Actin filament labels for localizing protein components in large complexes viewed by electron microscopy.
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.
Stroupe
M Elizabeth
ME
Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
Xu
Chen
C
Goode
Bruce L
BL
Grigorieff
Nikolaus
N
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry; D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry*; D007438:Introns; D008840:Microfilament Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure; D021122:Protein Subunits; D012322:RNA Precursors / Q000737:chemistry*; D017381:Spliceosomes / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology
D000199:Actins; D000345:Affinity Labels; D001426:Bacterial Proteins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D012322:RNA Precursors; C061260:RuvB protein, Bacteria; C402385:spir protein, Drosophila