Journal: bioRxiv

Article Title: Affinity purification contaminants identified by cryo-EM and mass spectrometry

doi: 10.64898/2026.03.19.712978

Figure Lengend Snippet: Protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) was identified as a contaminant of anti-FLAG M2 resin by cryo-EM. (A) Selected 2D classes illustrate how the contaminant appeared during cryo-EM data preprocessing. (B) A 3D volume with a reported resolution of 4.53 Å was generated from 9243 particles. The approximate dimensions of the volume (measured along its principal axes) are shown. (C) Four copies of an atomic model of PRMT5:MEP50 (PDB:8cyi) fit well into the generated volume. (D) A Coomassie-stained gel shows the elution fractions after purification of a preparation of the type II receptor construct (85 kDa doublet band), with a faint band corresponding to PRMT5 (expected molecular weight: 73 kDa) indicated with a purple arrowhead. MEP50 (33 kDa) was also identified by LC-MS/MS, but is not visible on the gel, likely due to low concentration. M: marker, E: elution.

Article Snippet: Clarified lysate was incubated with anti-FLAG M2 resin (Merck, A2220), pre-equilibrated in wash buffer (100 mM Tris-HCl, pH 8, 150 mM NaCl, 0.02% DDM /0.002% CHS), for 1 hour at 4 °C with gentle mixing by rotation.

Techniques: Cryo-EM Sample Prep, Generated, Staining, Purification, Construct, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Marker

Journal: bioRxiv

Article Title: Affinity purification contaminants identified by cryo-EM and mass spectrometry

doi: 10.64898/2026.03.19.712978

Figure Lengend Snippet: AlphaFold3 prediction of anti-FLAG M2 Fab bound to PRMT5:MEP50 suggests that the Fab may interact with PRMT5 at a non-linear surface epitope. (A) AlphaFold3 was used to generate a model of 4 chains of PRMT5 (purple), 4 chains of MEP50 (yellow) and the anti-FLAG M2 Fab heavy (dim grey) and light (light grey) chains . The overall predicted structure had an ipTM score of 0.78 and a pTM score of 0.81. (B) The PRMT5:MEP50 complex and the Fab complex were each predicted with very high confidence, with pLDDT > 90 (dark blue) for most residues. (B) The chains of the PRMT5:MEP50 complex were positioned with reasonable confidence relative to each other (mean predicted aligned error (PAE) of 12 Å). Similarly, the relative position of the Fab chains was reasonably confidently predicted (mean PAE of 8 Å). However, the interaction between the Fab (chains i and j) and PRMT5 (chain a) is not confidently predicted, with high mean PAE of around 30 Å. (C) The surface of PRMT5:MEP50 is highly charged, which may explain its recognition by the antibody against the highly charged FLAG-tag.

Article Snippet: Clarified lysate was incubated with anti-FLAG M2 resin (Merck, A2220), pre-equilibrated in wash buffer (100 mM Tris-HCl, pH 8, 150 mM NaCl, 0.02% DDM /0.002% CHS), for 1 hour at 4 °C with gentle mixing by rotation.

Techniques: FLAG-tag