user manual - ArachnoServer
Contents
1. BLAST against Peptide Sequences Using blastall program blastp Filter query sequence DUST with blastn SEG with others L Blast Figure 23 BLAST form in ArachnoServer populated with a toxin from the database Note that SEG and DUST filtering is off by default Alternatively under the Sequences section of each toxin card each sequence can be sent to the BLAST search form and run using a single click This is a convenient way of quickly identifying toxins similar to those already in the database For protein sequences both the full sequence including the signal and propeptide sequence where available or just the mature toxin sequence can be selected as the BLAST query As toxins are uniquely identified by their mature toxin sequence this second option provides a mechanism to enrich the alignment results for mature toxins only Due to the often very short length of mature toxin sequences when running BLAST on toxin data we recommend you leave the low compositional complexity filter off DUST for BLASTn and SEG for other BLAST programs The BLAST results will be formatted in HTML and contain links to ArachnoServer toxin cards for those toxins that are most similar to the query sequence If those toxins also have links to UniProtKB Swiss Prot these will also be available in the BLAST result Figure 24 20 Home search 7 Browse 7 BLast BLASTP 2 2 16 Mar 25 2007 Reference Altschul Stephen F2. lt and characters are encoded as amp gt and amp lt whereas PDF exported data is designed to be readable in report format FASTA sequences can be exported for all molecule types or for specific types peptide DNA or mRNA Browsing ArachnoServer f Browse The Browse tab is always available at the top of the page Toxins can be browsed by any of four categories Araneae taxonomy Molecular Targets Posttranslational Modifications and Phyletic Specificity Each category creates a different browsing tree on the right hand side of the screen for easy selection of toxins Figure 10 is a screenshot of the tree that is visible when browsing ArachnoServer according to Araneae spider taxonomy As with all browse trees the taxonomy tree only contains species for which there are corresponding toxins in the database As can be seen selecting a family of spiders will display a table of all toxins characterised from a genus within that family in this case the genus Apomastus within the family Cyrtaucheniidae FASTA sequences can be exported from the Browse results and clicking on the toxin name will open the Toxin Card for that particular toxin within a new browser tab see the section below entitled Each toxin has its own Toxin Card for more information Browse Toxins help Show toxins by ontology Araneae Taxonomy Peo z y me Name Synonym Genus Species ae L aE Araneae 748 U cyrta
3. and the year the toxin was discovered note that this has been carefully curated from literature information and may differ from the year the toxin was deposited in a public database For most toxins ArachnoServer also displays a high resolution image of the source spider The legend below the photo lists the photographer and provides the prosoma length for scale Click on the thumbnail image to view a high resolution version Images are free for academic use according to the creative commons noncommercial license Schematic of the mature toxin Below the general information for the toxin is a dynamically generated image displaying information about the mature toxin It shows where available the disulfide bond framework pharmacophore residues and posttranslational modifications Figure 13 12 Mousing over the sequence will popup the residue number and amino acid while mousing over a posttranslational modification will popup the residue number and type of modification not shown Note that all residue numbers displayed begin from the Start of the mature toxin sequence 2 g L5 16 L 28 ag RYLFGGCKTTS DCQKHLGCKFRDKYCAWDFT FS 22 24 f l 16 Cysteine on 23 A pharmacophore experimentally determined disulfide bond fexperimentally determined post translational modification A pharmacophore by homology disulfide bond by homology Cy3 disulfide bond predicted Average Reduced M
4. Ga a a al X help f Molecular target unknown w actinopoditoxin Mb1a w missulenatoxin Mb1a Missulena bradleyi Transporter w agstonvAeta uragatonin iA Ta Membrane Perturbation w agatoxin Aa 1b N w agatoxin IB Agelenopsis aperta Lectin binds carbohydrate terminal fragment H Enzyme Inhibitor w agatoxin Aa2a N w agatoxin IIA Agelenopsis aperta H Enzymatic Activity terminal fragment Channel Ion channel emt wagon mA _ Acid sensing ion channel ASIC Calcium channel voltage gated vertebrate H Calcium cha Ita ted invertebrat u agatoxin Aa3c N w agatoxin IIIC Agelenopsis aperta a re AF rear a opera terminal fragment thet art patel isc Potassium channel calcium activated vertebrate terminal fragment _ Sodium channel voltage gated vertebrate emo weapon IB SR E Sodium channel voltage gated para type invertebrate Transient receptor potential TRP cation channel Potassium channel voltage gated archaebacterium Mechanosensitive ion channel Transmitter Gated Channel weuntonnceta osma owemu al hota waracoonnAria Armee heats watcctoinari Awe Ce waracoomnate ar O heath waraoonnart Ara O bs weona wating Ale hoax watznondy Haderyew ions toate waracoontite Hadovce awe wroraonntivia waracoion
5. Mi S te a RRO N ay With the publicly available toxin information and the curated data sets described above the curation team then work through each toxin from within the curation interface ascribing a toxin name and exhaustively collecting and summarising information from literature searches and patent information with data from public databases Taxonomic source species for toxins in the public data set are matched or corrected with the current species from the World Spider Catalog and annotated with a unique Life Science Identifier LSID High resolution photos are added to spider species if available Names for peptide toxins defined as those smaller than 10 kDa are ascribed using the rational nomenclature described by King et al 2008 which is Summarized at http www venomics org nomenclature while sphingomyelinase nomenclature is derived from Binford et al 2009 All known synonyms are included as well Once a toxin is curated it is published to the public interface and then becomes searchable Toxin s can at any stage be added or further curated by the team as additional information becomes available Searching ArachnoServer ArachnoServer supports both basic and advanced searches In the banner at the top of every page is the basic search field Figure 3 Search terms entered here query toxin names including synonyms taxonomic information including historic taxonomy and common names of spiders If no searc
6. from the spider Hadronyche versuta ATGAATACTGCTACATGTTTCATCGTTCTTTTGGATGTGGCGACTGTCATCGGAGGCATT GAAGCAGGAGAATCTGATATGAGAAAAGATGTCATGGGATTATTTCGCCGAGCTATTTGC ACTGGAGCCGACAGACCGTGCGCGGCGTGCTGCCCGTGCTGCCCAGGGACCTCGTGCAAA GCAGAATCAAACGGTGTTTCTTATTGCAGGAAAGACGAACCTTGAATCCCAACTCAAATC ATTTCCCTTCGCATGATATTTTCTATTAATGTCTTTTATCGTTGCAAGGAAATGTCAATG GTTATGTTGAACGTTATGTAAAACGTTGACTATGAAATAAAATGTGAACAATAATTAAAT ACCTGAAAAAAAAAAAAAAAAAAAAAA gt as x hexatoxin Hvlc_ 3 cDNA sequence XenFW393 for Full BLAST insecticidal toxin x hexatoxin Hvlc from the spider Hadronyche versuta ATGAATACTGCTACATGTTTCATCGTTCTTTTGGTTGTGGCGACTGTCATCGGAGGCATT GAAGCAGGAGAATTTGATATGAGAAAAGATGTCATGGGATTATTTCGCCGAGCTATTTGC ACTGGAGCCGACAGACCGTGCGCGGCGTGCTGCCCGTGCTGCCCAGGGACCTCGTGCAAA GCAGAATCAAACGGTGTTTCTTATTGCAGGAAAGACGAACCTTGAATCCCAACTCAAATC ATTTCCCTTCGCATGATATTTTCTATTAATGTCTTTTATCGTTGCAAGGAAATGTCAATG GTTATGTTGAACGTTATGTAAAACGTTGACTATGAAATAAAATGTGAACAATAATTAAAT CCCTTAAAAAAAAAAAAAAAAAAAAC Figure 20 Sequences stored in ArachnoServer for the toxin k hexatoxin Hvic some mRNA sequences have been excluded for brevity All sequences can be directly sent to the BLAST page from here For protein sequences either the full sequence or just the mature toxin sequence can be used as the search query Toxin synonyms This section displays all known synonyms for the toxin as well as the current recommended names and abbreviations For example the toxin k hexatoxin Hvlc has the following s
7. lethal and neurotoxin paralytic biological activities If we changed the first or to and then this search would return zero results as expected as species cannot belong to more than one taxonomic family Many combinations of these search terms are possible Exporting search results and toxin sequences in FASTA format Search results are returned in a paginated table Only general information is displayed for each toxin within the search results table to minimise clutter on the screen At the bottom of each search result page are export options Error Reference source not found U gt agatoxin Aoth Agel_07 Agelena orientalis U gt agatoxin Ao1l Agel_ 08 Agelena orientalis 748 toxins found displaying 1 to 15 FirstPrev 1 2 3 4 5 6 Next Last Export options 42 XML AJ PDF CaP a Fasta Sequences Peptide DNA mRNA P am THE UNIVERSITY at CQ ran B EN o QUEENSLAND Ea r a i Figure 9 Export options available for search results M B Institute for Molecular Bioscience Data can be exported in both PDF and XML formats Exporting data from any result page for a multi page set of search results will export all data in the current search results Additional information about each toxin is provided in the PDF export description synonyms and taxonomic family XML exported data is designed to be reformatted programmatically and may contain some HTML embedded codes for example
8. scientists clinicians and structural biologists At the time of writing ArachnoServer makes available 748 manually curated spider toxin records As more spider toxins are discovered or known toxins further characterised the curation team carefully collates this additional information into the database As can be seen in Figure 1 the rate of discovery of spider toxins is increasing exponentially Cumulative Toxins Deposited by Year 900 800 400 300 200 s nil puu 1985 1990 1995 2000 2005 Year Number of Toxins Figure 1 Cumulative number of spider toxins deposited into public databases since 1984 We are committed to maintaining the most up to date and accurate repository of spider toxin information for the scientific community We use ArachnoServer in our daily research and we hope you find it useful too Should you have any questions comments or feedback please contact us at support afab org What data is stored in ArachnoServer and how is it curated The starting point for data curation is the automated collection of all publically available sequence and annotation information for spider toxins from UniprotKB SwissProt INSDC The International Nucleotide Sequence Database Collection DDBJ EMBL and GenBank and the Protein Data Bank PDB We join these data sets with the assistance of Sequence Retrieval System SRS into a single non redundant set typically containing peptide sequences nucleotide mRNA seque
9. search and find that a single toxin has antimicrobial activity on Arthrobacter globiformis as well as a solved PDB structure Alternatively we might be interested not in antimicrobial toxins with solved structures but those discovered at a particular time for example the year 2006 We can do this by changing the number of solved PDB structures to Toxin Discovery Year then choosing equals gt 2006 Search In this case ArachnoServer returns 8 toxins Advanced search fields Table 1 lists searchable fields and provides a brief description of each Table 1 Database fields searchable using ArachnoServer s advanced search interface Field Field Data Description Category Type Toxin Name Text Free text to search all the recommended toxin names in the database Toxin names are ascribed using the rational nomenclature described in King et al 2008 Toxin Description Free text describing the toxin Generic Toxin Group List Toxins are grouped by taxonomic family The toxin group is used when naming the toxin see King et al 2008 and http www venomics org nomenclature Toxin Discovery Number The date the toxin was discovered note this has been Year carefully curated and may differ from the date the toxin was Toxin Meaga originally deposited in the public database Toxin Synonym Text ArachnoServer keeps a record of all names used to describe toxins including abbreviations and these
10. spider names Historic Taxonomy Text Many spider species have been taxonomically reclassified in the past and this process is ongoing ArachnoServer uses Species historic taxonomy data from the World Spider Catalog to make it possible to search for toxins using outdated taxonomy Journal Article Title Text Search for a toxin according to words in the title of citations in Semea TST citeconorthceonn ooie ne oreraa i All authors of journal citations are stored in ArachnoServer You can search for toxins published by a particular author using this field article publication or range of years Deposition Authors ArachnoServer also stores the names of those who originally deposited the toxin sequence in UniProtKB Swiss Prot or the original INSDC database Patent ID Text In cases where a toxin has been patented and this information is publicly available you can search using the patent ID and this field Information Average Reduced Number Search based on the mass of the mature toxin in daltons Da mass Average Most databases provide the mass of the reduced form of Oxidised mass proteins However venom proteins are often rich in cysteines Monoisotopic and these cysteines are invariably found in the oxidized form Reduced mass and as disulfide bonds Thus for venom toxins the oxidized mass Monoisotopic is usually more relevant ArachnoServer thus provides the Oxidised mass mass of both the fully reduced and fully
11. II ArachnoServer SPIDER TOXIN DATABASE ArachnoServer Manual a US orr ENE 2 UTOT en ees eee Ce ee eee E E EE ANE N E NA 2 IN EFOQUCUION ocscararccroranmisiesesarenncvenneemnneniennbeeneesenneieteeeiemunieeoenmneees 3 What data is stored in ArachnoServer and how is it Curated csscsssssscsssssceees 3 Searching ArachnoServer esessssssesecesessesecesesescsoececesssseoeoesesseseosoesesesoeoesesesesoesesese 5 A e COG aaa NE E E E TEA E A 5 Advanced Search PICIOS sisriiseiiiirani i anri anA EA E AA EA EEEE 7 Negating search terms and specifying numeric ranges sss sssssssssssssssrssenrrnssnrrnssnrensnnnnnennnnennnenennnnn 8 Joining search clauses using boolean operators and grouping search clauses 8 Exporting search results and toxin sequences in FASTA format s sssseessssssssresrerrrsrresrerreesrsrens 9 Browsing Arachno Servel pineda a EEEE 10 Each toxin has its own Toxin Card sssssssssesssssosessososeoseseoeossososecsossssossseosessoses 11 General toxin informatio i ssisississio iana a aE emit 12 Schematic of the mature toxin vcesarantvnanatisnesscraterautentaiapicaineaniiaioiesarnasolanemdndniyentnnnindieniatin 12 Toxin card le cecigsc 0 0l eee ae a ee a ee en eee 13 TOT OIA ee te cess estes rete ete sansa E a doce E EE A E E aes 14 POOGEN qt aa atten nen eee Tera ere renter terra mrt were sree Martone ae see rte Taree reer 14 DOCODOSS GCCCSSIONS A nnn wanna E 15 TCC QUOT CPV COS aenn czars ee
12. Thomas L Madden Alejandro A Sch ffer Jinghui Zhang Zheng Zhang Webb Miller and David J Lipman 1997 Gapped BLAST and PSI BLAST a new generation of protein database search programs Nucleic Acids Res 25 3389 3402 Query as Ul agatoxin Talb sp 046167 Paralytic insecticidal toxin TaITX 2 from the spider Tegenaria agrestis 68 letters Database arachnoserver 897 sequences 79 211 total letters SOEMTOHING sc ccccccccccncccccccncscccecesancsccescccseccccceces done Score E Sequences producing significant alignments bits Value as Ul agatoxin Talb sp 046167 Paralytic insecticidal toxin 152 3e 40 as Ul agatoxin Talc sp 046168 Paralytic insecticidal toxin 149 2e 39 as Ul agatoxin Tala sp 046166 Paralytic insecticidal toxin 145 2e 38 as Alpha latrotoxin associated LMWP sp P49125 Low molecular 33 2e 04 as delta ctenitoxin Pnla_2 sp P59368 Toxin from venom of the 25 0 076 as delta ctenitoxin Pnla 1 sp 2108421A Toxin from venom of t 23 0 22 as delta ctenitoxinNenlb sp P84034 Toxin from venom of the s 23 0 22 as U15 ctenitoxin Cola sp P85264 Toxin from venom of the spi 22 0 38 as U14 lycotoxin Lslc sp 36DD39 Toxin from venom of the spi 22 0 49 as U12 ctenitoxin Cola sp P85265 Toxin from venom of the spi 22 0 49 as Ul ctenitoxin Pnla sp P61229 Toxin from venom of the spid 22 0 49 as alpha Latrotoxin associated LMWP2 sp Q4U4N3 720 Low molecular 22 0 64 as U14
13. ainst Escherichia coli We can further limit the number of toxins by any number of criteria For example a medicinal chemist may be interested in only those toxins with a solved structure To find this subset of toxins add an extra search clause choose And Number of solved PDB structures greater than or equal to gt gt 1 then click the Search button This limits the results to only three toxins Figure 6 Search Toxins help Biological Activities include Antimicrobial grouping and Phyletic Specificities include Escherichia coli 2C and Number of solved PDB structures s fons 1 Search Reset 3 toxins found displaying all toxins Name Synonym Genus Species Number of PDBs as EAn laa M ctenitoxin Cs 1a Cupiennin 1a Cupiennius salei M theraphotoxin Gria GsMTx4 Grammostola rosea f M zodatoxin Lt2a Latarcin 2a Lachesana tarabaevi 3 toxins found displaying all toxins Export options gt XML AJ PDF Get All Fasta Sequences Figure 6 The results of adding an extra search term to the advanced search query that further limits the results to show only toxins with a solved PDB structure The advanced search interface is extremely flexible For example with the above query we could change the species from Escherichia coli to Arthrobacter globiformis click
14. ass 4120 73 Average Oxidised Mass 4114 66 Monoisotopic Reduced Mass 4117 78 Monoisotopic Oxidised Mass 4111 74 Figure 13 Dynamically generated image of the mature toxin sequence showing location of disulfide bonds horizontal blue lines and pharmacophore residues green triangles Several categories of evidence for disulfide bond annotations are visually displayed In the figure above all disulfide bonds have been experimentally determined However if this is not the case they will appear as either defined by homology or predicted see the key below the sequence where each evidence level is displayed slightly differently in the image Likewise two categories of evidence for pharmacophore are displayed experimentally determined and by homology The mature toxin displayed above does not have any identified posttranslational modifications ArachnoServer also displays information on the mass of the mature toxin sequence within the sequence image Masses displayed are the average and monoisotopic reduced mass as well as the average and monoisotopic oxidized masses This information is also searchable from the advanced search interface Toxin card sub categories To minimise clutter on the screen and allow for fast access to required information sub categories of toxin data have been collapsed within the toxin card Figure 14 s Taxonomy Biological Activity s Accessions Literature Referen
15. can be searched using this field Toxin Accession Text Use this field to search for a toxin by its accession number in Number one of the INSDC databases or other data repository such as Gene Ontology or InterPro PDB structures number of solved structures submitted to PDB Biological Activities of species is sourced from the NCBI Taxonomy Phyla Effective When known search for toxins with specific ED50 LD50 and Dosage ED50 PD50 in consistent units of pmol g All literature units have Biological Lethal Dosage been converted to pmol g to allow for easy comparison of Activity LD50 and Paralytic toxins Dosage PD50 Activities known biological activities Molecular Targets modifications modification Number of Number Search for toxins with a specific number of posttranslational Protein Posttranslational modifications Information modifications Number of Disulfide Number Search according to the number of disulfide bonds in a toxin Bonds regardless of whether they are classified as experimentally determined predicted or by homology to another toxin List Search the toxins according to the species of spider from which they derive Species names are sourced from the World Spider Catalog Species with an asterisk next to them also have downloadable photos available Source Family List Search the database for toxins from spiders belonging to a Species specific taxonomic family Taxonomy Search the database according to common
16. ces s Protein Information s Sequences s Toxin Synonyms Toxin Structure Figure 14 Sub categories of information available for a typical toxin Clicking the category heading will make the information from this category appear Each category is expandable by clicking the category heading If data is not available for a toxin from a particular category the category will not appear for example if no solved PDB structure exists then the Toxin Structure category will not appear Following is a description of the information contained within each of the category 13 Taxonomy This section lists the current and historic taxonomy sourced from the World Spider Catalog Figure 15 shows an example As can be seen in this example Grammostola rosea has a long history of taxonomic reclassification In cases where researchers may not be aware of these changes or when information from the literature is outdated the advanced search interface of ArachnoServer enables searching for toxins using this historic taxonomy The current taxonomic family Theraphosidae for this example in blue links to the World Spider Catalog entry for that family Taxonomy Current Taxonomy Historic Taxonomy Phylum __Athropod omens Grammostola argentinensis Genus Gammstle Sarena pa Figure 15 Taxonomy Section of the toxin card showing the current and historic taxonomy both sourced from the World Spider Catalog Biological activ
17. cle or information about the patent Literature References Original Deposition References Swartz K J MacKinnon R Neuron 15 941 949 1995 An inhibitor of the Kv2 1 potassium channel isolated from the venom of a Chilean tarantula Swartz K J MacKinnon R Neuron 18 665 67 1997 ifi ti Swartz K J MacKinnon R Neuron 18 675 682 1997 Mapping the receptor site for hanatoxin a gating modifier of voltage dependent K channels Li Smerin Y Swartz K J Proc Natl Acad Sci U S A 95 8585 8589 1998 Gating modifier toxins reveal a conserved structural motif in voltage gated Ca2 and K channels Takahashi H Kim J I Min H J Sato K Swartz K J Shimada I J Mol Biol 297 771 780 2000 Solution structure of hanatoxin1 a gating modifier of voltage dependent K channels common surface features of modifier toxins Other References Garrett S A Mueller A L Sanguinetti M C Perspectives in Drug Discovery and Design 15 16 71 81 1999 Peptide toxins isolated from spider venom that modulate gating of voltage dependent K channels Li Smerin Y Swartz K J J Gen Physiol 115 ee Figure 17 Example literature table from the toxin card for k theraphotoxin Gr1a Each citation is linked to its Pubmed record where available Literature references include research articles details about sequence deposition dates and patent information The references are normally divided into two groups those for the
18. earch results 9 Figure 10 Browsing ArachnoServer by Araneae taxonomy 10 Figure 11 Browsing ArachnoServer using the molecular target ontology to 11 Figure 12 Toxin card for k theraphotoxin Gr1a All information about this toxin is summarised on this page eae aE ev eT a ae EE al EEE Oty Oe SECT Figure 13 Dynamically generated image of the mature toxin sequence showing location of 13 Figure 14 Sub categories of information available for a typical toxin Clicking 13 Figure 15 Taxonomy Section of the toxin card showing the current and historic 14 Figure 16 The Biological Activity section of a toxin card displays information 14 Figure 17 Example literature table from the toxin card for k theraphotoxin Gr1la 16 Figure 18 Protein information for the toxin U2 agatoxin Aolg from Agelena orientalis showing the signal propeptide and mature toxin sequences as well the C terminal Gly residue that is excised in the process of C terminal amidation The protein information section also lists locations and available evidence for disulfide bonds and posttranslational modifications 17 Figure 19 Protein information for the toxin k hexatoxin Hv1c Hadronyche versuta showing multiple transcripts with slightly different signal and propeptide sequences 17 Figure 20 Sequences stored in ArachnoServer for the toxin k hexatoxin Hv1c some mRNA sequences have been excluded for brevity All sequences can be directly sent to the BLAST page from here For
19. ei http www arachnoserver org toxincard html id AS000292 21 The non zero digits are retrieved from the toxin ID found in the URL for all toxins then the ID is padded with zeros in front to make a six digit number Toxin IDs in ArachnoServer are persistent and will never change for that toxin UniProtKB Swiss Prot mapping to ArachnoServer Data from ArachnoServer is now being used to assist the curators at UniProtKB Swiss Prot to provide the most up to date descriptions for spider toxin characterisation The rational toxin names recommended in ArachnoServer are also being adopted by UniProtKB Swiss Prot and are directly searchable from their interface You can map UniProtKB Swiss Prot identifiers to ArachnoServer toxin entries or vice versa by using the UniProtKB Swiss Prot ID Mapping Service just select the ArachnoServer database under the Organism Specific Databases section 22
20. enitoxin Cs 1a Cusieriin 1a Cupiennius salei M ctenitoxin Cs 1b Cupiennin 1b Cupiennius salei M ctenitoxin Cs 1c Cupiennin 1c Cupiennius salei M ctenitoxin Cs1d_ Cupiennin 1d Cupiennius salei Figure 4 Using the advanced search interface to search for toxins with antimicrobial activity We can further limit the list of toxins by refining the search to include only those with known activity on Escherichia coli From the search interface click the plus button to create a new search clause and then choose And gt Phyletic Specificities gt include Escherichia coli Then click the Search button This time the search returns only 11 toxins the first three of which are shown in Figure 5 Search Toxins help Biological Activities include Antimicrobial grouping and Phyletic Specificities include Escherichia coli gt Search Reset 11 toxins found displaying all toxins Name Synonym Genus Species M ctenitoxin Cs 1a Cupiennin 1a Cupiennius salei M ctenitoxin Cs1d pereen 1d Cupiennius salei M theraphotoxin Gria GsMTx4 Grammostola rosea M zodatoxin Lt1a Latarcin 1 Lachesana tarabaevi Figure 5 The results of adding an extra search term to the advanced search query to limit the results to only those toxins with antimicrobial biological activity ag
21. epc E catty E E E E deepest ce tea 16 Ng GOLGI EAM LLY ROL a A A E IA oe E en eee ener ee ent ee eee tee 16 E O c A N N E NE A ere oar anne A Nee nem Tone er mere pre renner A Serre reer erry eer Ts 17 TOT IIT SUM OVNI ecru pe cee sessed aves est aes ee seas aaa a ths ass A EA meee 18 VORA Ea 63 00 a A A E nen N RC E E E E ree 19 Running similarity searches sesesessssesesesesesosoececesssosoececssesoececesesosoeosoesesossesesessee 20 Linking to ArachnoServer s esesesesessssesecesessscesesecesesosoecesesosseoececesosoececesesossesesessee 21 UniProtKB Swiss Prot mapping to ArachnoServer ssssssssssssssssseeccosssesessseeeosssss 22 THE UNIVERSITY z iQ 7 aa M B Institute for Molecular Bioscience In collaboration with iQ FAB List of Figures Figure 1 Cumulative number of spider toxins deposited into public databases since 1984 3 Figure 2 Data sets integrated into ArachnoServer 4 Figure 3 The basic search field which is available on the banner of every page 5 Figure 4 Using the advanced search interface to search for toxins with antimicrobial activity 5 Figure 5 The results of adding an extra search term to the advanced search query to limit 6 Figure 6 The results of adding an extra search term to the advanced search query 6 Figure 7 Radio buttons used to group search clauses 8 Figure 8 An example of joining search clauses together using the grouping radio buttons g Figure 9 Export options available for s
22. formation available on the structure of spider toxin genes ArachnoServer currently holds 926 protein sequences 632 mRNA sequences and 29 DNA sequences The sequences section is a convenient location to perform similarity searches of ArachnoServer for related toxins More information on this is provided in the section below on Running Similarity Searches 17 Sequences gt as x hexatoxin Hvlc 1 sp P82228 Insecticidal toxin from Full BLAST BLAST mature Blue Mountains funnel web spider Hadronyche versuta toxin only AICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP gt as x hexatoxin Hvlc_2 Translation x hexatoxin Hvlc Full BLAST BLAST mature insecticidal toxin XenFW386 from Blue Mountains funnel web toxin only spider Hadronyche versuta MNTATCFIVLLDVATVIGGIEAGES DMRKDVMGLFRRAICTGADRPCAACCPCCPGTSCK AESNGVSYCRKDEP gt as k hexatoxin Hvlc_3 Translation x hexatoxin Hvlc Full BLAST BLAST mature insecticidal toxin XenFW393 from Blue Mountains funnel web toxin only spider Hadronyche versuta MNTATCFIVLLVVATVIGGIEAGEFDMRKDVMGLFRRAICTGADRPCAACCPCCPGTSCK AESNGVSYCRKDEP gt as x hexatoxin Hvlc_ 4 Translation x hexatoxin Hvlc Full BLAST BLAST mature insecticidal toxin XenFW402 from Blue Mountains funnel web toxin only spider Hadronyche versuta MNTATCFIVFLVVATVIGGIEAGES DMRKDVMGLFRRAICTGADRPCAACCPCCPGTSCK AESNGVSYCRKDEP RNA Sequences gt as Kk hexatoxin Hvlc 2 cDNA sequence XenFW386 for Full BLAST insecticidal toxin x hexatoxin Hvlc
23. gs can be specified using the radio buttons at the right hand side of the search clauses and look like this grouping Figure 7 Radio buttons used to group search clauses Groups are defined vertically in the radio buttons By default each search clause is placed into its own group as in Figure 7 In such a case each clause will be joined with the clause immediately preceding it and immediately following it if those clauses exist If you would like to change this behaviour you can choose to group the clauses together Consider the example in Figure 8 Search Toxins help Family 44 is need Hexathelidae 44 grouping or a4 Family H is pd Theraphosidae B C and bd Biological Activities B include Neurotoxin Lethal b d 2 O O or F Biological Activities b include 14 Neurotoxin Paralytic F 20 BC DIG Search Reset Figure 8 An example of joining search clauses together using the grouping radio buttons This search is asking show me all toxins from the families Hexathelidae or Theraphosidae which display a biological activity of either neurotoxin lethal or neurotoxin paralytic The four search clauses are grouped together into two groups the first group specifies the Araneae families and the second the biological activities It s easy to see that if we changed the second or to and then this search would return only those toxins in either family with both neurotoxin
24. h term is entered and the search button is clicked the search will return all toxins in the database funnel web search Figure 3 The basic search field which is available on the banner of every page Advanced searches For more specific queries ArachnoServer provides an advanced search interface Context specific fields are dynamically arranged on the page and search keys populated in drop down lists of relevant data from the database Search clauses can be grouped then joined using boolean operators such as or and and A large selection of database fields can be searched including all basic search fields as well as additional features such as biological activity posttranslational modifications literature references and counts of various data fields e g the number of disulfide bonds in a toxin The figures below show an advanced search being constructed In this search we are particularly interested in finding toxins that have antimicrobial activity Select Biological Activities gt include Antimicrobial then click the Search button This returns 40 toxins from the database although only the first four toxins are displayed for brevity in Figure 4 Search Toxins help Biological Activities include Antimicrobial grouping Search Reset 40 toxins found displaying 1 to 15 FirstPrev 1 2 3 Next Last Name Synonym Genus Species M ct
25. iivia asronyere vena ohoatxintive watacotonniwie Hadenyehe vema Figure 11 Browsing ArachnoServer using the molecular target ontology to identify all toxins that target invertebrate voltage gated calcium channels Each toxin has its own Toxin Card Each toxin in the database is provided with its own page or toxin card Toxin cards are accessed from the results page of any Search or Browse or directly if the ArachnoServer toxin ID is known The toxin card summarises all available information for this toxin in a single place It has been designed to minimise information clutter by collapsing categories of information that are likely to be only of interest to particular groups of researchers As an example Figure 12 shows the toxin card for x theraphotoxin Gr1la The following sections explain 11 I ArachnoServer SPIDER TOXIN DATABASE Home 7 search 7 srowse BLAST Toxin Name k theraphotoxin Gria Source Species Grammostola rosea Chilean rose tarantula Toxin Group Theraphotoxin Description The toxin inhibits Kv2 1 and Kv4 2 voltage gated potassium channels but not Shaker IR Kv1 1 Kv1 3 Kv1 6 Kv2 1 Kv3 1 and eag channels Inhibits also voltage gated calcium channels Cav2 1 with a lower affinity compared to Kv channels The toxin acts as a gating modifier by shifting channel openings to more depolarized voltages and multiple toxin molecules can simultaneously Sex female Carapace length 28mm bind
26. ity This section reports all biological activities for example neurotoxin lectin antimicrobial and others as well as known molecular targets Where available and relevant the EDso ICs0 and Ka values are reported for each molecular target accompanied with a comment Figure 16 Molecular Target EDO iC50 Ka Phamacophore Comment Potassium channel Inhibition of Kv2 1 expressed in oocytes by voltage gated native toxin mixture of k TRTX Gria and K vertebrate Ky2 1 TRTX Gria The Kd for synthetic k TRTX Gria drk1 is 160 nM The Kd values are based on the assumption of four equivalent and independent toxin binding sites per channel Calcium channel i Inhibition of Ca2 1 expressed in oocytes by voltage gated native toxin vertebrate Cay2 1 Potassium channel Inhibition of Kv4 2 expressed in oocytes by voltage gated recombinant toxin vertebrate Ky4 2 Figure 16 The Biological Activity section of a toxin card displays information about known biological activities and molecular targets 14 The list of biological activities in ArachnoServer is under constant curation Table 2 lists the biological activities currently recognised in ArachnoServer and the number of toxins associated with each activity Table 2 List of biological activities recognized in ArachnoServer at the time of writing with the number of toxins currently associated with that particular activity Antiparasitic 9 o 2 Protease activit
27. lycotoxin Lsla_3 sp B6DD38 Toxin from venom of the s 21 0 84 as U34 theraphotoxin Cjla sp B1P1J2 Toxin from venom of the 21 1 1 as U14 lycotoxin Lslb 2 sp 36DD37 Toxin from venom of the s 20 1 4 as U14 lycotoxin Lslb_ 1 sp B6DD36 Toxin from venom of the sS 20 1 4 as U14 lycotoxin Lsla_2 sp B6DD35 Toxin from venom of the S 20 1 4 as U14 lycotoxin Lsla_1 sp B6DD34 Toxin from venom of the s 20 1 4 as U2 ctenitoxin Pkla sp P83905 Toxin from venom of the spid 20 1 4 as U13 ctenitoxin Cola sp P85263 Toxin from venom of the spi 20 1 9 as omeqa lvcotoxin Gsp 2671 1blsp A9XDGO Presumed Cav2 1 bloc 20 2 4 Figure 24 BLAST results as formatted in ArachnoServer Each hit is linked to the corresponding toxin card in the database as well as to Uni ProtKB Swiss Prot records where available The links in the score column provide a quick mechanism to access a text representation of the alignment for a particular hit BLAST databases are stored on the server and are updated every 15 minutes to ensure that the similarity searches most accurately represent the curated data in ArachnoServer Linking to ArachnoServer ArachnoServer entries are externally identifiable by accessions with the letters AS followed by six digits These accessions can be used to link to ArachnoServer for example the following hypertext reference links to the toxin w ctenitoxin Cs1la from the wandering spider Cupiennius sal
28. nces and protein structures when available for each individual toxin During this process we record all other database identifiers for example NCBI taxonomy codes Gene Ontology classifications PROSITE and Pfam accessions and others see the Database accessions section for more information in the ArachnoServer database We also keep records of all literature references sequence and structure feature annotations for example known locations of disulfide bonds and descriptions of toxins ArachnoServer stores additional data sets summarized in Figure 2 These include e The World Spider Catalog e NCBI Taxonomy for association of a toxin s phyletic specificity to a consistent taxonomic ontology e A molecular target ontology we specifically created based on the channel and receptor subtype definitions and nomenclature recommended by the International Union of Basic amp Clinical Pharmacology IUPHAR e Lists of post translational modifications sourced from ConoServer with some additions biological activities sequence features spider common names and even units used to describe LDso PDso0 and ICso values e A repository of high resolution photos for spiders with toxins in the database e M ya n eS NCBI UniProt 2P B PROTEIN DATA BANK Guo iw of basic and clinical pharmacology a gt f D iiis p D A T Kg 5 3Taxonomy Figure 2 Data sets integrated into ArachnoServer Th et M A 2
29. ng the embedded molecular structure viewer Jmol Multiple structures have been solved for this toxin each of these can be viewed by clicking the appropriate button displaying the PDB accession number at the top of the structure viewer 19 Many alternative viewing options are available in Jmol so long as you have a three button mouse Right click the viewing panel to open a menu then select the view and style you prefer Alternative surfaces can also be added For a full description of what Jmol can offer please visit their web site http www jmol org Running similarity searches ArachnoServer makes available all toxin sequences for similarity searches using BLAST Basic Local Alignment Search Tool Click the BLAST tab and then paste into the text area your sequence of interest Select the type of query sequence Protein or DNA and the database to search against either Peptide or Nucleic Sequences Once both of these selections have been made you can select the BLAST program to use For a description of BLAST programs see here All main BLAST programs are available in ArachnoServer Figure 23 shows the BLAST interface and some of the available options BLAST Toxins Paste input sequence in FASTA format here gt as U1 agatoxin Talb sp 046167 Paralytic insecticidal toxin TalTX 2 from the spider Tegenaria agrestis MKLOLMICLVLLPCFFCEPDEICRARMT NKEFT YKSNVCNNCGDQVAACEAECFRNDVYT ACHEAQKG Query sequence type Protein
30. original deposition and additional references Where possible each of the citations will link to the PubMed page for that article ArachnoServer currently contains over 1650 citations sourced from more than 400 distinct journal articles in 80 different journals excluding patent records and references to original deposition details in public databases All of these literature articles including the names of original deposition authors as well as patent information are directly searchable from the advanced search interface Protein information This section contains detailed information on the primary structure of each toxin such as schematics of the signal propeptide and mature toxin sequences if known the disulfide bond scaffold and any posttranslational modifications Figure 18 16 Protein Information MKAIISLLLISAMVFSMIEAVPYVEEGLOLFEGERGCLPHNRFCNALSGPRCCSGLRCKELSIWDSRCLG SignalSequence Propeptide Sequence Excision Mature Toxin Disulfide Bonds Posttranslational modifications Residue italien Type Symbol 34 C terminal amidation By similarity NH2 if f Left Right Residue Residue 2 18 By homology 9 23 By homology S ee By homology Evidence Figure 18 Protein information for the toxin U2 agatoxin Ao1g from Agelena orientalis showing the signal propeptide and mature toxin sequences as well the C terminal Gly residue that is excised in the process of C te
31. oxidized forms of the toxin Moreover ArachnoServer also provides the monoisotopic mass which is used in mass spectrometry as well as the more commonly used average mass The advanced search enables searches using any of these mass categories Mature Toxin Mass Negating search terms and specifying numeric ranges We have tried to make the advanced search as intuitive as possible by providing an interface that closely resembles natural language while being specific enough to drill down into the database quickly and accurately For example if text can be used to specify a search term we provide like not like is and is not where terms specified in conjunction with like and not like are wrapped with wild card characters Numeric data can be specified using a range with equals not equals greater than or equal to and less than or equal to Note that only the first field is required for numeric data but a full range can be specified using both available fields Joining search clauses using boolean operators and grouping search clauses The advanced search interface allows for up to 6 fields to be joined in a single search clause using boolean operators To add a search field press the plus button on the far right of the screen Search clauses are joined with one of three boolean terms and or and but not Search clauses can also be grouped together Groupin
32. protein sequences either the full sequence or just the mature toxin sequence can be used as the search query ___ 18 Figure 21 A list of synonyms for k hexatoxin Hv1c 18 Figure 22 Structure PDB 1RYG of the toxin u theraphotoxin Hhn1b viewed in ArachnoServer using the embedded molecular structure viewer Jmol Multiple structures have been solved for this toxin each of these can be viewed by clicking the appropriate button displaying the PDB accession number at the top of the structure viewer 19 Figure 23 BLAST form in ArachnoServer populated with a toxin from 20 Figure 24 BLAST results as formatted in ArachnoServer Each hit is linked to the corresponding __ 21 List of Tables Table 1 Database fields searchable using ArachnoServer s advanced search interface urssssserssseresseneees 7 Table 2 List of biological activities recognized in ArachnoServer at the time of writing with the number of toxins currently associated with that particular activity wessssssssssssseesssssssssnssssssssssesssienssesneessneens 15 Table 3 List of all external databases with entries linked to toxins in ArachnoServer s s s 15 Introduction ArachnoServer provides a single source of high quality manually curated information about proteinaceous spider toxins The database has been designed specifically for the research needs of a wide rage of biological scientists including pharmacologists neuroscientists toxinologists medicinal chemists ion channel
33. rminal amidation The protein information section also lists locations and available evidence for disulfide bonds and posttranslational modifications In some spiders different prepropeptide transcripts can encode the same mature toxin i e the transcripts have the same mature toxin sequence but have at least one difference in the signal or propeptide region As each toxin in ArachnoServer is uniquely defined by its mature toxin sequence all such transcripts will be stored on the same toxin card The Protein Information tab provides a quick schematic overview of the range of transcripts encoding the same mature toxin Figure 19 illustrates one such example AICTGADRPCAACCPCCPGTSCKAESNGYS CRKDEP MNTATCFIVLLDVA TVIGGIEAGESDMRKDVMCLFRRAICTGADRPCAACCPCCPGTSCKAESNGYS YCRKDEP MNTATCFIVLLYVA T IGGIEAGEFDMRKDYMGLFRRAICTGADRPCAACCPCCPCTSCKAESNGYS YCRKDEP MNTATCFIVFLYVA TYIGGIEAGESDMRKDVMCLFRRAICTGADRPCAACCPCCPGTSCKAESNGYS Y CRKDEP SignalSequence Propeptide Sequence Excision Mature Toxin Figure 19 Protein information for the toxin k hexatoxin Hvic Hadronyche versuta showing multiple transcripts with slightly different signal and propeptide sequences Sequences All available sequences for a given toxin will be displayed in this section such as protein sequences either with or without signal and propeptide sequences as well as mRNA and DNA sequences if known Figure 20 Note that at the time of writing there is very little in
34. to a single channel The toxin binding sites are situated in a a helical region Photo courtesy of Bastian Rast on the C terminus of segment S3 of the channel 1273 F274 E277 and at least Use of photo governed by creative 20 25 A away from the central pore The toxin partitions into the membrane and E EO O interacts with the voltage sensor paddle Discovered 1995 This toxin last updated on Jun 02 2009 F 2 g 15 L6 2L 28 ECRYLFGGCKT TSDCCKHLGCKFRDKYCAW DFT FS 4 22 240 ai a A E 23 A pharmacophore experimentally determined l disulfide bond iexperimentally determined post translational modification A pharmacophore by homology disulfide bond by homology Cy3 disulfide bond predicted Average Reduced Mass 4120 73 Average Oxidised Mass 4114 68 Monoisotopic Reduced Mass 4117 78 Monoisotopic Oxidised Mass 4111 74 s Taxonomy Biological Activity s Accessions s Literature References f Protein Information Sequences Toxin Synonyms s Toxin Structure ZIQ F A B Erun I M B Institute for Molecular Bioscience Figure 12 Toxin card for k theraphotoxin Gr1a All information about this toxin is summarised on this page General toxin information General information about a toxin is displayed at the top of the card this includes the toxin name source species and the common name the group this toxin belongs to a general description of the toxin
35. utoxin As1a _ Aptotoxin 1 Apomastus schlingeri Araneomorphae 482 g 7 i ji T 3 Mygalomorphae 266 _ U4 cyrtautoxin As 1b Aptotoxin 4 _Apomastus schlingeri pes a a chiens JE _ Actinopodidae 2 U cyrtautoxin As 1c Aptotoxin 6 Apomastus schlingeri 3 Cyrtaucheniidae 6 Ee TIE Eee TETERE EEES _ Apomastus 6 U cyrtautoxin As 1d _ Aptotoxin 9 Apomastus schlingeri H 3 Hexathelidae 67 U gt cyrtautoxin As 1a Aptotoxin 3 Apomastus schlingeri Nemesiidae 3 L a al _ Theraphosidae 188 _ U3 cyrtautoxin As1a _ Aptotoxin 7 Apomastus schlingeri Get All Fasta Sequences Figure 10 Browsing ArachnoServer by Araneae taxonomy Choosing a different ontology will reset the browse page and create a fresh browsing tree for the chosen ontology Browsing using the molecular target ontology is a convenient and powerful way to find all toxins that target for example a particular type or subtype of ion channel or receptor Figure 11 shows the result of browsing using the molecular target ontology to search for toxins that specifically target invertebrate voltage gated calcium channels The molecular target ontology which was developed specifically for ArachnoServer is based on the channel and receptor subtype definitions and nomenclature recommended by the International Union of Basic amp Clinical Pharmacology IUPHAR Browse Toxins Show toxins by ontology Molecular Targets
36. y 2 3 Protease inhibitor rr E Database accessions ArachnoServer stores all relevant and current external database cross references and accession numbers for curated toxins in the database These external entries can be accessed from the Accessions section in each toxin card Where possible each of the accessions is hyperlinked to their entry in the external database Table 3 lists all external databases ArachnoServer links to and the current URL for that database This list will continue to grow as more toxins are characterised and more databases store toxin related information Table 3 List of all external databases with entries linked to toxins in ArachnoServer EMBL _ __________ __ _ _ wwemblog S expasy org prosite Gene Ontology GO wwwegeneontology org o Z Gene3D gene3d biochem ucl ac uk Gene3D swift cmbi ru nl gv hssp PANTHER Ss WwWwpantherdb org Z o Z o PRINTS wwwbioinfmanchester ac uk dbbrowser PRINTS Protein Data Bank PDB www pdborg S SMART smart embl heidelberg de UniProt Knowledge Base www uniprotorg O O 15 Literature references Every toxin in ArachnoServer has been carefully curated using a wide variety of information Much of the additional information within the database is sourced from the reading and recording of results from journal articles and patents When this information is incorporated into the database the curator also records the citation for that arti
37. ynonyms in ArachnoServer Toxin Synonyms Synonym Te Karacan smem OOOO Sanus tacodaraooaniie Sron ACTA Figure 21 A list of synonyms for k hexatoxin Hvic 18 Recommended abbreviations are created using a four or five letter abbreviation of the generic toxin name as described at http www venomics org nomenclature All synonyms are searchable from the advanced search interface Toxin structure ArachnoServer also stores information on solved toxin structures that have been submitted to the Protein Data Bank PDB Currently 43 structures are available and these can be quickly identified using the advanced search field number of solved PDB structures To assist in the viewing of structures ArachnoServer has an embedded molecular structure viewer Jmol an open source Java viewer for chemical structures in 3D http www jmol org In order to use the viewer your browser must support java applets as do all modern web browsers Figure 22 shows one of the solved Structures of the toxin u theraphotoxin Hhnib from the Chinese Black Earth Tiger tarantula Haplopelma hainanum To view an alternative structure simply select the appropriate button at the top of the viewer these buttons display the PDB accession number for the structure Toxin Structure Please click a structure to view PDB LNIY POBLRYG PDB 1RYV Figure 22 Structure PDB 1RYG of the toxin p theraphotoxin Hhn1b viewed in ArachnoServer usi
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