Extended Data Fig. 2: Data-processing pipeline for the cryo-EM movies of CSA-VAR2CSA NF54 complex.

a, Flow chart showing the image-processing pipeline for the cryo-EM data of VAR2CSA starting with 6,196 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Data were processed in cryoSPARC. Full frame motion correction was done by cryoSPARC’s own implementation. CTF estimation for each micrograph was calculated with Gctf. Particles were autopicked from each micrograph with the blob picker from cryoSPARC and then sorted by 2D classification for two rounds. The twelve highest-populated classes with clear features from the 2D classification are shown. The dataset contained 858,299 particles. A subset of particles was used to generate an ab initio map in cryoSPARC. Particles were classified into 5 classes using the low-pass-filtered (30 Å) ab initio map as a template. Class 1 was selected with 157,702 particles to conduct NU-refinement and generated a 3.87 Å map. A mask covering DBL5ɛ and DBL6ɛ domains were then used to perform local refinement and generated a 4.88 Å map. Class 1 and Class 4 which have a clear core density were selected again with 299,571 particles to conduct NU-refinement and generated a 3.5 Å map, local refinement improved the resolution of the core to 3.36 Å. Scale bar: 10 nm. Analysis was performed three times independently with similar results. b, Gold-standard FSC curves are shown. The dotted line represents the 0.143 FSC cut-off. c, Angular distribution calculated in cryoSPARC for particle projections of the full-length protein (right) and the core (left). Heat map shows number of particles for each viewing angle. d,e and f, Local resolution of the core (d), full length (e) and arm region (f) in two views. The representation of colors for different resolution are shown on the right. g, FSC calculated between the refined structures and the full map. h, Representative cryo-EM densities from the core machinery map. i, Representative cryo-EM densities from the arm with DBL5ɛ and DBL6ɛ model docked in.