9722353 Stewart Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques provides a powerful way to examine the structure of large macromolecular assemblies. Specific research objectives include cryo-EM studies of the ribonucleoprotein vault particle and alpha-crystallin, a molecular chaperone. Vault studies will include a three-dimensional reconstruction of the intact particle, difference imaging analysis with ribonuclease treated particles to reveal the location of the internal RNA, and structural mapping using truncated forms of the major vault protein. Alpha-crystallin studies will include reconstruction of native and cross-linked recombinant human (B crystallin multimers and (B crystallin complexed with lactalbumin, a large target protein. Specific educational objectives include: mentoring graduate students and postdoctoral fellows, strengthening multi-departmental graduate education at UCLA, and volunteering in science outreach programs for minority and high school students. Cryo-EM often represents the only feasible structural approach for large (1-400 million Dalton) complexes if the sample has proven resistant to crystallization, as have both the vault and alpha-crystallin. Thus these studies will enable structure/function analysis of a ribonucleoprotein particle conserved across species as diverse as humans and the slime mold Dictyostelium, and provide insight into the quaternary structure of alpha-crystallin and its mechanism of action in binding partially denatured proteins. Integration of the research and educational goals will be accomplished by teaching novel structural methods such as cryo-EM and image reconstruction to upper level undergraduates and graduate students, as well as mentoring graduate students in the laboratory. ***
