Extended Data Figure 3: Analysis of MRJP1 via gel-filtration and mass spectrometry.

a, Gel-filtration analysis (HiLoad 16/600 Superdex 200 pg column) of MRJP1 present in the flow through (FT) (blue line) and eluting with 300 mM NaCl (red line) from the cation exchange chromatography (SP sepharose). In 20 mM sodium citrate pH 4.0, 150 mM NaCl (upper panel) MRJP1 is present as higher oligomers (FT fraction) and as monomer (300 mM NaCl elution fraction). In 50 mM potassium phosphate pH 7.0, 150 mM NaCl, the higher oligomers detected in the FT fraction dissociate mainly into hexamer (~90%) and minor portions of dodecamer (~5%), monomer (~4%) and probably trimer (~1%). Monomeric MRJP1 detected in the citrate buffer is in phosphate buffer mostly present as monomer (~91%) but also as hexamer (~9%). The different elution volumes of monomeric MRJP1 in pH 4.0 (84 ml = 57 kDa) and pH 7.0 (82 ml = 43 kDa) may result from different hydrodynamic volumes of the protein in the respective buffers. As the hydrodynamic volume appears to change largely between pH 4.0 and 7.0, this suggests that the structure of the monomer is different at the different pH values. The column was calibrated with ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa) (dashed black line) and the void volume determined with blue dextran (2,000 kDa) (solid black line). b, MRJP1 has been identified with 68.8% sequence coverage (red amino acids) by mass spectrometry. Mass deviation between measured and theoretical masses was not exceeding 5 p.p.m. and 0.02 Da for precursor and fragment ions, respectively.