Extended Data Fig. 2: Data processing and 3D reconstruction flowchart for mature CA hexamer and CypA/CA complex.

a, Individual tubes are cropped from motion-corrected micrographs in EMAN2. b, Tubes are sorted based on their diameters. c, The FFT of each tube is calculated and used to determine helical symmetry. In these data, 12 different helical symmetries exist within a 3 nm diameter variation (red box in b). d, Segments from the same helical symmetry tubes are classified in RELION. e, Iterative Real Space Helical Reconstruction and RELION refinement for each helical symmetry are performed. f, Symmetry expansion and localized reconstruction of individual hexamers (dashed circle) from each tube segment are performed, and C2 symmetry is applied. g, After symmetry expansion, the CA density was subtracted, leaving only the local region of CA and CypA for further classification, as highlighted in the dashed magenta box in e). h, Localized reconstruction was performed to calculate the position of each sub-particle and the extracted sub-particles were subject to 3D-classification into 10 classes without alignment, with a regularization T factor of 10 and resolution limit of 8 Å. i, Classes showing clear features of CypA and CA density were selected, further classified into Mode 1 (one CypA, blue or orange) and Mode 2 (2 CypAs, blue-cyan or orange-pink). j, The Mode 2 classes were aligned and averaged (right), whereas CypA above dimer interface from both Mode 1 and Mode 2 were aligned and averaged (left) to yield the final density maps from each helical symmetry.