Verschueren E.,University of California at San Francisco |
Verschueren E.,California Institute for Quantitative Biomedical science |
Von Dollen J.,University of California at San Francisco |
Von Dollen J.,California Institute for Quantitative Biomedical science |
And 7 more authors.
Current Protocols in Bioinformatics | Year: 2015
High-throughput Affinity Purification Mass Spectrometry (AP-MS) experiments can identify a large number of protein interactions, but only a fraction of these interactions are biologically relevant. Here, we describe a comprehensive computational strategy to process raw AP-MS data, perform quality controls, and prioritize biologically relevant bait-prey pairs in a set of replicated APMS experiments with Mass spectrometry interaction STatistics (MiST). The MiST score is a linear combination of prey quantity (abundance), abundance invariability across repeated experiments (reproducibility), and prey uniqueness relative to other baits (specificity). We describe how to run the full MiST analysis pipeline in an R environment and discuss a number of configurable options that allow the lay user to convert any large-scale AP-MS data into an interpretable, biologically relevant protein-protein interaction network. © 2015 by John Wiley & Sons, Inc.
Luo J.,Institute for Systems Biology |
Cimermancic P.,California Institute for Quantitative Biomedical science |
Viswanath S.,California Institute for Quantitative Biomedical science |
Ebmeier C.C.,University of Colorado at Boulder |
And 10 more authors.
Molecular Cell | Year: 2015
TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit. Luo et al. used an integrative approach to define the molecular architecture of both human and yeast general transcription and DNA repair factor TFIIH. They identified several conserved topological features that can explain how TFIIH enzymatic activities are regulated. © 2015 Elsevier Inc.