STRUCTURE OF COMPLEMENT POLY-C9 DETERMINED IN PROJECTION BY CRYOELECTRON MICROSCOPY AND SINGLE-PARTICLE ANALYSIS

The ring-like complement 'lesions' found on membranes of complement ly sed cells comprise a complex of components C5b through C9 that coalesc e to form hollow cylinders which penetrate the membrane bilayer and cr eate lytic pores. Walls of these C5b-9 membrane attack complex cylinde rs may consist primarily of the C9 component, since samples of purifie d, isolated C9 can polymerize into cylindrical structures which appear identical with the fully assembled C5b-9 complex. The structure of th ese poly-C9 molecules has been investigated using the techniques of cr yo-electron microscopy and single particle analysis. Sets of single po ly-C9 particles viewed as rings were selected from cryo-EM images, the n particles were aligned and treated by correspondence analysis to ide ntify the principle interparticle similarities and variations. The hig hest ranking variation found was the presence or absence of a dense in ner ring of protein density. Other important variations were interpret ed as different types of particle tilt. These results were used in sel ecting a subgroup of untilted particles for averaging and symmetry ana lysis. The rotational power spectrum of the initial average suggested 13-fold symmetry. The 13-fold symmetry was used to select and group pa rticles for further analysis. Individual particles were 13-fold rotati onal averaged and those with enhanced peripheral features were placed into either a right-handed subgroup or into a left-handed subgroup bas ed on orientation of the peripheral features. Particles within each gr oup were aligned and averaged, and a poly-C9 structure was produced wh ich shows important structural details and from which the C9 monomer s tructure can be deduced. The poly-C9 structure contains a dense inner ring of diameter between 113-181 Angstrom and which is modulated into 13 discrete peaks with peak-to-peak separation of approx. 35 Angstrom. The dense inner ring is surrounded by a less dense, concentric outer rim extending to 254 Angstrom diameter. The outer rim contains project ions that are contiguous with the inner peaks but are skewed relative to the ring radius to produce the appearance of a pin-wheel. These pro jections correspond with the peripheral features picked up in the rota tionally averaged individual particles; the left- or right-handed orie ntation of projections may result from the up/down orientation of indi vidual particles in ice. The C9 monomer structure within the cylinder is suggested by the density distribution. The monomer would be a rod w ith diameter of 35 Angstrom, oriented parallel to the cylinder axis an d would be roughly perpendicular to a membrane. A portion of the C9 mo nomer would form the projecting outer rim. In side views of the comple x the outer rim density would be the torus structure seen distal to th e membrane-inserting region. These results indicating an extended C9 s tructure are consistent with C9 unfolding in the transition from monom er to poly-C9 complex.