3-DIMENSIONAL STRUCTURE OF A SINGLE FILAMENT IN THE LIMULUS ACROSOMALBUNDLE - SCRUIN BINDS TO HOMOLOGOUS HELIX-LOOP-BETA MOTIFS IN ACTIN

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal fila ment with 28 actin and 28 scruin molecules in 13 helical turns. A nove l computational procedure was developed to extract single columns of s uperimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scr uin molecule is organized into two domains which contact two actin sub units in different strands of the same actin filament. A correlation o f Holmes' actin filament model to the density in our acrosomal filamen t map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scr uin makes extensive interactions with helix-loop-beta motifs in subdom ain 3 of one actin subunit and in subdomain 1 of a consecutive actin s ubunit along the genetic filament helix. These two actin subdomains ar e structurally homologous and are closely spaced along the actin filam ent. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-i dentical positions across two consecutive actin subunits.