UniProt UniProt http://www.uniprot.org micevski-2014-2 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 Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation micevski-2014-1 in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation micevski-2014-4 in vitro In vitro enzymatic footprint enzymatic footprinting enzymatic footprint weight by comassie molecular weight estimation by coomasie staining weight by comassie Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. Proteins were degraded by addition of thermolysin, complex formation protected proteins from the activity of this protease d.dougan@latrobe.edu.au Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation micevski-2014-3 ecoli Escherichia coli (strain K12) pull down pull down western blot western blot Immuno blot Immuno blot Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation irad_ecoli Anti-adapter protein IraD iraD yjiD b4326 JW5782 protein protein ecoli Escherichia coli (strain K12) MMRQSLQAVLPEISGNKTSSLRKSVCSDLLTLFNSPHSALPSLLVSGMPEWQVHNPSDKHLQSWYCRQLRSALLFHEPRIAALQVNLKEAYCHTLAISLEIMLYHDDEPLTFDLVWDNGGWRSATLENVS rssb_ecoli Regulator of RpoS rssB hnr sprE ychL JW1223 b1235 protein protein ecoli Escherichia coli (strain K12) MTQPLVGKQILIVEDEQVFRSLLDSWFSSLGATTVLAADGVDALELLGGFTPDLMICDIAMPRMNGLKLLEHIRNRGDQTPVLVISATENMADIAKALRLGVEDVLLKPVKDLNRLREMVFACLYPSMFNSRVEEEERLFRDWDAMVDNPAAAAKLLQELQPPVQQVISHCRVNYRQLVAADKPGLVLDIAALSENDLAFYCLDVTRAGHNGVLAALLLRALFNGLLQEQLAHQNQRLPELGALLKQVNHLLRQANLPGQFPLLVGYYHRELKNLILVSAGLNATLNTGEHQVQISNGVPLGTLGNAYLNQLSQRCDAWQCQIWGTGGRLRLMLSAE rpos_ecoli RNA polymerase sigma factor RpoS Sigma-38 rpoS appR katF nur otsX sigS b2741 JW5437 Sigma S protein protein ecoli Escherichia coli (strain K12) MSQNTLKVHDLNEDAEFDENGVEVFDEKALVEQEPSDNDLAEEELLSQGATQRVLDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVASRRRMIESNLRLVVKIARRYGNRGLALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKELNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKALLDILADEKENGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLREILQTQGLNIEALFRE irap_ecoli Anti-adapter protein IraP iraP b0382 JW0373 yaiB protein protein ecoli Escherichia coli (strain K12) MKNLIAELLFKLAQKEEESKELCAQVEALEIIVTAMLRNMAQNDQQRLIDQVEGALYEVKPDASIPDDDTELLRDYVKKLLKHPRQ iram_ecoli Anti-adapter protein IraM iraM elb1 elbA ycgW Enhancing lycopene biosynthesis protein 1 b1160 JW1147 protein protein ecoli Escherichia coli (strain K12) MKWIVIDTVIQPTCGISFSAIWGNMKMIIWYQSTIFLPPGSIFTPVKSGIILKDKEYPITIYHIAPFNKDLWSLLKSSQECPPGESKITNKCLHNSCIIKICPYGLK irad-rssb-2 3636049 N/A 3636053 unspecified role unspecified role neutral component neutral component purified purified 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 3636054 unspecified role unspecified role neutral component neutral component purified purified 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) direct interaction direct interaction false false false Fig. 2D rpos-rssb-1 3636049 N/A 3636054 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) N/A 3636055 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 2F Addition of iraD caused dissociation of this complex irad-1 3636049 N/A 3636053 unspecified role unspecified role neutral component neutral component purified purified 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. 2D irad-rssb-1 3636050 N/A 3636053 unspecified role unspecified role bait bait purified purified 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 3636054 unspecified role unspecified role prey prey purified purified 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) direct interaction direct interaction false false false Fig. 2C irap-rssb-1 3636050 N/A 3636056 unspecified role unspecified role bait bait purified purified 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 ecoli Escherichia coli (strain K12) N/A 3636054 unspecified role unspecified role prey prey purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0AEV1:p.Asp58Pro mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P0AEV1:p.Asp58Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 3C, 3F Addition of iraD caused dissociation of this complex irad-rssb-3 3636051 N/A 3636053 unspecified role unspecified role neutral component neutral component purified purified 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 3636054 unspecified role unspecified role neutral component neutral component purified purified 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) direct interaction direct interaction false false false Fig. 5A, 5B irap-rssb-2 3636051 N/A 3636056 unspecified role unspecified role neutral component neutral component purified purified 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 ecoli Escherichia coli (strain K12) N/A 3636054 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 5C Addition of iraD caused dissociation of this complex iram-rssb-1 3636052 N/A 3636057 unspecified role unspecified role bait bait purified purified 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 N/A 3636054 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate region sufficient to bind sufficient binding region sufficient to bind 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 physical association physical association false false false Fig. 4