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Part 2: Multiple sequence alignment of protein sequences
Overview
Well done! In the first task you probably figured out that our unknown protein was a bovine rhodopsin – the photosensitive molecule of the retina. Now, we want to learn more about the proteins of this macromolecular family and see if bioinformatics can help us to find information concerning their biochemistry and evolution.
For this purpose, we are going to create a multiple sequence alignment. This means that we will use many amino acid sequences (which you could get, for example, by extracting information from BLAST results) and align them in such a way that the similar residues are always below each other.
You can find a set of prepared sequences in the tab “Sequences”. The first one is the bovine rhodopsin which you already worked with in Part 1. We will now use the EMBL-EBI MUSCLE multiple sequences alignment tool to create the alignment.
Your task
Proceed as described below:
1. Using the sequences in the “Sequences” tab, align the 24 different opsins for comparison using the EMBL-EBI MUSCLE multiple sequences alignment tool.
2. Follow the instructions in the “MUSCLE” tab and try to answer the questions in the “Questions” tab.
Sequences
Your input sequences:
>Bos_rho MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA
>Homo_rho MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSRYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIAFLICWVPYASVAFYIFTHQGSNFGPIFMTIPAFFAKSAAIYNPVIYIMMNKQFRNCMLTTICCGKNPLGDDEASATVSKTETSQVAPA
>Homo_melan MNPPSGPRVPPSPTQEPSCMATPAPPSWWDSSQSSISSLGRLPSISPTAPGTWAAAWVPLPTVDVPDHAHYTLGTVILLVGLTGMLGNLTVIYTFCRSRSLRTPANMFIINLAVSDFLMSFTQAPVFFTSSLYKQWLFGETGCEFYAFCGALFGISSMITLTAIALDRYLVITRPLATFGVASKRRAAFVLLGVWLYALAWSLPPFFGWSAYVPEGLLTSCSWDYMSFTPAVRAYTMLLCCFVFFLPLLIIIYCYIFIFRAIRETGRALQTFGACKGNGESLWQRQRLQSECKMAKIMLLVILLFVLSWAPYSAVALVAFAGYAHVLTPYMSSVPAVIAKASAIHNPIIYAITHPKYRVAIAQHLPCLGVLLGVSRRHSRPYPSYRSTHRSTLTSHTSNLSWISIRRRQESLGSESEVGWTHMEAAAVWGAAQQANGRSLYGQGLEDLEAKAPPRPQGHEAETPGKTKGLIPSQDPRM
>Octopus_rho MVESTTLVNQTWWYNPTVDIHPHWAKFDPIPDAVYYSVGIFIGVVGIIGILGNGVVIYLFSKTKSLQTPANMFIINLAMSDLSFSAINGFPLKTISAFMKKWIFGKVACQLYGLLGGIFGFMSINTMAMISIDRYNVIGRPMAASKKMSHRRAFLMIIFVWMWSIVWSVGPVFNWGAYVPEGILTSCSFDYLSTDPSTRSFILCMYFCGFMLPIIIIAFCYFNIVMSVSNHEKEMAAMAKRLNAKELRKAQAGASAEMKLAKISMVIITQFMLSWSPYAIIALLAQFGPAEWVTPYAAELPVLFAKASAIHNPIVYSVSHPKFREAIQTTFPWLLTCCQFDEKECEDANDAEEEVVASERGGESRDAAQMKEMMAMMQKMQAQQAAYQPPPPPQGYPPQGYPPQGAYPPPQGYPPQGYPPQGYPPQGYPPQGAPPQVEAPQGAPPQGVDNQAYQA
>Mizuhopecten_Gqops MADNKSTLPGLPDINGTLNRSMTPNTGWEGPYDMSVHLHWTQFPPVTEEWHYIIGVYITIVGLLGIMGNTTVVYIFSNTKSLRSPSNLFVVNLAVSDLIFSAVNGFPLLTVSSFHQKWIFGSLFCQLYGFVGGVFGLMSINTLTAISIDRYVVITKPLQASQTMTRRKVHLMIVIVWVLSILLSIPPFFGWGAYIPEGFQTSCTFDYLTKTARTRTYIVVLYLFGFLIPLIIIGVCYVLIIRGVRRHDQKMLTITRSMKTEDARANNKRARSELRISKIAMTVTCLFIISWSPYAIIALIAQFGPAHWITPLVSELPMMLAKSSSMHNPVVYALSHPKFRKALYQRVPWLFCCCKPKEKADFRTSVCSKRSVTRTESVNSDVSSVISNLSDSTTTLGLTSEGATRANRETSFRRSVSIIKGDEDPCTHPDTFLLAYKEVEVGNLFDMTDDQNRRDSNLHSLYIPTRVQHRPTTQSLGTTPGGVYIVDNGQRVNGLTFNS
>Drosophila_rh6 MASLHPPSFAYMRDGRNLSLAESVPAEIMHMVDPYWYQWPPLEPMWFGIIGFVIAILGTMSLAGNFIVMYIFTSSKGLRTPSNMFVVNLAFSDFMMMFTMFPPVVLNGFYGTWIMGPFLCELYGMFGSLFGCVSIWSMTLIAYDRYCVIVKGMARKPLTATAAVLRLMVVWTICGAWALMPLFGWNRYVPEGNMTACGTDYFAKDWWNRSYIIVYSLWVYLTPLLTIIFSYWHIMKAVAAHEKAMREQAKKMNVASLRNSEADKSKAIEIKLAKVALTTISLWFFAWTPYTIINYAGIFESMHLSPLSTICGSVFAKANAVCNPIVYGLSHPKYKQVLREKMPCLACGKDDLTSDSRTQATAEISESQA
>Drosophila_rh2 MERSHLPETPFDLAHSGPRFQAQSSGNGSVLDNVLPDMAHLVNPYWSRFAPMDPMMSKILGLFTLAIMIISCCGNGVVVYIFGGTKSLRTPANLLVLNLAFSDFCMMASQSPVMIINFYYETWVLGPLWCDIYAGCGSLFGCVSIWSMCMIAFDRYNVIVKGINGTPMTIKTSIMKILFIWMMAVFWTVMPLIGWSAYVPEGNLTACSIDYMTRMWNPRSYLITYSLFVYYTPLFLICYSYWFIIAAVAAHEKAMREQAKKMNVKSLRSSEDCDKSAEGKLAKVALTTISLWFMAWTPYLVICYFGLFKIDGLTPLTTIWGATFAKTSAVYNPIVYGISHPKYRIVLKEKCPMCVFGNTDEPKPDAPASDTETTSEADSKA
>Xenopus_melanops MDLGKTVEYGTHRQDAIAQIDVPDQVLYTIGSFILIIGSVGIIGNMLVLYAFYRNKKLRTAPNYFIINLAISDFLMSATQAPVCFLSSLHREWILGDIGCNVYAFCGALFGITSMMTLLAISINRYIVITKPLQSIQWSSKKRTSQIIVLVWMYSLMWSLAPLLGWSSYVPEGLRISCTWDYVTSTMSNRSYTMMLCCCVFFIPLIVISHCYLFMFLAIRSTGRNVQKLGSYGRQSFLSQSMKNEWKMAKIAFVIIIVFVLSWSPYACVTLIAWAGHGKSLTPYSKTVPAVIAKASAIYNPIIYGIIHPKYRETIHKTVPCLRFLIREPKKDIFESSVRGSIYGRQSASRKKNSFISTVSTAETVSSHIWDNTPNGHWDRKSLSQTMSNLCSPLLQDPNSSHTLEQTLTWPDDPSPKEILLPSSLKSVTYPIGLESIVKDEHTNNSCVRNHRVDKSGGLDWIINATLPRIVIIPTSESNISETKEEHDNNSEEKSKRTEEEEDFFNFHVDTSLLNLEGLNSSTDLYEVVERFLS
>Platynereis_rops MSRSEVLVPGSMSLDGLLTTAHPIGNDSIETILHPYWQQFDIENTIPDSWHYAVAAWMTFFGILGVSGNLLVVWTFLKTKSLRTAPNMLLVNLAIGDMAFSAINGFPLLTISSINKRWVWGKLWRELYAFVGGIFGLMSINTLAWIAIDRFYVITNPLGAAQTMTKKRAFIILTIIWANASLWALAPFFGWGAYIPEGFQTSCTYDYLTQDMNNYTYVLGMYLFGFIFPVAIIFFCYLGIVRAIFAHHAEMMATAKRMGANTGKADADKKSEIQIAKVAAMTIGTFMLSWTPYAVVGVFGMIKPHSEMFIHPLLAEIPVMMAKASARYNPIIYALSHPKFRAEIDKHFPWLLCCCKPKPKAQLPSSTTKGSIASKTEADTSV
>Danio_melanops MSHHSSWRGHHCAPGDINCTAGFKESLGSRNYKLLHVPVHGPTHSHHHDPPHPFPTVDVPDHAHYIIGSVILIVGITGVIGNALVVYVFCRSRTLRTAGNMFIVNLAVADFLMSVTQSPVFFAASLHRRWVFGERPCELYAFCGALFGICSMMTLTAIAADRCLAITQPLALVSRVSRRKAGAVFVVVWLYSLGWSLPPFFGWSAYVPEGLQTSCSWDYMTFTPSVRAYTILLFVFVFFIPLGIIGSCYFAIFQTIRAAGKEIRELDCGETHKVYERMQNEWKMAKVALVVILLFIISWSPYSVVALTATAGYSHFLTPYMNSVPAVIAKASAIHNPIIYAITHPKYRVAIARYIPVLRPILRVKEKDLRSSFSSGSVSSRRPTLTSHQCSLGVSMGNAARANGRWGKTRLSSASDSDSCWTESEADGSSVSSLTFGRRVSTEISTDTVILSPGSSVSNASGQKSERAHKVVSVPVPSITFETDAADGESLSDGKALLGGN
>Sepia_rho MGRDIPDNETWWYNPTMEVHPHWKQFNQVPDAVYYSLGIFIGICGIIGCTGNGIVIYLFTKTKSLQTPANMFIINLAFSDFTFSLVNGFPLMTISCFIKKWVFGMAACKVYGFIGGIFGLMSIMTMSMISIDRYNVIGRPMAASKKMSHRRAFLMIIFVWMWSTLWSIGPIFGWGAYVLEGVLCNCSFDYITRDSATRSNIVCMYIFAFCFPILIIFFCYFNIVMAVSNHEKEMAAMAKRLNAKELRKAQAGASAEMKLAKISIVIVTQFLLSWSPYAVVALLAQFGPIEWVTPYAAQLPVMFAKASAIHNPLIYSVSHPKFREAIAENFPWIITCCQFDEKEVEDDKDAETEIPATEQSGGESADAAQMKEMMAMMQKMQQQQAAYPPQGAYPPQGGYPPQGYPPPPAQGGYPPQGYPPPPQGYPPAQGYPPQGYPPPQGAPPQGAPPQAAPPQGVDNQAYQA
>Takifugu_TMT MIVSNVSLSGCAGVNGAVCAAEGHQAGGSDRSTLTPTGNLVVSVFLGFIGTFGLVNNLLVLVLFCRYKMLRSPINLLLMNISISDLLVCVLGTPFSFAASTQGRWLIGEAGCVWYGFANSLFGVVSLISLAVLSFERYSTMMTPTEADPSNYCKVCLGITLSWVYSLVWTVPPLFGWSSYGPEGPGTTCSVNWTAKTTNSISYIICLFVFCLIVPFLVIVFCYGKLLCAIRQVSGINASTSRKREQRVLCMVVIMVICYLLCWLPYGVVALLATFGPPDLVTPEASIIPSVLAKSSTVINPIIYVFMNKQFYRCFLALLCCQDPRSGSSMKSSSKVATKAKGVTPTGQRRTDLLYMVASLGRPAATIPQLGPSFDATNDFTKPPSSDTIKPVVVSLAAHCDG
>Gallus_LWops MSSNSSQAPPNGTPGPFDGPQWPYQAPQSTYVGVAVLMGTVVACASVVNGLVIVVSICYKKLRSPLNYILVNLAVADLLVTLCGSSVSLSNNINGFFVFGRRMCELEGFMVSLTGIVGLWSLAILALERYVVVCRPLGDFQFQRRHAVSGCAFTWGWALLWSTPPLLGWSSYVPEGLRTSCGPNWYTGGSNNNSYILSLFVTCFVLPLSLILFSYTNLLLTLRAAAAQQKEADTTQRAEREVTRMVIVMVMAFLLCWLPYSTFALVVATHKGIIIQPVLASLPSYFSKTATVYNPIIYVFMNKQFQSCLLEMLCCGYQPQRTGKASPGTPGPHADVTAAGLRNKVMPAHPV
>Xenopus_Parietops MDGNSTTPGIAVNLTVMPTIFPRSGYSILSFLMFLNAVFSICNNAIVILVTLKHPQLRNPINIFILNLSFSDLMMALCGTTIVVSTNYHGYFYLGKQFCIFQGFAVNYFGIVSLWSLTLLAYERYNVVCEPIGALKLSTKRGYQGLVFIWLFCLFWAIAPLFGWSSYGPEGVQTSCSIGWEERSWSNYSYIISYFLTCFIIPVGIIGFSYGSILRSLHQLNRKIEQQGGKTNPREEKRVVIMVLFMVLAFLICWLPYTVFALIVVINPQLYISPLAATLPTYFAKTSPVYNPIIYIFLNKQFRTYAVQCLTCGHINLDSLEEDTESVSAQAENMLTPKTNQVAPA
>Danio_MW4 MNGTEGNNFYIPLSNRTGLARSPYEYPQYYLAEPWQFKLLAVYMFFLICLGFPINGLTLLVTAQHKKLRQPLNFILVNLAVAGTIMVCFGFTVTFYTAINGYFVLGPTGCAIEGFMATLGGEVALWSLVVLAVERYIVVCKPMGSFKFSASHAFAGCAFTWVMAMACAAPPLVGWPRYIPEGMQCSCGPDYYTLNPEYNNESYVLYMFICHFILPVTIIFFTYGRLVCTVKAAAAQQQESESTQKAEREVTRMVILMVLGFLIAWTPYATVAAWIFFNKGAAFSAQFMAVPAFFSKTSALYNPVIYVLLNKQFRNCMLTTLFCGKNPLGDDESSTVSTSKTEVSSVSPA
>Danio_extraocular MNGTEGPNFYVPMSNRTGLVRSPFEEPQYYLAEPWQFSLLAAYMLFLILGSFPINALTLYVTVQHKKLRTPLNYILLNLAVADLFMVLGGFTVTLYTALHGYFLLGVTGCNIEGFFATLGGEIALWSLVVLAIERYIVVCKPMSTFRFGEKHAIIGVGFTWVMALTCAVPPLLGWSRYIPEGMQCSCGIDYYTPKPEVHNTSFVIYMFILHFSIPLLIIFFCYSRLLCTVRAAAAQQQESETTQRAEREVTRMVVVMVIAFLVCWVPYASVAWYIFANQGAEFGPVFMTVPAFFAKSAALYNPVIYIMLNRQFRNCMLSTVCCGKNPLAEDESSSAVSSKTQSSVVSSAQVSPA
>Latimeria_Rh2 MNGTEGMNFYVPLSNRTGLVRSPFEYTQYYLAEPWKFSVLCAYMFLLIILGFPINFLTLLVTFKHKKLRQPLNYILVNLAVASLFMVVFGFTVTFYSSLNGYFVLGPMGCAMEGFFATLGGQVALWSLVVLAIERYIVVCKPMGNFRFASSHAIMGIAFTWIMALACAAPPLVGWSRYIPEGLQCSCGPDYYTLNPDFHNESYVMYLFLVHFLLPIIIIFFTYGRLICKVKEAAAQQQESASTQKAEKEVTRMVILMVIGFLTAWVPYASAAFWIFCNRGAEFTATLMTVPAFFSKSSCLFNPIIYVLLNKQFRNCMITTLCCGKNPLGDDDTSSAVSQSKTDVSSVSSSQVSPA
>Xenopus_green_ops MSKGRPDLRMEMPDEFYVPIPLETTNISSLSPFLVPQTHLGTPGIFMSISAFMLFTIIFGFPLNLLTIICTVKYKKLRSHLNYILVNLAVANLIVICFGSTTAFYSFSQMYFSLGTLACKIEGFTATLGGIIGLWSLAVVAFERFLVICKPMGNFTFRESHAVLGCILTWVIGLVAAIPPLLGWSRYIPEGLQCSCGPDWYTVNNKWNNESYVLFLFCFCFGFPLAIIVFSYGRLLLALHAVAKQQEQSATTQKAEREVTRMVIVMVVGFLVCWLPYASFALWAVTHRGELFDLRMSSVPSVFSKASTVYNPFIYIFMNRQFRSCMMKMIFCGKNPLGDDEETSVSGSTQVSSVSSSQIAPS
>Xenopus_violet_ops MLEEEDFYLFKNVSNVSPFDGPQYHIAPKWAFTLQAIFMGMVFLIGTPLNFIVLLVTIKYKKLRQPLNYILVNITVGGFLMCIFSIFPVFVSSSQGYFFFGRIACSIDAFVGTLTGLVTGWSLAFLAFERYIVICKPMGNFNFSSSHALAVVICTWIIGIVVSVPPFLGWSRYMPEGLQCSCGPDWYTVGTKYRSEYYTWFIFIFCFVIPLSLICFSYGRLLGALRAVAAQQQESASTQKAEREVSRMVIFMVGSFCLCYVPYAAMAMYMVTNRNHGLDLRLVTIPAFFSKSSCVYNPIIYSFMNKQFRGCIMETVCGRPMSDDSSVSSTSQRTEVSTVSSSQVSPA
>Gallus_blue_ops MHPPRPTTDLPEDFYIPMALDAPNITALSPFLVPQTHLGSPGLFRAMAAFMFLLIALGVPINTLTIFCTARFRKLRSHLNYILVNLALANLLVILVGSTTACYSFSQMYFALGPTACKIEGFAATLGGMVSLWSLAVVAFERFLVICKPLGNFTFRGSHAVLGCVATWVLGFVASAPPLFGWSRYIPEGLQCSCGPDWYTTDNKWHNESYVLFLFTFCFGVPLAIIVFSYGRLLITLRAVARQQEQSATTQKADREVTKMVVVMVLGFLVCWAPYTAFALWVVTHRGRSFEVGLASIPSVFSKSSTVYNPVIYVLMNKQFRSCMLKLLFCGRSPFGDDEDVSGSSQATQVSSVSSSHVAPA
>Danio_LWops MAEHWGDAIYAARRKGDETTREAMFTYTNSNNTKDPFEGPNYHIAPRWVYNVATVWMFFVVVASTFTNGLVLVATAKFKKLRHPLNWILVNLAIADLGETLFASTISVINQFFGYFILGHPMCIFEGYTVSVCGIAALWSLTVISWERWVVVCKPFGNVKFDAKWASAGIIFSWVWAAAWCAPPIFGWSRYWPHGLKTSCGPDVFSGSEDPGVQSYMVVLMITCCIIPLAIIILCYIAVYLAIHAVAQQQKDSESTQKAEKEVSRMVVVMIFAYCFCWGPYTFFACFAAANPGYAFHPLAAAMPAYFAKSATIYNPVIYVFMNRQFRVCIMQLFGKKVDDGSEVSTSKTEVSSVAPA
>Danio_SWops MDAWAVQFGNASKVSPFEGEQYHIAPKWAFYLQAAFMGFVFIVGTPMNGIVLFVTMKYKKLRQPLNYILVNISLAGFIFDTFSVSQVSVCAARGYYSLGYTLCSMEAAMGSIAGLVTGWSLAVLAFERYVVICKPFGSFKFGQGQAVGAVVFTWIIGTACATPPFFGWSRYIPEGLGTACGPDWYTKSEEYNSESYTYFLLITCFMMPMTIIIFSYSQLLGALRAVAAQQAESESTQKAEREVSRMVVVMVGSFVLCYAPYAVTAMYFANSDEPNKDYRLVAIPAFFSKSSSVYNPLIYAFMNKQFNACIMETVFGKKIDESSEVSSKTETSSVSA
>Homo_MWops MAQQWSLQRLAGRHPQDSYEDSTQSSIFTYTNSNSTRGPFEGPNYHIAPRWVYHLTSVWMIFVVIASVFTNGLVLAATMKFKKLRHPLNWILVNLAVADLAETVIASTISVVNQVYGYFVLGHPMCVLEGYTVSLCGITGLWSLAIISWERWMVVCKPFGNVRFDAKLAIVGIAFSWIWAAVWTAPPIFGWSRYWPHGLKTSCGPDVFSGSSYPGVQSYMIVLMVTCCITPLSIIVLCYLQVWLAIRAVAKQQKESESTQKAEKEVTRMVVVMVLAFCFCWGPYAFFACFAAANPGYPFHPLMAALPAFFAKSATIYNPVIYVFMNRQFRNCILQLFGKKVDDGSELSSASKTEVSSVSSVSPA
>Danio_mel_rec1A MFMNGSSLNSSALDPSEQALQRPPWVTTTLGCFLIFTIVVDILGNLLVIFSVYRNKKLQNAGNIFVVSLAVADLVVAIYPYPLVLTSIFHRGWNLGYMHCQISGFLMGVSVIGSIFNITGIAINCYCYICHSLKYDKLYSDKNSVCYVLLIWALTVLAIVPNLFVGSLQYDPRVYSCTFEQSASSAYTIAVVFFHFILPIMIVTYCYLRIWVLVIQVRRRVKPDNRPKITPHDVRNFVTMFVVFVLFAVCWAPLNFIGLAVAISPERVVPLIPEWLFVASYFMAYFNSCLNAIVYGVLNQNFRREYKRIVVSVCTARIFFGESSNEAQERLKSKPSPLMTNNNQVKVDSV
MUSCLE
1. Paste all your amino acid sequences (including greater-than symbols and names) at once into the input box below. (Alternatively, upload a FASTA file containing the sequences from your computer.)
2. Select ClustalW as output format.
3. Submit the sequences for alignment.
4. The multiple opsin sequences have now been aligned. You can have a look at the results in the “Alignment” tab and, for a better view, click on “Show Colors”.
5. To do a more advanced analysis, we will use the JalView programme to look at the alignment and to edit it. To do this, right-click on the “Results Summary” tab and open the “Results Summary” in a new browser tab (“Open Link in New Tab”). In the new browser tab open the alignment using “JalView”. Please note that the new browser tab is only required for JalView to stay open throughout the activity; all future activity steps can still be performed in your initial browser tab.
6. Ensure you can see all of the 24 sequences below each other by increasing the height of the window (use the cursor of your mouse to pull the window upwards).
In JalView you can select different colouring criteria for the alignment (go to “Colour” to select them). For example, you can select colouring based on percentage identity or hydrophobicity of the individual amino acid residues.
7. On the bottom of the JalView window, you see alignment scores which are represented by bars of sequence identity/similarity. Do you notice any peculiarities? Try and answer the questions in the “Questions” tab.
Note: please do not close the JalView window after you finish this part of the activity; you will need it in the next exercise.
Questions
1. What can you deduce from the alignment?
2. Are there any sequence stretches more conserved than others? If yes, how many can you identify? What is their biochemical nature? (Are they rather basic, acidic, hydrophobic or hydrophilic?)
3. Are there any conserved residues or short motives which are present in all the sequences? Be careful, one of the sequences – “Danio_mel_rec1A” – is is not an opsin but an evolutionary related molecule (which will be relevant for the next part of the activity).
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