IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ schmalen-2014-5 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-9 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro chromatography chromatography technology column chromatography chromatography ms participant Identification by mass spectrometry ms participant Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-1 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-2 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. An X-ray fluorescence scan was used to confirm the presence of zinc achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-3 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro biophysical biophysical predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. Inductively coupled plasma atomic emission spectroscopy (ICP-AES), also referred to as inductively coupled plasma optical emission spectrometry (ICP-OES), is an analytical technique used for the detection of trace metals. It is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The zinc-specific wavelength = 213.857 nm achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-4 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-6 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro comig non denat gel comigration in non denaturing gel electrophoresis comig non denat gel weight by staining molecular weight estimation by staining weight by staining Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-7 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro biophysical biophysical predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. Melting temperature determination by thermofluor analysis achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-8 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip tag visualisation tag visualisation Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation schmalen-2014-10 Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Schmalen I., Reischl S., Wallach T., Klemz R., Grudziecki A., Prabu J.R., Benda C., Kramer A., Wolf E. achim.kramer@charite.de, evawolf1@uni-mainz.de Cell (0092-8674) 2014 Only protein-protein interactions imex curation zinc(2+) zinc(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) cry1_mouse Cryptochrome-1 Cry1 protein protein mouse Mus musculus Mouse MGVNAVHWFRKGLRLHDNPALKECIQGADTIRCVYILDPWFAGSSNVGINRWRFLLQCLEDLDANLRKLNSRLFVIRGQPADVFPRLFKEWNITKLSIEYDSEPFGKERDAAIKKLATEAGVEVIVRISHTLYDLDKIIELNGGQPPLTYKRFQTLVSKMEPLEMPADTITSDVIGKCMTPLSDDHDEKYGVPSLEELGFDTDGLSSAVWPGGETEALTRLERHLERKAWVANFERPRMNANSLLASPTGLSPYLRFGCLSCRLFYFKLTDLYKKVKKNSSPPLSLYGQLLWREFFYTAATNNPRFDKMEGNPICVQIPWDKNPEALAKWAEGRTGFPWIDAIMTQLRQEGWIHHLARHAVACFLTRGDLWISWEEGMKVFEELLLDADWSINAGSWMWLSCSSFFQQFFHCYCPVGFGRRTDPNGDYIRRYLPVLRGFPAKYIYDPWNAPEGIQKVAKCLIGVNYPKPMVNHAEASRLNIERMKQIYQQLSRYRGLGLLASVPSNSNGNGGLMGYAPGENVPSCSSSGNGGLMGYAPGENVPSCSGGNCSQGSGILHYAHGDSQQTHSLKQGRSSAGTGLSSGKRPSQEEDAQSVGPKVQRQSSN per2_mouse Period circadian protein homolog 2 Per2 Circadian clock protein PERIOD 2 protein protein mouse Mus musculus Mouse MNGYVDFSPSPTSPTKEPGAPQPTQAVLQEDVDMSSGSSGNENCSTGRDSQGSDCDDNGKELRMLVESSNTHPSPDDAFRLMMTEAEHNPSTSGCSSEQSAKADAHKELIRTLKELKVHLPADKKAKGKASTLATLKYALRSVKQVKANEEYYQLLMSSESQPCSVDVPSYSMEQVEGITSEYIVKNADMFAVAVSLVSGKILYISNQVASIFHCKKDAFSDAKFVEFLAPHDVSVFHSYTTPYKLPPWSVCSGLDSFTQECMEEKSFFCRVSVGKHHENEIRYQPFRMTPYLVKVQEQQGAESQLCCLLLAERVHSGYEAPRIPPEKRIFTTTHTPNCLFQAVDERAVPLLGYLPQDLIETPVLVQLHPSDRPLMLAIHKKILQAGGQPFDYSPIRFRTRNGEYITLDTSWSSFINPWSRKISFIIGRHKVRVGPLNEDVFAAPPCPEEKTPHPSVQELTEQIHRLLMQPVPHSGSSGYGSLGSNGSHEHLMSQTSSSDSNGQEESHRRRSGIFKTSGKIQTKSHVSHESGGQKEASVAEMQSSPPAQVKAVTTIERDSSGASLPKASFPEELAYKNQPPCSYQQISCLDSVIRYLESCSEAATLKRKCEFPANIPSRKATVSPGLHSGEAARPSKVTSHTEVSAHLSSLTLPGKAESVVSLTSQCSYSSTIVHVGDKKPQPELETVEDMASGPESLDGAAGGLSQEKGPLQKLGLTKEVLAAHTQKEEQGFLQRFREVSRLSALQAHCQNYLQERSRAQASDRGLRNTSGLESSWKKTGKNRKLKSKRVKTRDSSESTGSGGPVSHRPPLMGLNATAWSPSDTSQSSCPSAPFPTAVPAYPLPVFQAPGIVSTPGTVVAPPAATHTGFTMPVVPMGTQPEFAVQPLPFAAPLAPVMAFMLPSYPFPPATPNLPQAFLPSQPHFPAHPTLASEITPASQAEFPSRTSTLRQPCACPVTPPAGTVALGRASPPLFQSRGSSPLQLNLLQLEEAPEGSTGAAGTLGTTGTAASGLDCTSGTSRDRQPKAPPTCNEPSDTQNSDAISTSSDLLNLLLGEDLCSATGSALSRSGASATSDSLGSSSLGFGTSQSGAGSSDTSHTSKYFGSIDSSENNHKAKMIPDTEESEQFIKYVLQDPIWLLMANTDDSIMMTYQLPSRDLQAVLKEDQEKLKLLQRSQPRFTEGQRRELREVHPWVHTGGLPTAIDVTGCVYCESEEKGNICLPYEEDSPSPGLCDTSEAKEEEGEQLTGPRIEAQT cry1-per2-10 1565232 n/a 1565242 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 n/a 1565244 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 1565250 1565248 direct interaction direct interaction false false false 1565232 6B cry1-per2-11 1565232 n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 n/a 1565244 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 1565255 1565253 direct interaction direct interaction false false false 1565232 5B cry1-per2-12 1565233 n/a 1565244 unspecified role unspecified role neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1565243 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 1565262 1565259 direct interaction direct interaction false false false Supp Fig. 6 cry1-per2-12 1565233 n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 n/a 1565244 unspecified role unspecified role neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1565242 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 1565269 1565265 direct interaction direct interaction false false false Supp. Fig. 6 The presence of zinc in the complexes was analyzed in parallel by atomic absorption spectroscopy (ICP-OES) cry1-per2-2 1565234 n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 n/a 1565244 unspecified role unspecified role neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 1565277 1565273 direct interaction direct interaction false false false Supp. Table 1 2.45A Rworking = 0.19 Rfree = 0.23 cry1-per2-4 1565235 n/a 1565242 unspecified role unspecified role neutral component neutral component n/a 1565244 unspecified role unspecified role neutral component neutral component n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1565243 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 1565285 1565282 direct interaction direct interaction false false false Supp. Fig. 4C cry1-per2-5 1565236 n/a 1565242 unspecified role unspecified role neutral component neutral component n/a 1565243 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. n/a 1565244 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) 1565292 1565290 direct interaction direct interaction false false false Supp. Fig. 4D, 7 Only copurified CRY/PER2 complex binds zinc, while single mCRY1 and mPER2 proteins are zinc-free in solution cry1-per2-6 1565237 n/a 1565243 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. n/a 1565244 unspecified role unspecified role bait bait n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false O54943:p.[Asp1167Arg;Phe1181Arg;His1193Glu] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O54943:p.Asp1167Arg mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O54943:p.[Asp1167Arg;His1193Glu] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) 1565299 1565296 physical association physical association false false false Fig. 5A cry1-per2-8 1565238 n/a 1565244 unspecified role unspecified role neutral component neutral component O54943:p.[Asp1167Arg;His1193Glu] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O54943:p.Asp1167Arg mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false O54943:p.[Asp1167Arg;Phe1181Arg;His1193Glu] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 1565310 1565307 direct interaction direct interaction false false false Fig. 5C, 5D cry1-per2-9 1565239 n/a 1565244 unspecified role unspecified role neutral component neutral component O54943:p.[Cys1210Ala;Cys1213Ala] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. 1565316 1565314 direct interaction direct interaction false false false Fig. 6A Zinc binding stabilizes the purified CRY1/PER2 complex as demonstrated by the higher melting temperature of the wild-type complex compared to the zinc-free CRY1/PER2(cys1210ala/cys1213ala) mutant complex per2-zinc_2__ 1565240 n/a 1565244 unspecified role unspecified role bait bait O54943:p.[Cys1210Ala;Cys1213Ala] mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false n-terminal v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. n/a 1565243 unspecified role unspecified role ancillary ancillary P97784:p.[Cys412Ala;Cys414Ala] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. n/a 1565242 unspecified role unspecified role prey prey region radiolabelled radiolabel radiolabeled radiolabelled undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig. 7C, Supp. Fig. 5B cry1-per2-1 1565241 n/a 1565244 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 n/a 1565243 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false trini-high_5 Trichoplusia ni cell line from eggs high_5 Insect cell line from Trichoplusia ni egg cells Invitrogen supplies this cell line. Invitrogen supply this cell line. Stoichiometry: 1.0 1565329 1565327 direct interaction direct interaction false false false Supp Table 2 Unbound C-terminal mPER2 region Is mainly unstructured; upon binding to mCRY1-PHR, it adopts a helical fold.