45 research outputs found
Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy
Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens
Detection of Alpha-Rod Protein Repeats Using a Neural Network and Application to Huntingtin
A growing number of solved protein structures display an elongated structural
domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel
alpha-helices. Alpha-rods are flexible and expose a large surface, which makes
them suitable for protein interaction. Although most likely originating by
tandem duplication of a two-helix unit, their detection using sequence
similarity between repeats is poor. Here, we show that alpha-rod repeats can be
detected using a neural network. The network detects more repeats than are
identified by domain databases using multiple profiles, with a low level of
false positives (<10%). We identify alpha-rod repeats in
approximately 0.4% of proteins in eukaryotic genomes. We then
investigate the results for all human proteins, identifying alpha-rod repeats
for the first time in six protein families, including proteins STAG1-3, SERAC1,
and PSMD1-2 & 5. We also characterize a short version of these repeats
in eight protein families of Archaeal, Bacterial, and Fungal species. Finally,
we demonstrate the utility of these predictions in directing experimental work
to demarcate three alpha-rods in huntingtin, a protein mutated in
Huntington's disease. Using yeast two hybrid analysis and an
immunoprecipitation technique, we show that the huntingtin fragments containing
alpha-rods associate with each other. This is the first definition of domains in
huntingtin and the first validation of predicted interactions between fragments
of huntingtin, which sets up directions toward functional characterization of
this protein. An implementation of the repeat detection algorithm is available
as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized
using BiasViz, a graphic tool for representation of multiple sequence
alignments
Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma
Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis. Here, we describe mutations occurring exclusively at arginine-625 in splicing factor 3B subunit 1 (SF3B1) in low-grade uveal melanomas with good prognosis. Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutation, and these mutations denote a distinct molecular subset of uveal melanomas
Exon, intron and splice site locations in the spliceosomal B complex
In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5′ and 3′ exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The gold-labelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process