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EBI web resources II:
Ensembl and InterPro
Yanbin Yin
http://www.ebi.ac.uk/training/online/course/
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Homework3
• Gotohttp://www.ebi.ac.uk/interpro/training.html andfinishthe
secondonlinetrainingcourse“Introductiontoproteinclassification
attheEBI”andthenanswerthefollowingquestions:
– Whatisthedifferencebetweenaproteinfamilyandaprotein
domain?
– Canaproteinbelongtomultiplefamiliesorcontainmultipledomains?
– Whatareproteinsequencefeatures?Examples?
– Whatisaproteinsignature?Whatisitusedfor?
– Whatarethemajorsignaturetypes?
– IsPROSITEasequencepatterndatabaseoraprofiledatabase?What
aboutPfam?
– Whatisthedefinitionof“annotation”?
• Inyourreport,answerthesequestionsandalsoincludethescreen
shotofthepage(s)thatsupportyouranswer.
Dueon10/7(sendbyemail)
Officehour:
Tue,ThuandFri2-4pm,MO325A
Oremail:[email protected]
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Outline
• Introtogenomeannotation
• Proteinfamily/domaindatabases
– InterPro,Pfam,Superfamilyetc.
• Genomebrowser
– Ensembl
• HandsonPractice
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Genomeannotation
• Predictgenes(wherearethegenes?)
– proteincoding
– RNAcoding
• Functionannotation(Whatarethesegenes?)
– SearchagainstUniProt orNCBI-nr(GenPept)
– Searchagainstproteinfamily/domaindatabases
– SearchagainstPathwaydatabases
Function vocabularies
defined in
GeneOntology
Proteinscanbeclassifiedintogroups accordingtosequenceorstructuralsimilarity.These
groups oftencontainwellcharacterizedproteins whosefunction isknown.Thus,whena
novelprotein isidentified, itsfunctional properties canbeproposed basedonthegroup to
whichitispredictedtobelong.
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Superfamily
Gene3D
SCOP
CATH
PDB
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InterPro components
1. CATH/Gene3D
2. PANTHER
3. PIRSF
4. Pfam
5. PRINTS
6. ProDom
7. PROSITE
8. SMART
9. SUPERFAMILY
10. TIGRFAMs
11. HAMAP
UniversityCollege, London, UK
Universityof Southern California,CA,USA
ProteinInformation Resource,Georgetown University,USA
Wellcome TrustSangerInstitute,Hinxton, UK
Universityof Manchester,UK
PRABIVilleurbanne, France
SwissInstituteofBioinformatics(SIB),Geneva,Switzerland
EMBL,Heidelberg, Germany
Universityof Bristol,UK
J.CraigVenterInstitute,Rockville,MD,US
SwissInstituteofBioinformatics(SIB),Geneva,Switzerland
CDDcomponents
Pfam,SMART,TIGRFAM,
COG,KOG, PRK,CD,LOAD
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MostUniProt proteinsare
annotatedwithatleastone
InterPro signature
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Proteinfamiliesareoftenarrangedintohierarchies,withproteinsthatsharea
common ancestorsubdivided intosmaller, morecloselyrelatedgroups. Theterms
superfamily (describing alargegroup ofdistantlyrelatedproteins) andsubfamily
(describing asmallgroup ofcloselyrelatedproteins) aresometimesusedinthis
context
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ProteinClassification
Nearlyallproteinshavestructuralsimilaritieswithother proteins and, insomeofthesecases,
shareacommon evolutionary origin. Proteinsareclassifiedtoreflectboth structuraland
evolutionary relatedness.Manylevelsexistinthehierarchy,but theprincipal levelsarefamily,
superfamily andfold, describedbelow.
Family:Clearevolutionarilyrelationship
Proteinsclusteredtogetherintofamiliesareclearlyevolutionarily related.Generally,this
meansthatpairwiseresidue identitiesbetweentheproteins are30%andgreater.
Superfamily:Probablecommonevolutionaryorigin
Proteinsthathavelowsequenceidentities, butwhosestructuralandfunctional features
suggestthatacommon evolutionary origin isprobable areplacedtogetherinsuperfamilies.
Fold:Majorstructuralsimilarity
Proteinsaredefined ashavingacommon foldiftheyhavethesamemajorsecondary
structuresinthesamearrangementandwiththesametopologicalconnections. Different
proteins withthesamefoldoften haveperipheralelementsofsecondary structureandturn
regions thatdifferinsizeandconformation. Proteinsplacedtogether inthesamefold category
maynothaveacommon evolutionary origin: thestructuralsimilaritiescouldarisejustfrom
thephysicsandchemistryofproteins favoringcertainpackingarrangementsandchain
topologies.
http://scop.mrc-lmb.cam.ac.uk/scop/intro.html
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PDB
Structure
Superfamily
Gene3D
Pfam
SMART
ProSite
Function
(literature)
SCOP
CATH
Protein
Sequence
UniProt
GenPept
Evolution
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http://www.cathdb.info/
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fold~class– superfamily ~clan– family– subfamily – domainsequence
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Family- anddomain-based classificationsarenotalwaysstraightforwardandcan
overlap, sinceproteinsaresometimes assignedtofamiliesbyvirtueofthedomain(s)
theycontain.Anexampleofthiskind ofcomplexityisoutlined below
Domaincomposition ofphospholipase D1,whichisanenzymethatbreaksdown
phosphatidylcholine. Theprotein containsaPX(phox) domain thatisinvolvedin
binding phosphatidylinositol, aPH(pleckstrin homology) domainthathasarolein
targetingtheenzymetoparticularlocationswithinthecell,andtwoPLD
(phospholipase D)domains responsible fortheprotein’scatalyticactivity
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Sequence featuresdifferfrom domainsinthattheyareusuallyquitesmall(often onlya
fewaminoacidslong), whereasdomains represententirestructuralorfunctional unitsof
theprotein(seeFigure).Sequence featuresareoftennestedwithindomains – aprotein
kinasedomain, forexample,usuallycontainsaproteinkinaseactivesite
Sequences featuresaregroups of aminoacidsthatconfercertaincharacteristicsupon a
protein, andmaybeimportant foritsoverallfunction.Suchfeaturesinclude:
activesites,whichcontainamino acidsinvolvedincatalyticactivity.
binding sites,containing aminoacidsthataredirectlyinvolvedinbinding moleculesorions.
post-translationalmodification (PTM)sites,whichcontainresiduesknown tobechemically
modified (phosphorylated, palmitoylated, acetylated,etc)aftertheprocessofprotein
translation.
repeats,whicharetypicallyshort aminoacidsequencesthatarerepeatedwithinaprotein,
andmayconferbinding orstructuralproperties upon it.
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Handsonexercise1:search
againstproteinfamilydatabases
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http://www.ebi.ac.uk/interpro/
http://cys.bios.niu.edu/yyin/teach/PBB/csl-pr.fa,putthefirstsequence inthesearchbox
HitSearch;takeabout1min
ReadmoreaboutInterPro
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http://www.ebi.ac.uk/interpro/release_notes.html
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ClicktolinktoInterPro pageofthisdomain
Clicktolinktoindividual databasewebsite
Theseare
individual
family/domain
matchesnot
integratedin
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Thisislinked fromtheprevious page:theInterPro pagetodescribeIPR029044
ScientificliteratureforthisIPRfamily
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http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
NCBI’sConserved DomainDatabase(CDD):equivalentto
InterPro ofEBI,much faster,but integratelessmember
databases
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Genomebrowser:ENSEMBL
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http://www.ensembl.org/
TheEnsembl projectaimstoautomatically annotate genomesequences,integrate
thesedatawithother biologicalinformation andtomaketheresults freelyavailable
togeneticists,molecular biologists,bioinformaticians andthewiderresearch
community. Ensembl isjointly headedbyDr StephenSearleattheWellcome Trust
SangerInstitute andDr PaulFlicek attheEuropeanBioinformatics Institute (EBI).
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Whatdoweneedingenomebrowsers?
TomakethebareDNAsequence,itsproperties,andtheassociatedannotations
moreaccessiblethroughgraphicalinterface.
Genomebrowsersprovideaccesstolargeamountsofsequencedataviaagraphical
userinterface.Theyuseavisual,high-leveloverviewofcomplexdatainaformthat
canbegraspedataglanceandprovidethemeanstoexplorethedatainincreasing
resolutionfrommegabase scalesdowntothelevelofindividualelementsofthe
DNAsequence.
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Short tutorialvideosintroducing ENSEMBL
http://useast.ensembl.org/info/website/tutorials/index.html
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http://useast.ensembl.org/info/website/tutorials/index.html
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http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/data.shtml
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Nature491,56-65(01November2012)
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Nature458,719-724(9April2009)
NATURE|Vol 464|15April2010
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Whileausermaystartbrowsing foraparticulargene, theuserinterfacewilldisplaythe
areaof thegenome containing thegene, alongwithabroader contextofother
information availableintheregion ofthechromosome occupied bythegene.
Thisinformation isshown in“tracks,”witheachtrackshowing eitherthegenomic
sequencefrom aparticularspeciesoraparticularkindofannotation onthegene.The
tracksarealignedsothattheinformation about aparticularbaseinthesequenceislined
upandcanbeviewedeasily.
Inmodern browsers, theabundance ofcontextualinformation linked toagenomicregion
notonly helpstosatisfythemostdirectedsearch,butalsomakesavailableadepth of
contentthatfacilitatesintegrationofknowledge aboutgenes, geneexpression, regulatory
sequences, sequenceconservationbetweenspecies,andmanyother classesofdata.
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•Ensembl GenomeBrowsers:http://www.ensemblgenomes.org
•NCBIMapViewer:http://www.ncbi.nlm.nih.gov/mapview/
•UCSCGenome Browser:http://genome.ucsc.edu
Eachusesacentralizedmodel, wherethewebsiteprovides accesstoalargepublic
databaseofgenome dataformanyspeciesandalsointegratesspecializedtools,such
asBLASTatNCBIandEnsembl andBLATatUCSC.
Thepublic browsersprovideavaluableservicetotheresearchcommunity byproviding
tools forfreeaccesstowholegenome dataandbysupporting thecomplexandrobust
informaticsinfrastructure requiredtomakethedataaccessible
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Handsonexercise2:Ensembl
genesearch
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http://www.ensembl.org/
Clicktolinktohumanpage
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Put“livercancer”inthesearchboxandGo
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Thiskeywordsearchgiveseverything thatcontains“livercancer”
ClickonTableto
haveatableview
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Clickonthenumberstoonly
showgeneentries
Thiscoltellsthecategoryoftheentry
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Thisisthelistofgenes
Thefirsttwoentriesinthispageare
ncRNA genes.Let’strythe2nd one
ClickheretoshowthelistandselectLocation
andScoretoshowchromosome locationinfo
andscorerespectively
Scoreiscalculatedbasedonthequery:
howmuchtheannotationdescription
issimilartothesearchingkeyword
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(livercancer)
Nowit’sshowing theGene;therearealsoothertabs
Manythings canbeexplored
ThisisENSEMBLGeneID
LinktoNCBI
ThisisENSEMBLTranscriptID
Thisisisalong intergenic
non-coding RNA gene
Hereisthe
graphical
representation
ofthegene
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Let’stryaprotein-coding gene:LAT1,alsoknown asSLC7A5
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Clickhere
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Clicktoviewthesequencepage
Differentnamesofthegene
Thethreetranscripts
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Nowchecktheexpression
Clicktoopen ahelppagetoexplain
whatthesehighlights mean
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Differentgenome-wide expressionstudies
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Linkstoother
genome browsers
Zoomed inview
Thisiswherethegeneislocatedin
thewholechromosome view
Further
zoomed in
view
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ThisisthesameregionintheUCSCbrowser
PS:muchfasterandeasiertouse/understand thanENSEMBL(richerinfo?)
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Nextlecture:ExPASy andDTU
tools
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