214 226 00000 cSH ntSH s33 ncSH s35 s33 tWH tH 1s72 0 2374 G U 103 99999 cSH ntSH s33 ncSH s35 s33 tWH tH 1572 0 2391 G 1971 U 1972 A 1973 G 2009 A 2010 00000 cSH ntSH s33 ncSH s35 s33 tWH tH 1s72 0 2491 G 1292 U 1293 A 1294 G 911 A 912 00000 cSH ntSH s33 ncSH s35 s33 tWH tH 1s72 0 2714 G 953 U 954 A 955 A 1012 A 1013 00000 cSH ncSH s33 ncSH s35 ncHW tWH tH 1s72 0 4395 G 1543 U 1544 C 1545 C 1640 A 1641 00000 ncSH 1572 0 4644 G 706 C 707 A 708 G 720 A 721 00000 ns35 ntSH s33 ntSH s35 s33 tWH tH PPP Pp pS DNNNNNNHNHNNNNnNNNSEA n ol uo Q Nn am NNNNNNNNNNNNNNW jon on on ol an N on on an oo an N The columns following the Chain column indicate the basepairing or base stacking interactions between the nucleotides noted at the top of the column For instance in each of the candidates nucleotides 2 and 5 form a tWH trans Watson Crick Hoogsteen basepair The column headed Configuration indicates the configuration of each base whether anti A or syn S The final columns indicate the differences in nucleotide numbers between the indicated nucleotides This makes it easy to spot local v
21. MENU 10 Figure 1 bspayoptons File Edit View Insert Tools Desktop Window Help OSHS RQ E DE Next candidate Previous C a nr Add plot fad PO 1 42 702 2703 4269 12695 4226 9412690 27004 Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverso all marks Display marked only List to screen Write to PDB Sort by centrality Group Candidates show Alignment 10 Plot of 14 Discrepancy 1 12 Quit display If the user would like to view more than one Candidate a time they can add more figures by pressing the Add plot button on the menu When selecting a particular figure the user can press Next Candidate or Previous Candidate and the selected figure will change 80 MENU Bene Figure 1 DAE Figure 2 Display optons File Edit View Insert Tools Desktop Window Help File Edit View Insert Tools Desktop Window Help Nextcandidate C OSHS h RAS E DES a DgHs 429 E 080 G2701 42702 42703 A269 14U2693 42694 1372 G2701 A2702 42703 4269 12693 42694 1572 Previous Candidate Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to screen Write to PDB Sort by centrality Group Candidates Show Alignment 8 Plot 1 of 14 Discrepancy 1 1213e 15 8 Plot 1 of 14 Discrepancy 1 1213e 15 Quit display If the user would like to look at the surrounding bases of one of the Candidate motifs they can press the Larger Neighborhood bu
22. Z N13 NI4 NIS NIG Mask N11 Distance NTZ Interaction NIg NI4 NI5 NIG Search name Sarcin ricin Motif Search description Guaranteed Cutott 0 5 Relaxed Cutoff 0 5 Exclude Overlaps he Load previous results 2006 07 12 10 55 17 44 mat KS Generate Interaction Matrix Search Ready to Search Base phosphate interactions Specify that nucleotides 1 and j should make a base phosphate interaction by putting the text BPh in row 1 and column jJ This requires that the nucleotide in row 1 be a hydrogen donor and that the phosphate of the nucleotide in column j be an acceptor in a hydrogen bond between the two nucleotides Specify a particular base phosphate interaction with text such as 1BPh 2BPh 9BPh OBPh To reverse the roles of 1 and J use PhB 1PhB etc Near base phosphate interactions can be obtained with nBPh nl BPh n3PhB etc 6 The user can put sequence constraints on the search using the text boxes colored in cyan and are located on the bottom left of the diagonal For this search we are only using two constraints which include gt lt 2 and gt lt 4 The first greater than sign in each example represents that the row nucleotide should be after the column nucleotide sequentially The second less than signs represent the number of bulged base are allowed between the row and column nucleotides eec Structures to search FR3D GUI Geometric symbolic search f Pure symbolic search Number of NTs 6
23. didate Add plot Larger Neighborhood Toggle sugar Toggle display MarkUnmark current Reverse all marks Display marked only List to screen Write to PDB Sort by centrality Order by Similarity Show Alignment Show Scatterplot Navigate with Fig 99 Quit display Figure 99 Navigation window click here then click the Navigate button i ioj File Edit View Insert Tools Desktop Window Help al OSHS aana Ela a Discrepancies between all candidates ordered by by similarity ao S aoe DM E I Aligning candidates The user can look at the sequence alignment of each of the Candidates by pressing the Show Alignment button 1n the menu 43 D MENU eee Figure 1 bispayoptons File Edit View Insert Tools Desktop Window Help Deus kRAS EEE fad MO 1 2 P0242 T03 A269 12695 42694 lat Next candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverso all marks Display marked any List to screen Write to PDB Sort by centrality Group Candidatos Shaw Alig aK Quit display Plot 1 of 14 Discrepancy 1 1213e 15 The Candidate motifs along with their alignment will be printed in the command window as shown by the example below 1 23 456 1a72 0 0000 A2691 U2693 A2694 G2701 A2702 A2703 AGUA GAA lqvf 0 0138 A2691 U2693 A2694 G2701 A2702 A2703 AGUA GAA lavt 0 0954
24. e Overlaps 3 Ready to Search 8 For a Symbolic search the user does not need to specify a Guaranteed or Relaxed Cutoff emana ano ee o FR3D GUI Load previous results 2006 07 12 10 55 17 44 mat ES cd O Geometric symbolic search f Pure symbolic search 1034 0 1a9n Number of NTs 6 Generate Interaction Matrix E 1asz Configuration anti FSA anti SM anti pga anti BSM ami BSM ani SM 1b36 4b7f Mask N11 1br3 Distance NI2 hat Interaction NIg bs Search name Sarcin ricin Motif Search 1e7k Search description Symbolic Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Guaranteed Cutoff 0 5 Relaxed Cutoff 1 0 Exclude Overlaps C gt i Ready to Search 9 Using the drop down menu to the right of the Relaxed Cutoff text box the user can specify whether to Exclude Overlaps or Include Overlaps 28 Q BC Structures to search FR3D GUI Load previous results Geometric symbolic search f Pure symbolic search ns 2006 07 12 10 55 17 44 mat ES Number of NTs 6 Generate Interaction Matrix Configuration anti fg anti DOM anti BSM anti OM anti BSM anti BSM Ng NIZ NI3 NI4 NI5 NIB Mask tSH Distance tHW Interaction U a Search name Sarcin ricin Motif Search Search description Symbolic Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Guaranteed Cutoff 0 5 Relaxed Cutoff 1 0 clude Over ne erlaps Ready to Search 10 Select the P
25. e lines 2AW4 ALANY ls 7 2 1J5e Note that case does not matter In the FR3D GUI the list Nonredundant list will appear in the list of PDB files to search Selecting this list will include all named files in the search When the results of the search are saved all files in the list are saved by name so that when the results of the search are loaded again later the individual files that were searched will be highlighted so that it is clear which files were searched When using xSpecifyQuery to specify searches names of lists can appear for the PDB files to be searched Appendix FR3D includes additional programs that may be of interest These are a little harder to use however From the Matlab command prompt gt gt load PDB data this way gt gt File zAddNTData Nonredundant_list Specify searches in the Matlab program file xSpecifyQuery then execute the search using gt gt FR3D Searches are saved as usual and may be retrieved later using FR3D 45 References Leontis N B Stombaugh J and Westhof E 2002 Motif prediction in ribosomal RNAs Lessons and prospects for automated motif prediction in homologous RNA molecules Biochimie 84 961 973 Leontis N B and Westhof E 2001 Geometric nomenclature and classification of RNA base pairs RNA 7 499 512 Sarver M Zirbel C L Stombaugh J Mokdad A Leontis N B 2007 FR3D Finding Local and Composite Recurrent Structural Motifs in RNA
26. ectory to FR3D you can use the cd command to change the directory At the command prompt gt gt type FR3D to launch the graphical user interface Matlab 6 users The program has been lightly tested with Matlab 6 The mat data files distributed with the programs are saved in a Matlab 7 1 format In the FR3DSource folder delete PDBInfo mat and rename PDBInfo_version_6 mat as PDBInfo mat and similarly with PairExemplars mat Delete the mat files in PrecomputedData and download the pdb files from the Protein Data Bank and place in PDBFiles Using the compiled version of FR3D for the PC Installation First download and install the Matlab MCR installer 100 MB MCR stands for Matlab Component Runtime It lets you run compiled Matlab programs without purchasing Matlab You can read about it at the Matlab website Then download the latest Starter Package and follow the instructions above This will create a folder named FR3D and several subfolders The executable FR3D exe will be in the FR3D folder Running FR3D Double click the executable file FR3D exe to launch the graphical user interface to FR3D Step By Step Tutorial on Performing Motif Searches using FR3D In this tutorial we will take you through a step by step approach to performing a Sarcin ricin motif search using purely geometric symbolic and mixed parameters Each step will have a red arrow m which will direct the user to the step which is being explained For each search
27. ed of 6 NTs from 23S Haloarcula marismortul 1e7x F 180 Guaranteed Cutoff 0 5 Relaxed Cutoff 1 0 am ver Y ne e Ready to Search 12 Select the PDB files which you would like to search in for your particular motif 1 e Iqrs Iqvf Irc7 1s72 Ona PC by holding down the Control key on your keyboard one can select multiple files which are not consecutive in the list menu To do this on a Mac the user must hold down the Command or Open Apple key It is possible to make user defined lists of PDB files to facilitate specifying the PDB files to search see below ear FR3D GUI Load previous results Geometric symbolic search Pure symbolic search rurama to search y y 2006 07 12_10_55_17 44 mat H 1a7y a 1982 sic 2 i View query idos Query NTs 2701 2702 2703 2691 2693 2694 Read Query a a a a a iq9a Query Chains o Walo Walo ao Ralo lo RM a Configuration hed re HH A2702 A2703 1qbp e acu 1qt6 ex Mask igru Distance 1q Interaction A 0 18z1 Search name Sarcin ricin Motif 110d 1t0e Search description Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Guaranteed Cutoff 0 5 Relaxed Cutoft 1 0 Exclude Overlaps Ka Generate Interaction Matrix Search Searching Please wait press Ctrl C to interrupt 13 Perform the Search by pressing the Search Button Information about the progress of the search is displayed in the bottom right corner of the GUI
28. ef aro pay 1a34 Em jaga 1aq4 1asy 1asz 1aud 1av6 1b23 b36 b7f 1 1 1bmv ibys 1 cbs 1c0a 1c9s iccOd tcsl Ee tex EEE Sarcin ricin Motif Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortui 14 The user can specify the Relaxed Cutoff discrepancy using the text box labeled Relaxed Cutoff i e 1 0 This number must be equal to or greater than the Guaranteed Cutoff Using a larger value can allow for additional flexibility in searches without increasing runtime as much as increasing the guaranteed cutoff would do ean FR3D_GUI O To GEE Po amp 2707 2702 2703 2681 2685 2694 Read Query EM RMS BS Tam Po am SJ am PM am LS am PM am o ES ESSE SE22E8 ESE RISERS EE Sarcinricin Mot ee O O o K Se 15 Using the drop down menu to the right of the Relaxed Cutoff text box the user can specify whether to Exclude Overlaps or Include Overlaps 22 9 FR3D_GUI D Load previous results e Geometric symbolic search 3 Pure symbolic search Structures to search y id 2006 07 12_10_55_17 44 mat H wiv a i ssa Query PDB 1872 ref v View query ena 0 Query NTs 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix 1aq3 a a a a a a tag Query Chains o Wajo Aao Mo o o NH 1as Ta Contiguration anti FA anti BSM anti PSA anti DOM anti FSA anti FSA taud ue G2701 A2702 A2703 A2691 U2693 A2594 1b36 s em mask ee Fr Tlm ter oware a
29. er In this case we are using chain 0 809 FR3D GUI le To cr MW 270 2702 2703 2691 2699 2694 Read Query Generate Interaction Matix amo Galo Gio PAO PMS FM 1 1 1 1 1 1 1 1 1 1 1 1 1 1 b36 br3 eyi c a c9s cco csi cvj exo dot 1d0u 1d r 1096 1d9h 1ddi S888 6 Once the chains have been selected the user should press the Generate Interaction Matrix button The Interaction Matrix allows the user to impose certain types of constraints on the search A purely geometric search makes no such constraints Below we describe mixed geometric and symbolic searches and purely symbolic searches FR3D_GUI ee To icc Reasvey Cover rac QR RI MI ME MEM RR do cedo calo calo calo al ado dio di calo d cado 1 1 bmv cla c9s cco csl evo 1dot 1d0u id r 1096 1d9h 1day idfu 1di2 1dk1 idno idnt ido dah drz iduh tda idzs 1e6t 1e7k 1e7x 1cBo tec 7 The user should give a name to the search 1n the text box labeled Search name e g Sarcin ricin Motif This will become part of a filename so the name should not use characters such as 7 or NY because these have meanings in filenames and paths A0 FR3D_GUI y eee 2707 2702 2703 2681 2683 2698 nett RE o O ME MEM R EM BM Ml MM 8 The user can add more descriptive information about their search in the text box labeled Search description e g
30. eraction matrix will appear on the GUI eo osorerossrama OOOO Mo MM MM 3 The user can specify the glycosidic bond conformation anti or syn for each base in their search using the drop down menu labeled Configuration The order of the drop down menus corresponds to the same order as the Query nucleotides If the user does not want to restrict the conformation to either anti or syn they can leave the selection blank which means both conformations are allowed 25 IL AL FAL ALR 4 The user can impose a mask for their search by putting in nucleotide constraints in the text box in the Interaction Matrix which has a white background The program will take many types of masking letters i e A C G U R Y etc 060 FR3D GUI 2006 07 12 10 55 17 44 mat ba ees HE Generate Interaction Matrix EE ICE CE CE CE CE 5 The user can impose basepair constraints using the text boxes which are colored yellow and in the upper right half of the diagonal in the Interaction Matrix 1 e G2701 row forms a trans Sugar Edge Hoogsteen basepair tSH with A2794 column A2702 U2693 form trans Hoogsteen Watson Crick basepair HW and A2703 A2691 forms a trans Hoogsteen Hoogsteen basepair tHH 26 00 Structures to search y C gt FR3D GUI Geometric symbolic search f Pure symbolic search Number of NTs 6 Contiguration anti Ke anti HA anti be anti HA anti He anti HS NI NI
31. erous limit Type CAAM nc to get cVVWY and near co pairs cA Type ntrans to get all pairs nearest to a trans pair Type 35 for stacking in which the first base uses its 3 face and the second base uses its 5 face Similarly type s53 33 or s55 Type stack to allow all stacking interactions The prefixes n and work with stacking as above To specify that the nucleotides must match a certain pattern type for example city CG GC to get only CG or GC CVV pairs To require that two nucleotides make a base phosphate interaction enter BPR in the corresponding yellow box This will select pairs of nucleotides in which the first nucleotide s base is a hydrogen bond donor and the second nucleotide s phosphate is an acceptor To reverse the roles type PhB To specify particular base phosphate categories type UBPh 1BFh 26Ph 9BPh or OPREG 1PhE etc For near base phosphate interactions type nGPh nPh6 n1BPh n1Phe etc One can restrict to pairs that play a certain role in the secondary and tertiary structure For pairs that are nested type M or nested For pairs that cross nested interactions but involve nucleotides in the same branch of the RNA type local or L For long range or distant interactions between different branches of the RNA type long range distant O or LR Note that nested local and distant are mutually exclusive They can be negated with but local only returns distant Interac
32. ersus composite motifs In this case all of the candidates consist of two strands one corresponding to 2692 2694 the other corresponding to 2701 2702 Writing Candidates to a PDB File To write the Candidate motifs to a PDB file which can be view using 3D visualization tools the user can press the Write to PDB button in the menu 38 MENU aa sa Figure 1 Dispiyoptons File Edit View Insert Tools Desktop Window Help DHS k RAMS E UH q fad MO 1 2 do FOS 8269 12695 42694 lat Next candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverso all marks Display marked only List to screen Write to x Sort by centrality Group Candidatos show Alignment Quit display 5 Plot 1 of 14 Discrepancy 1 1213e6 15 After pressing the Write to PDB but the pdb filename will be displayed in the command window The following is an example of an output printed to the command window The file should be stored in the user s local working directory Wrote 2007 01 25 12 12 47 Sarcin Cand pdb 39 New Display Candidates Menu as of April 2009 MENU ox Display options Next candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to Screen Write to PDB Sor by centrality Order by Similarity Show Alignment Show Scatterplot Navigate with Fig 99
33. es 10 ssaroh x ci 2006 07 12 10 55 17 44 mat HH 1pwt iq2r Query PDB 1872 LH v View query 1a7y Query NTS 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix 1982 a a a a a 1986 Query Chains o Mo Ralo So o o RW Contiguration anti Hi anti HH anti a anti HS anti HH anti HH 1906 G2701 A2702 A2703 A2691 U2693 A2694 a eso a OS ee ee E eel pes Ejs a N taco Mask 62701 R a Distance A2702 ni 0 Interaction A2703 E N iat tau oil n tava U2693 Ln Es a aoe E s HS TU O a isi2 Search name Sarcin ricin Motif Search Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul Display Candidates 1t0d Guaranteed Cutott 0 5 Relaxed Cutott 1 0 Exclude Overlaps List Candidates 14 candidates found Conducting Symbolic searches with FR3D Here we illustrate the ability of FR3D to search for motifs based only on symbolic criteria such as desired basepairing base stacking nucleotide identity and sequential continuity constraints We assume the reader has read the previous sections and focus only on what is new to purely symbolic searches 1 Start by selecting Pure symbolic search Then the user must enter the number of nucleotides in the motif for which they want to search e g 6 24 ee0o 2006 07 12 10 55 17 44 mat 2 The user should now press the Generate Interaction Matrix button When this is pressed an Int
34. fs the user can mark each candidate they feel is what they are looking for Once marked they can list out just the marked candidates or view just the marked candidates The default is unmarked but the figure below shows that the query motif is marked This is a very useful when correlating it with some of the other tools such as writing pdb files sorting by centrality grouping candidates and showing an alignment For example the user marks 5 of 7 Candidate motifs and then wants to write them out to a pdb file only the marked Candidates will be written This idea applies to the other tools in the menu as well 35 O O O MENU Figure 1 pepayoptons File Edit View Insert Tools Desktop Window Help Next candidate 4 Drs ke RAC t E OES j i Previous Candidato Add plot Larger Neighborhood Toggle sugar al Cr Mark Unmark E Reverse all E Display mario GE 4 do are List to screen E 4 Write to PDB i Sort by centrality d Group Candidates Show Alignment Quit display Listing Candidates There are two methods to list the Candidates fad 01 42 P0242 03 4269 42695 42694 Iate Plot 1 of 14 Discrepancy 1 1213e 15 1 Using the FR3D GUI after performing a search and Candidates are found the user can press the List Candidates button located in the bottom right corner of the GUI 36 ear FR3D_GUI Load previous results Structures to search e Geometric symbolic search Pure symbolic search eee
35. htenkemesaciisatunesducnsdsiibaatbienbebnsaedabelesteomscendadteie sites 44 User mamitamed MSto EDB TIES srs tce sistas tease atercace rite aa des RC E a 45 PRPC 0 6 UDN PRE DDR RR MD E ER RAE ORA RED REAR RREO RD RR RR 45 FRE TORE INC eres RARE RARE REDE SN 46 Installation Running FR3D from Matlab Installation FR3D was written in Matlab version 7 1 and has been run successfully on PC Macintosh and UNIX platforms The easiest way to install it is to download the latest version of the Starter Package from http rna bgsu edu FR3D This will create a folder named FR3D and several subfolders It has pre computed data for four large PDB files results of a few FR3D searches and the FR3D source files data files and PC executables If you already have a FR3D installation download the current version of the Matlab code unzip and copy the new program files over the old ones in the FR3DSource folder If you have another folder on your computer with PDB files add that folder to Matlab s path File Set Path Add Folder Save The first time FR3D is asked to search a given PDB file it reads the text analyzes it and saves a data file in the subfolder PrecomputedData After that it will not need to re read the original PDB file New versions of FR3D may need to re analyze the 3D structure data stored in PrecomputedData to take advantage of new classifications Launching the Graphical User Interface GUI Start Matlab and change the working dir
36. m the Query motif this took an additional 70 seconds Only 14 candidates had discrepancy less than 0 5 the default cutoff discrepancy These candidates are listed in order of increasing discrepancy The Query motif is listed first with discrepancy 0 0000 Each of the five nucleotides is listed followed by a brief listing of the chains in which the nucleotides are found Note that the candidate with discrepancy 0 2374 was found in the 5S chain chain 9 The full display format includes information about the pairwise interactions between the nucleotides in each candidate and other information The output 1s quite wide so we use a very small font here Query Sarcin 5 nucleotide geometric Sarcin ricin motif with 5 nucleotides geometric search Found 137457 possibilities from 1s72 in 17 109 seconds Calculating discrepancy Seconds remaining 62 56 49 42 36 29 22 14 7 Found 14 candidates in the desired discrepancy range Removed highly overlapping candidates kept 14 Entire search took 87 8125 seconds or 1 4635 minutes Filename Discrepancy a 2 3 4 5 Chain 1 2 1 3 1 4 1 5 2 3 2 4 2 5 3 1572 0 0000 G 2692 U 2693 A 2694 G 2701 A 2702 00000 cSH ntSH s33 ncSH s35 s33 tWH H 3 5 4 5 Confi 1 2 1 s55 ncSH AAAAA 1572 0 0712 G 1370 U 1371 A 1372 G 2053 A 2054 00000 cSH ntSH s33 ncSH s35 s33 tWH tH 1s72 0 1080 G 381 U 382 1s72 0 1275 G 588 U 589 s55 ncSH AAAAA 1 s55 ncSH SAAAA 1 s55 ncSH SAAAA 383 G 406 A 407 00000 cSH ntSH s33 ncSH s35 s33 tWH t
37. milarly with C G U Type AC to allow only A or C or AGU to allow only A G or U Type G to exclude G jor type ACU You may use one of these standard abbreviations M AC R AG WHA S Ce Y HCU k l Y ACD H ACU OD AGU B COU N ACU Using no mask is the same as specifying ACGU or M Seltcontradictory specifications such as A A may give unpredictable results or empty searches specify the location weight for the given nucleotide by typing something of the form Lies In the box Location weights are normalized automatically so making them all equal to 5 0 has no effect on the search The default value if nothing is specified is 1 0 specify the angle weight for the given nucleotide by typing something of the form AMA In the box Angle weights are not automatically normalized the default is 1 0 Making angle weights larger than 1 0 increases the orientation error Thus candidates with low discrepancy will have bases more closely aligned than when using the default value of 1 0 for angle weight separate the mask location weight and angle weight by spaces 9 The user can impose basepair base stacking and base phosphate constraints using the text boxes which are colored yellow and in the upper right half of the diagonal in the Interaction Matrix 1 e G2701 row forms a trans Sugar Edge Hoogsteen basepair tSH with A2794 column A2702 U2693 form trans Hoogsteen Watson Crick basepair HW and A2703 A2691 forms a trans Ho
38. ogsteen Hoogsteen basepair tHH 90 FR3D GUI fe Geometric symbolic h gt Pure symbolic h s searc s searc Structures to search 7 d 2006 07 12_10_55_17 44 mat LH 168d 0 Query PDB 1872 v View query 1309 Query NTs l 2701 2702 2703 2691 2693 2694 l Read Query Generate Interaction Matrix E ia a tag Query Chains o Mo Salo Mo Mo Milo MH tas jaan Configuration anti PA anti BSM anti age anti BSM anti DS anti ESA laud Sara G2701 A2702 A2703 A2691 U2693 A2594 1b36 O 17 Mask G2701 R 1bmv r 1br3 Distance A2702 A toy Li d E interaction A ic0a 1c9s 1820 met a N A i cs 1evj U2693 U tev ee E icx E e Les e Me e 1dgr 1d96 1d9h iddy 1 idfu 1di2 idk1 idno idnt idnx idpl idaf 1dqh idrz iduh idul R E a Search name E Search 1e6t i 1e7k Search description 1e7x 2 1e80 Guaranteed Cutoff 0 5 Relaxed Cutoff 0 5 Exclude Overlaps HH ec r E Ready to Search For more information on using the basepair constraints the user can press the Interaction Button to the left of the Interaction Matrix 17 000 FR3D_GUI O To cr 2707 2702 2703 2691 2699 2694 Read Query RM RA RIO ME Cam pe amo PAY am P am pf am ff am pay CC This is the pop up help menu for the Basepair interactions 18 px Basepair base stacking base phosphate or letter pair constraint is specified above the diagonal
39. r A or C R for A or G W for A or U S for C or G Y for C or U K for G or U V for A C or G H for A C or U D for A G or U B for C G or U N for A C G or U Note that N is the default Itis not necessary to use these abbreviations however One may also exclude a given base using the syntax G for instance to exclude candidates with a G in the corresponding position The diagonal boxes are also the place to specify certain parameters that modify the definition of the geometric discrepancy These are described in the pop up window concerning the mask and in the article Sarver et al 2007 15 ea FR3D_GUI cl PE cr 2701 2702 2703 2691 2693 2604 ELI RN Ro E ME MEM art anti ar rr e SiS ee eee ee CE L Jo UML oO __ O I I I Se A a If the user would like to know more information on using masks they can press the help button labeled Mask S 000 FR3D_GUI Le ooo e serra mm ats 2707 2702 2703 2681 2683 2698 ELI RM Ballo BS MO MEM TCE CE CE CE CE CE ERERES do cedo n calo ana ali di e an n a a cd 1av6 1b23 1b36 b7t bmv br3 eyi cla c9s cco csl cvj ex0 1dot 1d0u id r d96 1d9h day dfu di2 dki dno dnt st A drz This is the pop up help menu when the Mask button is pressed 16 iol xi Nucleotide mask location weight and angle weight are specified inthe boxes on the diagonal For the mask type Ato allow only As si
40. ricin motif with 5 nucleotides geometric search a7 Found 137457 possibilities from 1s72 in 17 109 seconds Calculating discrepancy Seconds remaining 62 56 49 42 36 29 22 14 7 Found 14 candidates in the desired discrepancy range Removed highly overlapping candidates kept 14 Entire search took 87 8125 seconds or 1 4635 minutes Filename Discrepancy 1 2 3 4 5 Chain 1s72 0 0000 G 2692 U 2693 A 2694 G 2701 A 2702 00000 1s72 0 0712 G 1370 U 1371 A 1372 G 2053 A 2054 00000 1s72 0 1080 G 381 U 382 A 383 G 406 A 407 00000 1s72 0 1275 G 588 U 589 A 590 G 568 A 569 00000 1s72 0 1784 G 175 U 176 A 177 G 159 A 160 00000 1s72 0 1844 G 464 U 465 A 466 G 475 A 476 00000 1s72 0 1976 G 358 U 359 A 360 G 292 A 293 00000 1s72 0 2284 G 213 U 214 A 215 G 225 A 226 00000 1s72 0 2374 G 78 U 79 A 80 G 102 A 103 99999 1s72 0 2391 G 1971 U 1972 A 1973 G 2009 A 2010 00000 1s72 0 2491 G 1292 U1293 A 1294 G 911 A 912 00000 1572 0 2714 G 953 U 954 A 955 A 1012 A 1013 00000 1s72 0 4395 G 1543 U 1544 C 1545 C 1640 A 1641 00000 1572 0 4644 G 706 C 707 A 708 G 720 A 721 00000 The first lines tell details about the search process FR3D screens out possible candidates to reduce the number of candidates it has to consider in detail In this example it found 137457 five nucleotide motifs which could not be rejected based on the pairwise distances between their constituent nucleotides alone This took 17 seconds For each of these it calculated the geometric discrepancy fro
41. st Candidates 14 candidates found This pop up figure and menu appear after the user presses the Display Candidates button The order of the motifs is arranged according to the lowest discrepancy so the first Candidate should always be the Query motif MENU a Figure 1 vispayoptors File Edit View Insert Tools Desktop Window Help DHS k RAM By DE so G2701 A2702 42703 4269 12693 42694 Next candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to screen Write to PDB Sort by centrality Group Candidates show Alignment Quit display To look at the next Candidate in the user should press the Next Candidate button on the menu In the figure you can see the next lowest scoring Candidate motif 31 OO O MENU Display options Next coque Previous Candidato Add plat Larger Neighborhood Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to screen Write to PDB Sort by centrality Group Candidates show Alignment Quit display Figure 1 File Edit View Insert Tools Desktop Window Help rhe b RACH APEL G2701 42702 427034269 141269 1 quit To go back to a previous Candidate the user should press the Previous Candidate button on the menu Now the figure refers back to our first Candidate which is the Query motif 32 O O O
42. th FR3D We assume the reader has read the previous section on purely geometric searches and so we focus on what is new in a mixed search 1 The first step is to click on the radio button which says Geometric symbolic search 000 FR3D_GUI 2006 07 12_10_55_17 44 mat do cado n cado do lo on on an ado on a a cado o a dh alo calo a an ado ado lo selo aa SSESSERESSSRESEESRESEE2 FEER 2 The user must specify the PDB file e g 1s72 which contains the known motif from the drop down menu labeled Query PDB 809 FR3D GUI 12 3 The query nucleotides are entered into the text box labeled by Query NTs e g 2701 2702 2703 2691 2693 2694 ee 00 FR3D GUI 20060712 10 88 1744ma E MW 2707 2702 2703 2001 of rea 0 00 FR3D_GUI 2008 07 12 10 58 17 44 mat 1872 a d v 5 The user may use the drop down menus to select the chain in case of ambiguity 13 FR3D GUI cl CT ce 270 2702 2703 2691 2699 2694 ED MEIO amo Galo aio PAO PMS PM sia 1 1 1 1 aq4 1asy 1asz 1aud 1av6 1b23 1b36 1b7t 1bmv E 1 cbs 1c0a 1c9s 1cc0 tcsl tex 1dot 1d0u id r 1096 1d9h 6 The user should press the Generate Interaction Matrix button When this is pressed an Interaction matrix will appear on the GUI Now we describe how to focus the search by specifying symbolic constraints which must be met by each candidate Adding symbolic constraints
43. the text box labeled Search description i e Geometric Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortui Other comments about the search can be added here as well f x x O FR3D_GUI fe Geometric symbolic h Pure symbolic h s searc s searc cessar l id 2006 07 12 10 55 17 44 mat ES 168d Query PDB 1872 HJ V View query 0 Query NTs l 2701 2702 2703 2691 2693 2694 l Read Query Generate Interaction Matrix E 1a a a tags Query Chains o oO lo lo lo tas pet Configuration anti anti HH anti anti anti anti HH laud br G2701 A2702 A2703 A2691 U2693 A2694 1b36 5 1o71 Mask G2701 R EE E Es b O SHO 1br3 Distance A2702 A te e 6 RR No a 1c0a 5 as is Ea ds x s 1c9s 1820 eee ics Vy F1 F 7 U E veses DDD oe O TO icx o HH TRT Jaou tee O E M E A gt id r 1096 1d9h tay idfu idi2 1dk1 idno idnt idnx idpl idaf ise rz f iau u am Search name Sarcin ricin E Search 1e6t 1e7k Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula nA te7x z iodo Guaranteed Cutoft 0 5 Relaxed Cutort 0 5 Exclude Overlaps aa ec v Ready to Search 13 The user must set the Guaranteed Cutoff discrepancy using the text box labeled Guaranteed Cutoff i e 0 5 21 009 FR3D_GUI O To icc Mo SSS O dM Read Query RR OR CR O CR Fan E ame pep aro PE amo Pe amo
44. tions not nested ones To specify bases on the same strand that form ci pairs that flank a hairpin internal or junction loop type flank or F Note for internal and junction loops flanking nucleotides will be on the same strand one on each side of the internal or junction loop Such flanking nucleotides usually do not interact with one another Base phosphate interactions Specify that nucleotides 1 and j should make a base phosphate interaction by putting the text BPh in row 1 and column jJ This requires that the nucleotide in row 1 be a hydrogen donor and that the phosphate of the nucleotide in column j be an acceptor in a hydrogen bond between the two nucleotides Specify a particular base phosphate interaction with text such as 1BPh 2BPh 9BPh OBPh To reverse the roles of 1 and J use PhB 1PhB etc Near base phosphate interactions can be obtained with nBPh nl BPh n3PhB etc The user can put sequence constraints on the search using the text boxes colored in cyan and are located on the bottom left of the diagonal For this search we are only using two constraints which include gt lt 2 and gt lt 4 The first greater than sign in each example represents that the row nucleotide should be after the column nucleotide sequentially The second less than signs represent the number of bulged base are allowed between the row and column nucleotides 19 eA FR3D_GUI e n PT cscs 270 2702 2703 2691 269
45. tton In the figure it should be noted that the nucleotide list now includes the 33 neighboring bases By pressing the Large Neighborhood button several times 2 or 3 times the motif will go back to the original size of the Candidate motif O O MENU Display options Next candidate Previous Candidate Add plat Larger Neighb ama Toggle sugar Toggle display Mark Unmark current Reverse all marks Display marked only List to screen Write to POBE Sort by centrality Group Candidates Show Alignment Quit display Figure 1 File Edit View Insert Tools Desktop Window Help Ser IRECNO EEE fad PO 1 2 Odd USA 269 12693 263 B AMAS 26922615A 2700 lsfe 10 Plot 1 of 14 Discrepancy 1 1213e 15 If the sugars are impeding your visualization the user can press the Toggle sugar button to turn on or turn off the sugars as shown in the figure below 34 O O O MENU IOGA Figure 1 Display optons File Edit View Insert Tools Desktop Window Help enana Oels i RAC E OB ao Previous Candidate 2 FO 1 42 702 42 035 4269 1426935 42694 1572 Add plot Larger Neighborhood Toggle sug Geral Toggle display e e E P MarkvUnmark current Aero pe a A2703 ae i Reverse all marks Display marked only List ta screen Write ta PDE Sort by centrality Group Candidates show Alignment 5 Plot 1 of 14 Discrepancy 1 1213e 15 Quit display When analyzing the structures of the Candidate moti
46. we will be focusing on a sub motif of the Sarcin ricin shown below The six nucleotides are 2701 2702 2703 2691 2693 and 2694 from PDB file 1s72 The interactions involved are G2701 A2694 trans Sugar Edge Hoogsteen A2702 U2693 trans Hoogsteen Watson Crick and A2703 A2691 trans Hoogsteen Hoogsteen Query Motif 270103 lt 40 A A Cro U gt G A AS D o U 2600 23S H mansmortui PDB 1872 Leontis et al 2002 Leontis and Westhof 2001 Performing a Purely Geometric Search with FR3D 1 The first step is to click on the radio button which says Geometric symbolic search 00c FR3D_GUI O as vi i h P h Load previous results ometric symbolic searc ure symbolic searc span san a 2006 07 12 10 55 17 44 mat Ke 165d 0 Query PDB 157d a View query ea Query NTs 1 3 7 Read Query aq3 a E as md dh 4 see N i d d 28555 Hint Choose Query PDB and Query NTs then click Read Query repeat anytime to restar 2 The user must specify the PDB file which contains the known motif from the drop down menu labeled Query PDB e g 1s72 FR3D GUI 2006 07 12 10 55 17 44 mat a r o 3 The query nucleotides are entered into the text box labeled by Query NTs e g 2701 2702 2703 2691 2693 2694 Nucleotide numbers may be separated by commas spaces or semicolons A range of nucleotide numbers may be indicated with a colon as in 2701 270
47. with all other candidates 1s lowest will appear first and so on In this way the user can find the centroid for their Candidate motifs The centroid is often a good choice as the query motif for the next search In the figure below the centroid candidate is from 1s72 and is listed at the top of the graph List to Screen will list the candidates in this order gt menu TETES Display options Mest candidate Previous Candidate Add plot Larger Neighborhood Toggle sugar Toggle display File Edit View Insert Tools Desktop Window Help uM Dae hs k amp Aane 2 008 so Discrepancies between all candidates ordered by by centrality eer m MarkUnmark current ef mee EA Es Reverse all marks Display marked only List to screen Write to PDB Sort by centrality Order by Similarity 5 CLS Gi Kd a i T 7 E pia EL E Qo Gh i gr u m HE NR I Toi Show Alignment GAS Srowaigmeri CE oe Show Scatterplot Navigate with Fig 99 Gui display F cee i 1 a nae Order by Similarity Pressing the Order by Similarity button will re order the candidates in such a way that candidates which are geometrically similar to one another appear near each other in the list This makes it possible to identify clusters of candidates which share geometric features 42 gt menu six Display options Mest candidate Previous Can
48. z DD A Do mm E E ic9s teed ss J JL lnm tesi P i we ese TO icw icx E MM Tn JL Jj E am Search name Sarcin ricin Motif Search 1e7k Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul 1e80 Guaranteed Cutott 0 5 Relaxed Cutott 1 0 de O ne ps n Ready to Search 16 Select the PDB files which you would like to search in for your particular motif 1 e Iqrs lqvf Irc7 1s72 On a PC by holding down the Control key on your keyboard one can select multiple files which are not consecutive in the list menu To do this on a Mac the user must hold down the Command or Open Apple key If the user would like to select consecutive file in the list menu they should hold down the Shift Key PC Mac and select their files 0 0907 FR3D GUI Load previous results 2006 07 12 10 55 17 44 mat HH ger Query PDB 1872 is V View query ide Query NTs 2701 2702 2703 2691 2693 2694 Read Query Generate Interaction Matrix Query Chains o Mo Ralo lo o Ao RH Configuration anti HA anti hi anti Hd anti he anti hid anti Ka G2701 A2702 A2703 A2691 U2693 A2694 EE E E E wane ee DD Dm vereten vm lt OD Structures to search Geometric symbolic search Pure symbolic search 1982 Mask G2701 R Search name Sarcin ricin Motif Search Search description Sarcin ricin sub motif comprised of 6 NTs from 23S Haloarcula marismortul 1103 1t0d Guaranteed

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