UniProt UniProt http://www.uniprot.org Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 2017 imex curation Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. damian.ekiert@med.nyu.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column ms participant Identification by mass spectrometry ms participant Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. Complex purified affinity purification followed by gel filtration. damian.ekiert@med.nyu.edu Accepted 2018-JUL-20 AT 08:22 BST AT 08:22 BST by LIVIAP Cell 2017 Only protein-protein interactions imex curation imex curation Missing periplasmic regions added, tags were based on vector pET22b (+) - includes a C-termina l His tag. I disagree re methodology, pproteins bound to lipids are purified from Ecoli, lipids then separated off, then identified. I think the lipid marker are there as equivalent to mol wt markers on a protein gel.; I prioritised the gel filtration over the affinity purification so the logic follows through - in a gel filtration roles are neutral and the association argument only applies to affinity purification - gel filtration is always physical. I've changed the identification method to mol sieving and added as exp-mod to say first affinity purified if that helps make it clearer. Mock publication and experiment for abstract interactions that are not interaction evidences. Mock publication and experiment for abstract interactions that are not interaction evidences. unspecified method Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 2017 imex curation Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. damian.ekiert@med.nyu.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation ecoli Escherichia coli (strain K12) anti tag coip anti tag coimmunoprecipitation anti tag coip ms participant Identification by mass spectrometry ms participant Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. damian.ekiert@med.nyu.edu Accepted 2018-JUL-20 AT 08:22 BST AT 08:22 BST by LIVIAP Cell 2017 Only protein-protein interactions imex curation imex curation Missing periplasmic regions added, tags were based on vector pET22b (+) - includes a C-termina l His tag. I disagree re methodology, pproteins bound to lipids are purified from Ecoli, lipids then separated off, then identified. I think the lipid marker are there as equivalent to mol wt markers on a protein gel.; I prioritised the gel filtration over the affinity purification so the logic follows through - in a gel filtration roles are neutral and the association argument only applies to affinity purification - gel filtration is always physical. I've changed the identification method to mol sieving and added as exp-mod to say first affinity purified if that helps make it clearer. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 2017 imex curation Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. damian.ekiert@med.nyu.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro electron diffraction electron diffraction predetermined predetermined participant predetermined Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. Complex purified affinity purification followed by gel filtration. damian.ekiert@med.nyu.edu Accepted 2018-JUL-20 AT 08:22 BST AT 08:22 BST by LIVIAP Cell 2017 Only protein-protein interactions imex curation imex curation Missing periplasmic regions added, tags were based on vector pET22b (+) - includes a C-termina l His tag. I disagree re methodology, pproteins bound to lipids are purified from Ecoli, lipids then separated off, then identified. I think the lipid marker are there as equivalent to mol wt markers on a protein gel.; I prioritised the gel filtration over the affinity purification so the logic follows through - in a gel filtration roles are neutral and the association argument only applies to affinity purification - gel filtration is always physical. I've changed the identification method to mol sieving and added as exp-mod to say first affinity purified if that helps make it clearer. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 2017 imex curation Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. damian.ekiert@med.nyu.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro bio-layer interferometry bio-layer interferometry bli bli predetermined predetermined participant predetermined Ekiert DC., Bhabha G., Isom GL., Greenan G., Ovchinnikov S., Henderson IR., Cox JS., Vale RD. Complex purified affinity purification followed by gel filtration. damian.ekiert@med.nyu.edu Accepted 2018-JUL-20 AT 08:22 BST AT 08:22 BST by LIVIAP Cell 2017 Only protein-protein interactions imex curation imex curation Missing periplasmic regions added, tags were based on vector pET22b (+) - includes a C-termina l His tag. I disagree re methodology, pproteins bound to lipids are purified from Ecoli, lipids then separated off, then identified. I think the lipid marker are there as equivalent to mol wt markers on a protein gel.; I prioritised the gel filtration over the affinity purification so the logic follows through - in a gel filtration roles are neutral and the association argument only applies to affinity purification - gel filtration is always physical. I've changed the identification method to mol sieving and added as exp-mod to say first affinity purified if that helps make it clearer. mlae_ecoli Intermembrane phospholipid transport system permease protein MlaE mlaE yrbE b3194 JW3161 protein protein ecoli Escherichia coli (strain K12) MLLNALASLGHKGIKTLRTFGRAGLMLFNALVGKPEFRKHAPLLVRQLYNVGVLSMLIIVVSGVFIGMVLGLQGYLVLTTYSAETSLGMLVALSLLRELGPVVAALLFAGRAGSALTAEIGLMRATEQLSSMEMMAVDPLRRVISPRFWAGVISLPLLTVIFVAVGIWGGSLVGVSWKGIDSGFFWSAMQNAVDWRMDLVNCLIKSVVFAITVTWISLFNGYDAIPTSAGISRATTRTVVHSSLAVLGLDFVLTALMFGN 057409EA1E6E25EB mlad_ecoli Intermembrane phospholipid transport system binding protein MlaD b3193 mlaD yrbD JW3160 protein protein ecoli Escherichia coli (strain K12) MQTKKNEIWVGIFLLAALLAALFVCLKAANVTSIRTEPTYTLYATFDNIGGLKARSPVSIGGVVVGRVADITLDPKTYLPRVTLEIEQRYNHIPDTSSLSIRTSGLLGEQYLALNVGFEDPELGTAILKDGDTIQDTKSAMVLEDLIGQFLYGSKGDDNKNSGDAPAAAPGNNETTEPVGTTK mint 6BD8B922002EFD28 mlaf_ecoli Intermembrane phospholipid transport system ATP-binding protein MlaF mlaF b3195 JW3162 yrbF protein protein ecoli Escherichia coli (strain K12) MEQSVANLVDMRDVSFTRGNRCIFDNISLTVPRGKITAIMGPSGIGKTTLLRLIGGQIAPDHGEILFDGENIPAMSRSRLYTVRKRMSMLFQSGALFTDMNVFDNVAYPLREHTQLPAPLLHSTVMMKLEAVGLRGAAKLMPSELSGGMARRAALARAIALEPDLIMFDEPFVGQDPITMGVLVKLISELNSALGVTCVVVSHDVPEVLSIADHAWILADKKIVAHGSAQALQANPDPRVRQFLDGIADGPVPFRYPAGDYHADLLPGS 742EF8DEDA742CF2 742EF8DEDA742CF2 mlac_ecoli Intermembrane phospholipid transport system binding protein MlaC mlaC b3192 JW3159 yrbC protein protein ecoli Escherichia coli (strain K12) MFKRLMMVALLVIAPLSAATAADQTNPYKLMDEAAQKTFDRLKNEQPQIRANPDYLRTIVDQELLPYVQVKYAGALVLGQYYKSATPAQREAYFAAFREYLKQAYGQALAMYHGQTYQIAPEQPLGDKTIVPIRVTIIDPNGRPPVRLDFQWRKNSQTGNWQAYDMIAEGVSMITTKQNEWGTLLRTKGIDGLTAQLKSISQQKITLEEKK A8F22EDFB0830763 phosphatidylglycerol phosphatidylglycerol phosphatidylglycerols small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) mlaa_ecoli Intermembrane phospholipid transport system lipoprotein MlaA b2346 JW2343 mlaA vacJ protein protein ecoli Escherichia coli (strain K12) MKLRLSALALGTTLLVGCASSGTDQQGRSDPLEGFNRTMYNFNFNVLDPYIVRPVAVAWRDYVPQPARNGLSNFTGNLEEPAVMVNYFLQGDPYQGMVHFTRFFLNTILGMGGFIDVAGMANPKLQRTEPHRFGSTLGHYGVGYGPYVQLPFYGSFTLRDDGGDMADGFYPVLSWLTWPMSVGKWTLEGIETRAQLLDSDGLLRQSSDPYIMVREAYFQRHDFIANGGELKPQENPNAQAIQDDLKDIDSE F008CD42CB8C75B4 phosphatidylethanolamine phosphatidyl(amino)ethanols phosphatidylethanolamines small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ompf_ecoli Outer membrane porin F ompF cmlB tolF coa cry Porin OmpF Outer membrane protein 1A Outer membrane protein IA Outer membrane protein B b0929 JW0912 Outer membrane protein F protein protein ecoli Escherichia coli (strain K12) MMKRNILAVIVPALLVAGTANAAEIYNKDGNKVDLYGKAVGLHYFSKGNGENSYGGNGDMTYARLGFKGETQINSDLTGYGQWEYNFQGNNSEGADAQTGNKTRLAFAGLKYADVGSFDYGRNYGVVYDALGYTDMLPEFGGDTAYSDDFFVGRVGGVATYRNSNFFGLVDGLNFAVQYLGKNERDTARRSNGDGVGGSISYEYEGFGIVGAYGAADRTNLQEAQPLGNGKKAEQWATGLKYDANNIYLAANYGETRNATPITNKFTNTSGFANKTQDVLLVAQYQFDFGLRPSIAYTKSKAKDVEGIGDVDLVNYFEVGATYYFNKNMSTYVDYIINQIDSDNKLGVGSDDTVAVGIVYQF 3F0974D96DB65464 3F0974D96DB65464 mlab_ecoli Intermembrane phospholipid transport system binding protein MlaB mlaB yrbB JW5535 b3191 protein protein ecoli Escherichia coli (strain K12) MSESLSWMQTGDTLALSGELDQDVLLPLWEMREEAVKGITCIDLSRVSRVDTGGLALLLHLIDLAKKQGNNVTLQGVNDKVYTLAKLYNLPADVLPR BB71D8F0A23D0225 pqib_ecoli Intermembrane transport protein PqiB pqiB pqi5B b0951 Paraquat-inducible protein B JW0934 protein protein ecoli Escherichia coli (strain K12) MESNNGEAKIQKVKNWSPVWIFPIVTALIGAWVLFYHYSHQGPEVTLITANAEGIEGGKTTIKSRSVDVGVVESATLADDLTHVEIKARLNSGMEKLLHKDTVFWVVKPQIGREGISGLGTLLSGVYIELQPGAKGSKMDKYDLLDSPPLAPPDAKGIRVILDSKKAGQLSPGDPVLFRGYRVGSVETSTFDTQKRNISYQLFINAPYDRLVTNNVRFWKDSGIAVDLTSAGMRVEMGSLTTLLSGGVSFDVPEGLDLGQPVAPKTAFVLYDDQKSIQDSLYTDHIDYLMFFKDSVRGLQPGAPVEFRGIRLGTVSKVPFFAPNMRQTFNDDYRIPVLIRIEPERLKMQLGENADVVEHLGELLKRGLRGSLKTGNLVTGALYVDLDFYPNTPAITGIREFNGYQIIPTVSGGLAQIQQRLMEALDKINKLPLNPMIEQATSTLSESQRTMKNLQTTLDSMNKILASQSMQQLPTDMQSTLRELNRSMQGFQPGSAAYNKMVADMQRLDQVLRELQPVLKTLNEKSNALVFEAKDKKDPEPKRAKQ DIP protein P43671 original sequence version: null 2C75CEB22AB8F7AB yebt_ecoli Intermembrane transport protein YebT yebT b1834 JW1823 protein protein ecoli Escherichia coli (strain K12) MSQETPASTTEAQIKNKRRISPFWLLPFIALMIASWLIWDSYQDRGNTVTIDFMSADGIVPGRTPVRYQGVEVGTVQDISLSDDLRKIEVKVSIKSDMKDALREETQFWLVTPKASLAGVSGLDALVGGNYIGMMPGKGKEQDHFVALDTQPKYRLDNGDLMIHLQAPDLGSLNSGSLVYFRKIPVGKVYDYAINPNKQGVVIDVLIERRFTDLVKKGSRFWNVSGVDANVSISGAKVKLESLAALVNGAIAFDSPEESKPAEAEDTFGLYEDLAHSQRGVIIKLELPSGAGLTADSTPLMYQGLEVGQLTKLDLNPGGKVTGEMTVDPSVVTLLRENTRIELRNPKLSLSDANLSALLTGKTFELVPGDGEPRKEFVVVPGEKALLHEPDVLTLTLTAPESYGIDAGQPLILHGVQVGQVIDRKLTSKGVTFTVAIEPQHRELVKGDSKFVVNSRVDVKVGLDGVEFLGASASEWINGGIRILPGDKGEMKASYPLYANLEKALENSLSDLPTTTVSLSAETLPDVQAGSVVLYRKFEVGEVITVRPRANAFDIDLHIKPEYRNLLTSNSVFWAEGGAKVQLNGSGLTVQASPLSRALKGAISFDNLSGASASQRKGDKRILYASETAARAVGGQITLHAFDAGKLAVGMPIRYLGIDIGQIQTLDLITARNEVQAKAVLYPEYVQTFARGGTRFSVVTPQISAAGVEHLDTILQPYINVEPGRGNPRRDFELQEATITDSRYLDGLSIIVEAPEAGSLGIGTPVLFRGLEVGTVTGMTLGTLSDRVMIAMRISKRYQHLVRNNSVFWLASGYSLDFGLTGGVVKTGTFNQFIRGGIAFATPPGTPLAPKAQEGKHFLLQESEPKEWREWGTALPK DIP protein P76272 original sequence version: null 3F4585F094D2D05A mlad-phosphatidylethanolamine-1 3 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 12 unspecified role unspecified role prey prey physical association physical association Fig. 1C mlad-phosphatidylglycerol-1 3 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey physical association physical association Fig. 1C pqib-phosphatidylethanolamine-1 3 15 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 12 unspecified role unspecified role prey prey physical association physical association Fig. 1C pqib-phosphatidylglycerol-1 3 15 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey physical association physical association Fig. 1C yebt-phosphatidylethanolamine-1 3 16 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain 12 unspecified role unspecified role prey prey physical association physical association Fig. 1C yebt-phosphatidylglycerol-1 3 16 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 region sufficient to bind sufficient binding region sufficient to bind range range certain certain sequence position certain 10 unspecified role unspecified role prey prey physical association physical association Fig. 1C mlad-mlae-1 1 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell 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 14 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell 8 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell 6 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed physical association physical association Fig. 3C After affinity purification and gel filtration (Figure 3B), SDS-PAGE analysis of the purified MlaD sample revealed the presence of three additional proteins forming a stable complex with MlaD (Figure 3C) mlab-mlad-1 4 7 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 ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed ecoli Escherichia coli (strain K12) physical association physical association Fig. 3D We next sought to determine the architecture of the MlaFEDB complex by single particle cryo-electron microscopy (cryo-EM). mlac-mlafedb_ecoli-1 5 9 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 region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 65 unspecified role unspecified role prey prey purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 4F mlac-mlaa-1 5 9 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 region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey purified purified ompf binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role prey prey purified purified mlaa binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 75 72 physical association physical association Fig. 4G mlac-mlad-1 5 7 unspecified role unspecified role prey prey 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 ecoli Escherichia coli (strain K12) 9 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 region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 4G mlafedb_ecoli mlaB:mlaD:mlaE:mlaF MlaFEDB complex MlaFEDB lipid transport complex 7 unspecified role unspecified role 14 unspecified role unspecified role 8 enzyme enzyme 6 unspecified role unspecified role physical association physical association ecoli Escherichia coli (strain K12) stable complex stable complex physical interaction evidence used in manual assertion physical interaction evidence used in manual assertion IPI inferred from physical interaction Required for the maintenance of asymmetery of the outer membrane, with an outer leaflet rich in lipopolysaccharide and an inner leaflet composed of phospholipids. MlaC (P0ADV7) shuttles phospholipid substrates between the inner membrane MlaFEDB transporter and an outer membrane complex, diffusing across the periplasm to the MlaFEDB complex in the inner membrane, where ATP hydrolysis may facilitate extraction of the lipid from MlaC and/or the translocation of the lipid into the inner membrane. mlaD exists as a hexamer, forming a ring with a central hydrophobic pore. Accepted 2018-JUL-05 by LIVIAP