MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. EMBO J. (0261-4189) 2012 imex curation Suloway CJ., Rome ME., Clemons WM. Only protein-protein interactions in vitro In vitro light scattering light scattering predetermined predetermined participant predetermined Suloway CJ., Rome ME., Clemons WM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. EMBO J. (0261-4189) 2012 imex curation Suloway CJ., Rome ME., Clemons WM. Only protein-protein interactions in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Suloway CJ., Rome ME., Clemons WM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. EMBO J. (0261-4189) 2012 imex curation Suloway CJ., Rome ME., Clemons WM. Only protein-protein interactions ecoli Escherichia coli (strain K12) pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Suloway CJ., Rome ME., Clemons WM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation mtrb_metja Tetrahydromethanopterin S-methyltransferase subunit B mtrB N5-methyltetrahydromethanopterin--coenzyme M methyltransferase subunit B MJ0850 protein protein metja Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Methanococcus jannaschii MATYVFIDQNIPLVYTVETGVITKGFGDLLFVDVSPIEEQIKKLETLVDAYEHSLDPRYPPLNSFPNRDGVYAISGYFKSAFFGFWIGLGIMALLAIILGVKF mint 0797F2691A1CE10F arsa_metja Putative arsenical pump-driving ATPase Arsenical resistance ATPase Arsenite-translocating ATPase Arsenite-transporting ATPase MJ1142 protein protein metja Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Methanococcus jannaschii MLSKIKDSINSLRGITEKKLEKKDGTKYIMFGGKGGVGKTTMSAATGVYLAEKGLKVVIVSTDPAHSLRDIFEQEFGHEPTKVKGYDNLYVVEIDPQKAMEEYKEKLKAQIEENPFLGEMLEDQLEMAALSPGTDESAAFDVFLKYMDSNEFDVVIFDTAPTGHTLRFLGMPEVMDKYMTKLIKLRKQMSGFMKMMKKLLPFGGKDEDIDYDKMLEELEKMKERIVRARNILSDPERTAFRLVVIPEEMSILESERAMKALQKYGIPIDAVIVNQLIPEDVQCDFCRARRELQLKRLEMIKEKFGDKVIAYVPLLRTEAKGIETLKQIAKILYGEEEKEEQKIEQKVGQ mint 00041F603DFDD5A3 sece_metja Protein translocase subunit SecE secE Protein transport protein Sec61 gamma subunit homolog MJ0371 protein protein metja Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Methanococcus jannaschii MKTDFNQKIEQLKEFIEECRRVWLVLKKPTKDEYLAVAKVTALGISLLGIIGYIIHVPATYIKGILKPPTTPRV mint 10474AE938CC0ECD q5jif4_theko TK0994 protein protein theko Thermococcus kodakaraensis (strain ATCC BAA-918 / JCM 12380 / KOD1) Pyrococcus kodakaraensis (strain KOD1) MREFFLPKKDYRVVFFIGKGGVGKTTSSAAAAVALADKGYRTLIVSLDPAHNLGDVLMEKLSDKPKKIAENLYASELDMEKLIKSYLKHLEENLKHMYRYLTVINLEKYFEVLSFSPGIEEYATLEAVKEILMKGDEWDVIVFDTPPTGLTLRVLALPRISLIWTDKLIEIRRAILERRAAIANIHGEQEFVVEGERIKLPTKEEEDPVMKELKAYRKEVAFVESVLTDPDKTSVVAVMNPEMLPLYETERAYESLKKFRIPFNMIVMNKVFELKGEVPELKAKLEAQERVLMEVSEKFKGVDIVKIPIFPEEPRGVERLRELGGAIIGER mint 0A7A6AA740D72953 sc6b1_yeast Protein transport protein SBH1 Sec61 complex subunit SBH1 YER087BC YER087C-B SBH1 SEB1 Sec61 complex subunit beta protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSPTPPGGQRTLQKRKQGSSQKVAASAPKKNTNSNNSILKIYSDEATGLRVDPLVVLFLAVGFIFSVVALHVISKVAGKLF 23B0313EB37744FA mtra_metja Tetrahydromethanopterin S-methyltransferase subunit A mtrA N5-methyltetrahydromethanopterin--coenzyme M methyltransferase subunit A MJ0851 protein protein metja Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Methanococcus jannaschii MANKREPAPGWPIVSGEYVVGNPESCVGVVTLGSHGLEQACIDAGAAIAGPCHTENLGIEKVVANYISNPNIRFMILCGSEVQGHITGQCFKALWENGIGDDGGIIGAKGAIPFLENVNKEAVERFRRQIVEVVDLIDCEDIGKITQAIKECLSKDPGAIDEDPFIIELEGGKGGGEEEEGVIKPITPEMAIIESRMRLIGNEMCYNGLLAKWQAGYYNGKIQGIATGLFLMLLIMGILMF mint 155EAE6418E09B8D get3_yeast ATPase GET3 YDL100C GET3 ARR4 D2371 Arsenical pump-driving ATPase Arsenite-stimulated ATPase Guided entry of tail-anchored proteins 3 Golgi to ER traffic protein 3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MDLTVEPNLHSLITSTTHKWIFVGGKGGVGKTTSSCSIAIQMALSQPNKQFLLISTDPAHNLSDAFGEKFGKDARKVTGMNNLSCMEIDPSAALKDMNDMAVSRANNNGSDGQGDDLGSLLQGGALADLTGSIPGIDEALSFMEVMKHIKRQEQGEGETFDTVIFDTAPTGHTLRFLQLPNTLSKLLEKFGEITNKLGPMLNSFMGAGNVDISGKLNELKANVETIRQQFTDPDLTTFVCVCISEFLSLYETERLIQELISYDMDVNSIIVNQLLFAENDQEHNCKRCQARWKMQKKYLDQIDELYEDFHVVKMPLCAGEIRGLNNLTKFSQFLNKEYNPITDGKVIYELEDKE F795C359B5A4A461 F795C359B5A4A461 secg_metja Preprotein translocase subunit SecG secG Protein transport protein Sec61 subunit beta homolog MJ0912.1 protein protein metja Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) Methanococcus jannaschii MSKREETGLATSAGLIRYMDETFSKIRVKPEHVIGVTVAFVIIEAILTYGRFL mint D2D5DD22331BE2AF arsa-1 2 GET3 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Stoichiometry: 4.0 direct interaction direct interaction mint f1a f1b f2 sf2 st1 Coordinates and structure factors heve been deposited in the Protein Data Bank with accession code 3UG6 and 3UG7. arsa-secg 3 GET3 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Sec beta 11 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. arsa-sece 3 Sec E 6 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined GET3 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. arsa-mtra 3 GET3 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain mtrA 9 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. arsa-mtrb 3 mtrB 4 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined GET3 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. arsa-sbh1 3 GET3 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Sbh1 8 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. get3-sbh1 3 Sbh1 8 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined GET3 10 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. q5jif4-sbh1 3 GET3 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Sbh1 8 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. q5jif4-mtrb 3 mtrB 4 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined GET3 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. q5jif4-mtra 3 GET3 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined mtrA 9 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. q5jif4-sece 3 Sec E 6 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined GET3 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. q5jif4-secg 3 GET3 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Sec beta 11 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role prey prey partially purified partially purified mbp tag mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f3b mint ScGet3, MjGet3 or TkGet3 and TA proteins were co-expressed in BL21-Gold(DE3) E. coli. Soluble complexes were purified in two steps using amylose resin (NEB) and Ni-NTA resin. arsa-1 1 GET3 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q58542:p.Met193Asp mutation increasing interaction mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain M D Tue Jul 12 10:14:13 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation increasing' Wed May 02 17:21:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'met193asp' Tue Jun 30 16:35:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Met193Asp' binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Stoichiometry: 4.0 direct interaction direct interaction mint To test this, we dialysed the protein against a 1% (w/v) solution of the small micelle detergent N-octyl-b- D-glucopyranoside (b-OG) just above the critical micelle concentration. After overnight dialysis, the majority of the MjGet3 shifted to a peak consistent with a dimer (Figure 4D, compare green trace with black trace). When we then dialysed out the detergent, the protein returned to the tetramer (Figure 4D, black dashed trace) confirming that in these conditions this was the stable state. To test this directly, we introduced mutations in helix 8 of MjGet3 near the interface of the three-helix bundle (Figure 4C). Two of these, F192D and M196D, directly disrupt the hydrophobic interface and both of them shift predominantly to a dimer peak by SEC (Figure 4E, red and purple trace). A third mutation, M193D, was not at the interface (Figure 4C) and this mutant strongly favoured the tetramer (Figure 4E, orange trace). Partial deletion of helix 8 resulted in a loss of the tetramer peak as well (Figure 4E, yellow trace). f4d f4e f5b t1 get3-3 1 GET3 10 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 4.0 direct interaction direct interaction mint f4a f4b f5b t1 ScGet3 purified after expression in E. coli is predominantly a dimer by SEC; however, a small pool always purified as a tetramer (Figure 4A and B, solid blue trace). This tetramer pool was stable enough to be rerun over the column (Figure 4B, dashed line). We noted that the tetramer fraction would degrade over time to dimer, while we would never see conversion of the dimer to tetramer. get3-3 1 GET3 10 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 2.0 direct interaction direct interaction mint f4a f4b f5b t1 ScGet3 purified after expression in E. coli is predominantly a dimer by SEC; however, a small pool always purified as a tetramer (Figure 4A and B, solid blue trace). This tetramer pool was stable enough to be rerun over the column (Figure 4B, dashed line). We noted that the tetramer fraction would degrade over time to dimer, while we would never see conversion of the dimer to tetramer. arsa-1 1 GET3 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q58542:p.Phe192Asp mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F D Tue Jul 12 10:54:58 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:11:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe192asp' Tue Jun 30 19:55:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe192Asp' binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q58542:p.Met196Asp mutation increasing interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain M D Tue Jul 12 10:17:00 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation increasing' Wed May 02 17:10:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'met196asp' Tue Jun 30 18:58:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Met196Asp' direct interaction direct interaction mint To test this, we dialysed the protein against a 1% (w/v) solution of the small micelle detergent N-octyl-b- D-glucopyranoside (b-OG) just above the critical micelle concentration. After overnight dialysis, the majority of the MjGet3 shifted to a peak consistent with a dimer (Figure 4D, compare green trace with black trace). When we then dialysed out the detergent, the protein returned to the tetramer (Figure 4D, black dashed trace) confirming that in these conditions this was the stable state. To test this directly, we introduced mutations in helix 8 of MjGet3 near the interface of the three-helix bundle (Figure 4C). Two of these, F192D and M196D, directly disrupt the hydrophobic interface and both of them shift predominantly to a dimer peak by SEC (Figure 4E, red and purple trace). A third mutation, M193D, was not at the interface (Figure 4C) and this mutant strongly favoured the tetramer (Figure 4E, orange trace). Partial deletion of helix 8 resulted in a loss of the tetramer peak as well (Figure 4E, yellow trace). f4d f5b t1 f4e