Lumbricus terrestris erythrocruorin is a giant oxygen-transporting macromolecule in the blood of the common earth worm (worm "hemoglobin"). In our current study, we use specimens (kindly provided by Drs W.E. Royer and W.A. Hendrickson) embedded in vitreous ice (1) to avoid artefacts encountered with the negative stain preparation technigue used in previous studies (2-4).Although the molecular structure is well preserved in vitreous ice, the low contrast and high noise level in the micrographs represent a serious problem in image interpretation. Moreover, the molecules can exhibit many different orientations relative to the object plane of the microscope in this type of preparation. Existing techniques of analysis requiring alignment of the molecular views relative to one or more reference images often thus yield unsatisfactory results.We use a new method in which first rotation-, translation- and mirror invariant functions (5) are derived from the large set of input images, which functions are subsequently classified automatically using multivariate statistical techniques (6). The different molecular views in the data set can therewith be found unbiasedly (5). Within each class, all images are aligned relative to that member of the class which contributes least to the classes′ internal variance (6). This reference image is thus the most typical member of the class. Finally the aligned images from each class are averaged resulting in molecular views with enhanced statistical resolution.
The reassociation of Lumbricus terrestris hemoglobin dissociated at alkaline pH.