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

TitleActin filament labels for localizing protein components in large complexes viewed by electron microscopy
Publication TypeJournal Article
Year of Publication2009
AuthorsStroupe, ME, Xu C, Goode BL, Grigorieff N
Refereed DesignationRefereed
JournalRNA
Volume15
Pagination244-8
Date PublishedFeb
ISBN Number1469-9001 (Electronic)
Accession Number19095618
Keywords*Microscopy, Electron, Actins/chemistry, Affinity Labels/*chemistry, Animals, Bacterial Proteins/chemistry, Drosophila Proteins/*chemistry, Introns, Microfilament Proteins/*chemistry, Microfilaments/*chemistry, Multiprotein Complexes/*chemistry/ultrastructure, Protein Subunits, RNA Precursors/*chemistry, Spliceosomes/chemistry, Thermotoga maritima/enzymology
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.

URLhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19095618
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