Extended Data Fig. 5: Data-processing pipeline for the cryo-EM movies of apo VAR2CSA FCR3 and VAR2CSA FCR3_crosslink.

a, Flow chart showing the image-processing pipeline for the cryo-EM data of VAR2CSA starting with 100,108 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Processing was done within 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 to exclude bad particles. The twelve highest-populated classes with clear features from the 2D classification are shown. The dataset contained 783,088 particles. A subset of particles was used to generate an ab initio map in cryoSPARC. Particles were classified into 10 classes using the low-pass-filtered (30 Å) ab initio map as a template. Class 4 with a total of 271,442 particles was selected to conduct NU-refinement and generated a 4 Å map. A mask covering the arm region were then used to perform local refinement and generated a 4.69 Å map. The angular distribution calculated in cryoSPARC for particle projections are shown in heat map which shows number of particles for each viewing angle. Scale bar: 10 nm. Analysis was performed three times independently with similar results. b, Gold-standard FSC curves. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 4 Å (black) and 4.69 Å (blue) for the full length protein and arm region respectively. c, Local resolution of the full length VAR2CSA map in two views. The representation of colors for different resolution are shown on the right. d, Local resolution of the arm map in two views. The representation of colors for different resolution are shown on the right. e, Flow chart showing the image-processing pipeline for the cryo-EM data of crosslinked VAR2CSA starting with 4,739 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). All processing was done within cryoSPARC. Full frame motion correction was done by cryoSPARC’s own implementation and a sample. 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 to exclude bad particles. The twelve highest-populated classes with clear features from the 2D classification are shown. The dataset contained 505,409 particles. A subset of particles was used to generate an ab initio map in cryoSPARC. Particles were classified into 3 classes using the low-pass-filtered (30 Å) ab initio map as a template. Class 3 was selected to conduct NU-refinement and generated a 3.52 Å map. A mask covering the core was then used to perform local refinement and generated a 3.38 Å map. The angular distribution calculated in cryoSPARC for particle projections are shown in heat map which shows number of particles for each viewing angle. Scale bar: 10 nm. Analysis was performed three times independently with similar results. f, Gold-standard FSC curves. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 3.38 Å of the core. g, Local resolution of the crosslinked VAR2CSA FCR3 core map in two views. The representation of colors for different resolution are shown on the right.