Extended Data Fig. 1: Purification and crystal structure of ORF2p core.
From: Structures, functions and adaptations of the human LINE-1 ORF2 protein
a, Size exclusion chromatography (SEC) of recombinant ORF2p core (left, Superdex 200 increase 10/300 GL column, Cytiva) shows a homogenous and Gaussian peak corresponding to the expected retention time of a ~ 100 kDa monomer. SDS-PAGE analysis of peak fractions (right) shows the ORF2p core peak is >99% pure with contaminants and uncleaved MBP-ORF2p core removed in the void volume; a trace amount of uncleaved MBP-ORF2p remains in the preparation. b, In an ELISA-based reverse transcriptase assay (Roche), ORF2p core shows increased activity after SEC relative to heparin chromatography alone against an oligo(A) template. c, Comparison of ORF2p core crystal structure with AlphaFold model used for molecular replacement shows remarkable similarity, with a final root-mean-square deviation (RMSD) of 0.946 Å from the search model. ORF2p core comprises 46 secondary structural elements divided between 10 beta strands and 36 helices and is resolved from residues 251–1061 with gaps from 304–388, 799–803, 851–871, 905–912, and 923–927. d, 2Fo-Fc electron density map of the ORF2p core crystal with built model at a threshold of 2σ shows clear side chain density for important residues near the active site. The highlighted “gatekeeper” residue F605 sterically selects against ribonucleotides by clashing with the 2’-OH61, providing a rationale for ORF2p’s low RNA synthesis activity. e, Detailed view of key contacts between the primer and template and residues of the fingers (K541, K545, Q552), palm (F566, I567, P568, G569, M570, Q571, G660, P665) and wrist (Y878, K1047,G1048, I1050, S1051).
