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K E Y WORDS. Correspondence analysis, hierarchical ascendant classification, image analysis, electron microscopy. SUMMARY Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC.The method is applied to two sets of 204 and 177 images that show the 70s ribosome of Escherichia coli, in the range of overlap views as defined by A. Verschoor and co-workers, and to two sets of 480 and 496 images of the 50s subunit of E . coli depleted of L7/L12 proteins in the well-defined crown view. Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates. These classes appear to be related to different orientations on the specimen grid (in the case of the 70s particle) and to different conformational states (50s subunit).

Well-preserved structures of native and achymotrypsin-bound a2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native a2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the a2-macroglobulin-chymotrypsin conijugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of a2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex. a2-Macroglobulin (a2M) is one of the major antiproteases found in the plasma of vertebrates. It has the capacity to inhibit not only endoproteases normally present in the plasma but also proteases from other origins (1). a2M, isolated from human plasma, is a large glycoprotein (Mr, 725,000) composed of four identical subunits (Mr, 180,000) (2, 3). The quaternary structure consists of two noncovalently bound protomers; each protomer is made up of two subunits covalently linked by two disulfide bonds (4). When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5-7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11). The inactivated protease may form a covalently bound complex with a2M by reacting with an intramolecular thiol ester linkage between glutamine and cysteine residues. Even though there are four "bait" sites available, it has been proposed that a2M has two independent and identical proteinase binding sites (12)(13)(14). By using energy transfer experiments, Pochon et al. (12) demonstrated that the two sites are 4.4 nm apart. The binding of 2 mol of proteases per mol of a2M has been observed only with smaller proteases like trypsin and chymotrypsin (Mr, 25,000); larger proteases like plasmin (Mr, 81,000) display a 1:1 molar binding ratio (15).There have been conflicting reports relating the various structures of a2M obtained by electron microscopy to the native and protease bound forms. This discrepancy seems to have resulted from the study of a2M that had undergone proteolysis during its isolation (16). Early studies reported the presence of two forms (7,(17)(18)(19): an ovate structure shaped like two crescents facing each other with a bar in the center (the "closed" form) and a ...