A Two-Exposure Technique for Ice-Embedded Samples Successfully Reconstructs the Chlorocruorin Pigment of Sabella spallanzanii at 2.1 nm Resolution

Abstract

A technique for reconstructing ice-embedded macromolecules from electron micrographs taken at two specimen tilts (±23°) has been used to determine the structure of chlorocruorin isolated from the Polychaete annelid Sabella spallanzanii. Images of individual molecules were extracted in couples from two micrographs of the same field of view so each couple consists of two projections of the same molecule. One couple was used as a fixed reference for alignment. Different references yielded reconstructions with different orientations. These were merged to give a model against which the orientation of 1624 first-exposure images was refined to give a final reconstruction at 2.1 nm resolution. The structure of this hematic pigment, essentially the same as that for Lumbricus terrestris, is a bilayer structure with overall symmetry D6, containing six hollow groups per layer. A hollow group is formed by six globular masses and has approximate threefold symmetry. Other structural elements connect the two layers and the hollow groups in a layer. This non-globin material occupies about 15% of the total molecular volume. The results show that the double-exposure strategy, previously described by some of the authors and tested in computer simulations, performs well in real experiments and could be used to obtain preliminary reconstructions in a semiautomatic way.