Fig. 2.

MTH labelling of Spc42 at the yeast spindle pole body. (A) A diagram of SPB organization. Spc42 resides in the central core of the SPB roughly at the plane of the nuclear envelope. The protein’s C-terminus, where our MTH tag has been put, is at the outer of two layers visible in the EM. (B) A computational slice from a tomogram of wild-type yeast treated with gold, as described (Table S1, condition #3). This and all other tomographic slices presented are 5-nm thick. (C) A similar slice from a yeast cell expressing Spc42-2xMTH. Both these samples contained uranyl acetate in the freeze substitution to provide a general contrast of the biological material (Table S1, condition #3). Arrowhead indicates the region containing Spc42. (D) EM image of a 70-nm section cut from a yeast SPB in which Spc42, tagged with GFP at its C-terminus, has been labelled by indirect immuno-EM with colloidal gold. Bar = 100 nm. Movies of tomographic volumes corresponding to (B) and (C) can be found in the supplementary materials.