a, Cartoon depicting the ReconSil procedure, as performed to investigate the interactions between ORC and an asymmetric origin. Particles are picked on micrographs with a low signal-to-noise ratio. Two-dimensional averages are calculated. Averages are superposed to the raw micrographs, overlaid to the particles that contributed to their generation. For this purpose, particle coordinates are combined with alignment parameters derived from 2D classification. This approach yields a signal-enhanced view of single instances of molecular complexes bound to a flexible substrate (in this case, ORC binding to an entire origin of replication). b, Representative raw micrograph, 2D class averages positioned according to their constituent particles, and a micrograph of origins reconstituted in silico with positioned 2D class averages overlaid onto the original image. Instances boxed in black are selected, red are rejected. c, Left, origins might be rejected owing to local particle clustering and aggregation, or because they contain visible raw particles that could not be classified (and therefore are not matched by a high-quality 2D average). This assay used M.HpaII–nucleosome origins that permit measurement of the length of origins because both the M.HpaII roadblock (next to ACS-bound ORC) and the nucleosome can be reconstituted. The measurement of origins reconstituted in silico was performed using ImageJ. d, Comparison of raw negative-stain electron microscopy data and origins reconstituted in silico for representative OCCM-bound origins shown in Fig. 1g. e, Example of origins reconstituted in silico (and corresponding raw images) showing double hexamers recruited to nucleosome–M.HpaII origins. ORC frequently rebinds to the ACS on origins that contain double hexamers, but shows no fixed interaction with the C-terminal face of the loaded double hexamer.