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/ keyamura-2007-4 The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes. in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Keyamura K., Fujikawa N., Ishida T., Ozaki S., Su'etsugu M., Fujimitsu K., Kagawa W., Yokoyama S., Kurumizaka H., Katayama T. katayama@phar.kyushu-u.ac.jp,kurumizaka@waseda.jp Genes Dev. (0890-9369) 2007 Only protein-protein interactions imex curation keyamura-2007-2 The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Keyamura K., Fujikawa N., Ishida T., Ozaki S., Su'etsugu M., Fujimitsu K., Kagawa W., Yokoyama S., Kurumizaka H., Katayama T. katayama@phar.kyushu-u.ac.jp,kurumizaka@waseda.jp Genes Dev. (0890-9369) 2007 Only protein-protein interactions imex curation keyamura-2007-3 The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes. in vitro In vitro density sedimentation cosedimentation through density gradient density sedimentation weight by comassie molecular weight estimation by coomasie staining weight by comassie Keyamura K., Fujikawa N., Ishida T., Ozaki S., Su'etsugu M., Fujimitsu K., Kagawa W., Yokoyama S., Kurumizaka H., Katayama T. katayama@phar.kyushu-u.ac.jp,kurumizaka@waseda.jp Genes Dev. (0890-9369) 2007 Only protein-protein interactions imex curation keyamura-2007-1 The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Keyamura K., Fujikawa N., Ishida T., Ozaki S., Su'etsugu M., Fujimitsu K., Kagawa W., Yokoyama S., Kurumizaka H., Katayama T. katayama@phar.kyushu-u.ac.jp,kurumizaka@waseda.jp Genes Dev. (0890-9369) 2007 Only protein-protein interactions imex curation diaa_ecoli DnaA initiator-associating protein DiaA diaA b3149 JW3118 yraO protein protein ecoli Escherichia coli (strain K12) MQERIKACFTESIQTQIAAAEALPDAISRAAMTLVQSLLNGNKILCCGNGTSAANAQHFAASMINRFETERPSLPAIALNTDNVVLTAIANDRLHDEVYAKQVRALGHAGDVLLAISTRGNSRDIVKAVEAAVTRDMTIVALTGYDGGELAGLLGPQDVEIRIPSHRSARIQEMHMLTVNCLCDLIDNTLFPHQDD dnaa_ecoli Chromosomal replication initiator protein DnaA dnaA b3702 JW3679 protein protein ecoli Escherichia coli (strain K12) MSLSLWQQCLARLQDELPATEFSMWIRPLQAELSDNTLALYAPNRFVLDWVRDKYLNNINGLLTSFCGADAPQLRFEVGTKPVTQTPQAAVTSNVAAPAQVAQTQPQRAAPSTRSGWDNVPAPAEPTYRSNVNVKHTFDNFVEGKSNQLARAAARQVADNPGGAYNPLFLYGGTGLGKTHLLHAVGNGIMARKPNAKVVYMHSERFVQDMVKALQNNAIEEFKRYYRSVDALLIDDIQFFANKERSQEEFFHTFNALLEGNQQIILTSDRYPKEINGVEDRLKSRFGWGLTVAIEPPELETRVAILMKKADENDIRLPGEVAFFIAKRLRSNVRELEGALNRVIANANFTGRAITIDFVREALRDLLALQEKLVTIDNIQKTVAEYYKIKVADLLSKRRSRSVARPRQMAMALAKELTNHSLPEIGDAFGGRDHTTVLHACRKIEQLREESHDIKEDFSNLIRTLSS dnaa-diaa 1370689 n/a 1370693 unspecified role unspecified role prey prey P66817:p.Phe191Ser mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false P66817:p.Phe191Leu mutation disrupting mutation disrupting interaction mutation disrupting 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 P66817:p.Leu190Pro mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false P66817:p.Asn83Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P66817:p.Pro72Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P66817:p.Pro72Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P66817:p.Leu190Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false P66817:p.Lys101Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1370694 unspecified role unspecified role bait bait n-terminal biotin tag biotin 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) direct interaction direct interaction false false false Fig. 5 dnaa-diaa-1 1370689 n/a 1370693 unspecified role unspecified role prey prey P66817:p.Asn83Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing 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 P66817:p.Leu190Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false P66817:p.Ser52Phe mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1370694 unspecified role unspecified role bait bait n-terminal biotin tag biotin 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) direct interaction direct interaction false false false Fig. 6a This is a 2-stage anti-tag coip using a restricted amount of biotin-tagged DnaA in the first pulldown and excess amounts of untagged DnaA in the second pulldown before visualising the complex on SDS-PAGE. This allowed for several different DnaA molecules to be pulled down by one DiaA homotetramer. diaa-dnaa 1370689 n/a 1370694 unspecified role unspecified role prey prey n-terminal biotin tag biotin 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) Stoichiometry: 2.0 n/a 1370693 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 ecoli Escherichia coli (strain K12) Stoichiometry: 4.0 direct interaction direct interaction false false false Fig. 6b diaa homomultimer 1370690 n/a 1370693 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 ecoli Escherichia coli (strain K12) n/a 1370693 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig.s 3a diaa homodimer mut 1370690 n/a 1370693 unspecified role unspecified role neutral component neutral component P66817:p.Lys101Glu mutation mutation certain certain sequence position certain certain certain sequence position certain false P66817:p.Asn83Asp mutation mutation 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) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig.s 3a diaa homotetramer 1370691 n/a 1370693 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 ecoli Escherichia coli (strain K12) Stoichiometry: 4.0 direct interaction direct interaction false false false Fig.s 3c diaa homodimer mut 1370691 n/a 1370693 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 P66817:p.Lys101Glu mutation mutation certain certain sequence position certain certain certain sequence position certain false P66817:p.Asn83Asp mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig.s 3c diaa homotetramer 1370692 n/a 1370693 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 ecoli Escherichia coli (strain K12) Stoichiometry: 4.0 direct interaction direct interaction false false false Fig. 1b, c, S1 Rwork = 0.18; Rfree = 0.21 1.85A