NCBI Molecular Biology Resources A Field Guide part 2 September 30, 2004 ICGEB

Database Searching with Entrez Using limits and field restriction to find human MutL homolog Linking and neighboring with MutL Mapping SNPs onto structure and the genome

Global Entrez Search

Document Summaries: MutL[All Fields]

Entrez Nucleotides: Limits & Preview/Index Tabs

Entrez Nucleotides: Limits Accession All Fields Author Name EC/RN Number Feature key Filter Gene Name Issue Journal Name Keyword Modification Date Organism Page Number Primary Accession Properties Protein Name Publication Date SeqID String Sequence Length Substance Name Text Word Title Uid Volume Field Restriction MutL Exclude bulk sequences

Entrez Nucleotides: Limits MutL Title == Definition Exclude Bulk Sequences

Document Summaries: Limits

Adding Terms: Preview/Index Accession All Fields Author Name EC/RN Number Feature key Filter Gene Name Issue Journal Name Keyword Modification Date Organism Page Number Primary Accession Properties Protein Name Publication Date SeqID String Sequence Length Substance Name Text Word Title Uid Volume

Human MutL Search Results

Human MutL RefSeq GenBank Records

NM_000249: Links

Literature Links PubMed OMIM

NM_000249: PubMed Books

Books Link

OMIM: Human Disease Genes Conserved Domain

Sequence Links Nucleotide Protein

NM_000249: Related Sequences similarity Original GenBank mRNAs Original GenBank genomic Genome Project BAC

The Tax Browser NCBI’s Taxonomy Taxonomy Link The Tax Browser NCBI’s Taxonomy

Taxonomy Link

The Tax Browser Nucleotide Protein Structures Popset

Marsupial PopSets

Mammalian Phylogenetic Study

Batch Downloads

Batch Downloads: FASTA and GI list

Batch Entrez / Entrez-utilities

Links Between and Within Nodes Word weight Computational PubMed abstracts Taxonomy 3-D Structure 3 -D Structures VAST Genomes Phylogeny Computational Nucleotide sequences Protein sequences BLAST BLAST Computational Computational

Text Pubmed Sequence BLAST Structure VAST

BLAST® Basic Local Alignment Search Tool Why align sequences ? - because it is the best way to infer structure-function relationships for the unknown biomolecules Global vs local alignments BLAST basics MegaBLAST Discontiguous MegaBLAST

Global vs Local Alignment Seq 1 Seq 2 Global alignment Seq 1 Seq 2 Local alignment

Global vs Local Alignment Seq1: WHEREISWALTERNOW (16aa) Seq2: HEWASHEREBUTNOWISHERE (21aa) Global Seq1: 1 W--HEREISWALTERNOW 16 W HERE Seq2: 1 HEWASHEREBUTNOWISHERE 21 Local Seq1: 1 W--HERE 5 Seq1: 1 W--HERE 5 W HERE W HERE Seq2: 3 WASHERE 9 Seq2: 15 WISHERE 21

Basic Local Alignment Search Tool Calculates similarity for biological sequences Finds best local alignments Heuristic approach based on Smith-Waterman algorithm Searches for matching “words” and then extends the hits Uses statistical theory to determine if a match might have occurred by chance

Align program (Lipman and Pearson) Global Alignment Align program (Lipman and Pearson) Human: 15 IAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFH 84 +A + + + DL F K D+L I+ T+ W+ GR G IP+NYV + + +++ PW+ Worm: 63 VALFQYDARTDDDLSFKKDDILEILNDTQGDWWFARHKATGRTGYIPSNYVAREKSIES------QPWYF 125 Human: 85 GKITREQAERLLYPP--ETGLFLVRESTNYPGDYTLCVSCDGKVEHYRI-MYHASKLSIDEEVYFENLMQ 151 GK+ R AE+ L E G FLVR+S + D +L V + V+HYRI + H I F L Worm: 126 GKMRRIDAEKCLLHTLNEHGAFLVRDSESRQHDLSLSVRENDSVKHYRIQLDHGGYF-IARRRPFATLHD 194 Human: 152 LVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGN-KVA 220 L+ HY +ADGLC L P Y W ++ + ++L++ IG G+FG+V G + N VA Worm: 195 LIAHYQREADGLCVNLGAPCAKSEAPQTTTFTYDDQWEVDRRSVRLIRQIGAGQFGEVWEGRWNVNVPVA 264 Human: 221 VKCIK-NDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGD 289 VK +K A FLAEA +M +LRH L+ L V ++ + IVTE M + +L+ +L+ RGR Worm: 265 VKKLKAGTADPTDFLAEAQIMKKLRHPKLLSLYAVCTRDE-PILIVTELMQE-NLLTFLQRRGRQCQMPQ 332 Human: 290 CLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLT----KEASSTQDTG-KLPVKWTA 353 L++ S V M YLE NF+HRDLAARN+L++ K++DFGL KE TG + P+KWTA Worm: 333 -LVEISAQVAAGMAYLEEMNFIHRDLAARNILINNSLSVKIADFGLARILMKENEYEARTGARFPIKWTA 401 Human: 354 PEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWH 423 PEA +F+TKSDVWSFGILL EI +FGR+PYP + +V+ +V+ GY+M P GCP +Y++M+ CW Worm: 402 PEAANYNRFTTKSDVWSFGILLTEIVTFGRLPYPGMTNAEVLQQVDAGYRMPCPAGCPVTLYDIMQQCWR 471 Human: 424 LDAAMRPSFLQLREQLEHI 443 D RP+F L+ +LE + Worm: 472 SDPDKRPTFETLQWKLEDL 492 human M--------------SAIQ----------------------AAWPSGT------------ECIAKYNFHG M S .. AA SG. . .A ... . worm MGSCIGKEDPPPGATSPVHTSSTLGRESLPSHPRIPSIGPIAASSSGNTIDKNQNISQSANFVALFQYDA 1 20 40 60 Global alignments force a full-length comparison. In this example, the important domains are picked up by both methods, but looking at the first 15 a.a. of the query (60 a.a. of worm) shows how forcing an alignment in this region is not very helpful. 440 450 human REQLEHI--------KTHELHL . .:: . : ... worm QWKLEDLFNLDSSEYKEASINF 500

How BLAST Works Make a lookup table of all “words” in the query Scan the database for matching words Initiate extensions from these matches

Words Query: GTQITVEDLFYNIATRRKALKN GTQ TQI QIT ITV TVE VED Word Size = 3 Word size is adjustable 2 or 3 for protein ( 3 default) > 7 for blastn ( 11 default ) GTQ TQI QIT ITV TVE VED EDL DLF LFY … Make a lookup table of words Neighborhood Words LTV, MTV, ISV, LSV, etc.

Scan Database…Initiate Extensions Protein BLAST requires two hits GTQITVEDLFYNI <------ TVE FFN ------> two neighborhood words (threshold score) Nucleotide BLAST requires exact matches ATCGCCATGCTTAATTGGGCTT <------ CATGCTTAATT ------> exact word match

An Alignment That BLAST Can’t Find… 1 GAATATATGAAGACCAAGATTGCAGTCCTGCTGGCCTGAACCACGCTATTCTTGCTGTTG || | || || || | || || || || | ||| |||||| | | || | ||| | 1 GAGTGTACGATGAGCCCGAGTGTAGCAGTGAAGATCTGGACCACGGTGTACTCGTTGTCG 61 GTTACGGAACCGAGAATGGTAAAGACTACTGGATCATTAAGAACTCCTGGGGAGCCAGTT | || || || ||| || | |||||| || | |||||| ||||| | | 61 GCTATGGTGTTAAGGGTGGGAAGAAGTACTGGCTCGTCAAGAACAGCTGGGCTGAATCCT 121 GGGGTGAACAAGGTTATTTCAGGCTTGCTCGTGGTAAAAAC |||| || ||||| || || | | |||| || ||| 121 GGGGAGACCAAGGCTACATCCTTATGTCCCGTGACAACAAC Hypera postica cysteine proteinase mRNA vs Boophilus microplus cathepsin L-like proteinase precursor Reason: no contiguous exact match of 7 bp.

…but the corresponding amino acid sequences are conserved much better

Protein alignment looks good

…and they have the same domains, too

Local Alignment Statistics High scores of local alignments between two random sequences follow the Extreme Value Distribution Expect Value E = number of database hits you expect to find by chance size of database (applies to ungapped alignments) E = Kmne-S E = mn2-S’ K = scale for search space  = scale for scoring system S’ = bitscore = (S - lnK)/ln2 your score Alignments expected number of random hits Score

Scoring Systems - Nucleotides Identity matrix A G C T A +1 –3 –3 -3 G –3 +1 –3 -3 C –3 –3 +1 -3 T –3 –3 –3 +1 CAGGTAGCAAGCTTGCATGTCA || |||||||||||| ||||| raw score = 19-9 = 10 CACGTAGCAAGCTTG-GTGTCA

Scoring Systems - Proteins Position Independent Matrices PAM Matrices (Percent Accepted Mutation) Derived from observation; small dataset of alignments Implicit model of evolution All calculated from PAM1 PAM250 widely used BLOSUM Matrices (BLOck SUbstitution Matrices) Derived from observation; large dataset of highly conserved blocks Each matrix derived separately from blocks with a defined percent identity cutoff BLOSUM62 - default matrix for BLAST Position Specific Score Matrices (PSSMs) PSI- and RPS-BLAST

BLOSUM62 Common amino acids have low weights R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 X 0 -1 -1 -1 -2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1 A R N D C Q E G H I L K M F P S T W Y V X Positive for more likely substitutions Common amino acids have low weights Rare amino acids have high weights Negative for less likely substitutions

Options for Advanced Blast: Protein Example Entrez queries proteins all[Filter] NOT mammalia[Organism] green plants[Organism] srcdb refseq[Properties] Other advanced -W 2 word size –e 10000 expect value -v 2000 descriptions -b 2000 alignments Limit by taxon Mus musculus[Organism] Mammalia[Organism] Viridiplantae[Organism] Matrix Selection PAM30 -- most stringent BLOSUM45 -- least stringent

Options for Advanced Blasting: Nucleotide Example Entrez Queries nucleotide all[Filter] NOT mammalia[Organism] green plants[Organism] biomol mrna[Properties] biomol genomic[Properties] OtherAdvanced -W 7 word size –e 10000 expect value -v 2000 descriptions -b 2000 alignments

Homology Searches Find a homolog of human CSK in C. elegans Query = c-src tyrosine kinase (CSK) NP_004374 (450 aa) [Homo sapiens] Database = NCBI protein nr Entrez limit: Caenorhabditis elegans [ORGN] Program = BLASTP Query= >gi|4758078|ref|NP_004374.1| c-src tyrosine kinase [Homo sapiens] MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGKLPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELHL Hits to the Conserved Domain Database:

BLAST Graphical Overview SH3 SH2 tyr kinase domain

BLAST Alignments gi|7160701|emb|CAB04427.2| C. elegans KIN-22 protein (corresponding sequence F49B2.5) [Caenorhabditis elegans] gi|17508235|ref|NP_493502.1| Tyrosine kinase with SH2, SH3 and N myristoylation domains, Drosophila suppressor of pole hole homolog (57.5 kD) (kin-22) [Caenorhabditis elegans] Length = 507 Score = 290 bits (742), Expect = 1e-78 Identities = 170/440 (38%), Positives = 245/440 (55%), Gaps = 21/440 (4%) Pick one hit . . .

3D Domains SH2 SH3 TyrKc In this example, 3D Domains and Conserved Domains are similar, with Tyr kinase catalytic domain (CD) composed of 2 3D domains. Yellow is the catalytic loop.

Low Complexity Filtering Filtered Unfiltered sp|P27476|NSR1_YEAST NUCLEAR LOCALIZATION SEQUENCE BINDING PROTEIN (P67) Length = 414 Score = 40.2 bits (92), Expect = 0.013 Identities = 35/131 (26%), Positives = 56/131 (42%), Gaps = 4/131 (3%) Query: 362 STTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLS---SQPQAIVTEDKTD 418 S++S SSS+S SS + + ++S + + S S S+ + E K Sbjct: 29 SSSSSESSSSSSSSSESESESESESESSSSSSSSDSESSSSSSSDSESEAETKKEESKDS 88

Intermission?