Template matching and subtomogram averaging

Subtomograms of pyrenoid regions were binned once to a pixel size of 11.4 Å for localization of RuBisCO complexes. RuBisCO complexes were detected by template matching (Frangakis et al., 2002) using a spherical template with a diameter of 13.68 nm. A spherical template, which functioned well due to the homogeneity of pyrenoid composition and the roughly spherical shape of RuBisCO at the tomogram resolution, made it possible to discard the rotational search, dramatically increasing the localization speed and enabling iterative fine-tuning of parameter values. For template matching, the pyrenoid subtomograms were low-pass filtered using the Crowther criterion (Crowther et al., 1970) to the resolution of a 12 nm diameter particle with the same 2^° tilt increment as the tomographic tilt series. Cross-correlation peaks were exhaustively extracted, yielding a set of RuBisCO particle positions. Over 10,000 subvolumes (45.6 nm³) centered on these RuBisCO positions were selected from the unfiltered tomograms in order to analyze the local neighborhood around each RuBisCO complex. The subvolumes were subsequently aligned and averaged with the PyTom toolbox as previously described (Hrabe et al., 2012; Chen et al., 2013).