DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dong-2017-6 in vitro In vitro pull down pull down experimental particp experimental participant identification experimental particp Dong null., Guo M., Wang S., Zhu Y., Wang S., Xiong Z., Yang J., Xu Z., Huang Z. huangzhiwei@hit.edu.cn Nature 2017 imex curation Only protein-protein interactions imex curation dong-2017-5 in vitro In vitro mst microscale thermophoresis mst experimental particp experimental participant identification experimental particp Dong null., Guo M., Wang S., Zhu Y., Wang S., Xiong Z., Yang J., Xu Z., Huang Z. huangzhiwei@hit.edu.cn Nature 2017 imex curation Only protein-protein interactions imex curation dong-2017-3 in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column experimental particp experimental participant identification experimental particp Dong null., Guo M., Wang S., Zhu Y., Wang S., Xiong Z., Yang J., Xu Z., Huang Z. huangzhiwei@hit.edu.cn Nature 2017 imex curation Only protein-protein interactions imex curation dong-2017-4 in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction experimental particp experimental participant identification experimental particp Dong null., Guo M., Wang S., Zhu Y., Wang S., Xiong Z., Yang J., Xu Z., Huang Z. huangzhiwei@hit.edu.cn Nature 2017 imex curation Only protein-protein interactions imex curation urs000080e253_strpy_grna URS000080E253 gRNA grna guide rna guide RNA grna strpy Streptococcus pyogenes GGAAAUUAGGUGCGCUUGGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU cas9_strp1 CRISPR-associated endonuclease Cas9/Csn1 cas9 csn1 SPy_1046 SpCas9 SpyCas9 protein protein strp1 Streptococcus pyogenes serotype M1 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD a0a247d711_lismn acrIIA4 G3O21_003035 APD94_15065 GON91_02470 UI29_15095 protein protein lismn Listeria monocytogenes MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN a0a459ndx2_lismn acrIIA2 D4271_13320 D4D89_13255 B1N40_08540 B4X79_13415 E0I39_13150 FJU19_12475 G3R95_002503 AF006_07510 APD94_09090 D4C60_14490 D4D22_14105 D5M63_13385 E1V33_15495 protein protein lismn Listeria monocytogenes MTLTRAQKKYAEAMHEFINMVDDFEESTPDFAKEVLHDSDYVVITKNEKYAVALCSLSTDECEYDTNLYLDEKLVDYSTVDVNGVTYYINIVETNDIDDLEIATDEDEMKSGNQEIILKSELK cas9-a0a247d711_lismn-2 3554925 N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role ancillary ancillary in vitro translated in vitro translated protein in vitro translated in vitro In vitro N/A 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role bait bait purified purified gst tag gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst 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 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role prey prey purified purified ecoli Escherichia coli (strain K12) physical association physical association false false false Extended Data Figure 1 cas9-a0a247d711_lismn-1 3554925 N/A 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role bait bait purified purified gst tag gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst 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 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role prey prey purified purified ecoli Escherichia coli (strain K12) N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role ancillary ancillary in vitro translated in vitro translated protein in vitro translated in vitro In vitro physical association physical association false false false Extended Data Figure 1 a0a247d711_lismn-cas9-3 3554925 N/A 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role prey prey purified purified ecoli Escherichia coli (strain K12) N/A 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role bait bait purified purified A0A247D711:p.Asn12Thr N12T mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false A0A247D711:p.Gly38Ala G38A mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false gst tag gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false A0A247D711:p.Asp23Arg D23R mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role ancillary ancillary in vitro translated in vitro translated protein in vitro translated in vitro In vitro physical association physical association false false false Extended Data Figure 3b (AcrIIA4 mutant) a0a247d711_lismn-cas9-4 3554925 N/A 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role bait bait purified purified A0A247D711:p.Asn36Tyr N36Y mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false gst tag gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false A0A247D711:p.Asn39Arg N39R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false A0A247D711:p.Glu70Arg E70R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false A0A247D711:p.Asp69Arg D69R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false A0A247D711:p.[Asp14Arg;Glu40Arg] D14R, E40R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false A0A247D711:p.[Asn36Arg;Asp69Arg] N36R, D69R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing 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 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role prey prey purified purified ecoli Escherichia coli (strain K12) N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role ancillary ancillary in vitro translated in vitro translated protein in vitro translated in vitro In vitro physical association physical association false false false Extended Data Figure 3b (AcrIIA4 mutant) a0a247d711_lismn-cas9-5 3554925 N/A 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role prey prey purified purified Q99ZW2:p.[Arg976Tyr;Lys1200Tyr] R976Y, K1200Y mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q99ZW2:p.[Lys1200Tyr;Ser1216Arg] K1200Y,S1216R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Q99ZW2:p.Thr1138Tyr T1138Y mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q99ZW2:p.Asp1332Arg D1332R mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q99ZW2:p.Arg1335Tyr R1335Y mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q99ZW2:p.Ser1216Thr S1216T 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 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role bait bait purified purified gst tag gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst 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 3554929 predetermined predetermined participant predetermined unspecified role unspecified role ancillary ancillary in vitro translated in vitro translated protein in vitro translated in vitro In vitro physical association physical association false false false Extended Data Figure 3b (SpyCas9 mutant) a0a394vx67_lismn-cas9-1 3554926 N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component in vitro translated in vitro translated protein in vitro translated in vitro In vitro N/A 3554930 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) N/A 3554932 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4d a0a247d711_lismn-cas9-6 3554926 N/A 3554929 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component in vitro translated in vitro translated protein in vitro translated in vitro In vitro N/A 3554930 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) N/A 3554931 predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 4d a0a247d711_lismn-cas9-1 3554927 N/A 3554929 weight by bromide molecular weight estimation by bromide staining weight by bromide unspecified role unspecified role neutral component neutral component in vitro translated in vitro translated protein in vitro translated in vitro In vitro N/A 3554930 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) N/A 3554931 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) physical association physical association false false false Figure 1 a0a247d711_lismn-cas9-2 3554928 N/A 3554931 predetermined predetermined participant predetermined structure determination identification by structure determination identification by structure determination structure determination unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 N/A 3554930 predetermined predetermined participant predetermined structure determination identification by structure determination identification by structure determination structure determination unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 N/A 3554929 unspecified role unspecified role neutral component neutral component in vitro translated in vitro translated protein in vitro translated in vitro In vitro Stoichiometry: 1.0 physical association physical association false false false Figure 2, Extended Data Table 1