Actin filament labels for localizing protein components in large complexes viewed by electron microscopy

Abstract

Localizing specific components in three-dimensional reconstructions of protein complexes visualized in an electron microscope increases the scientific value of those structures. Subunits are often identified within the complex by labeling; however, unless the label produces directly visible features, it must be detected by computational comparison with unlabeled complex. To bypass this step, we generated a cloneable tag from the actin-nucleating protein Spire that produces a directly visible "pointer" to the subunit after actin polymerization. We have used this new label to identify the intron of the C complex spliceosome to its small domain by fusing the 10 kDa Spire moiety to the affinity label that binds recombinant stem loops in the pre-mRNA substrate and assembling an actin filament on the particle. Published by Cold Spring Harbor Laboratory Press. Copyright © 2009 RNA Society.