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. 2016;568:3-33.
doi: 10.1016/bs.mie.2015.09.024. Epub 2015 Nov 3.

How to Study Intermediate Filaments in Atomic Detail

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How to Study Intermediate Filaments in Atomic Detail

Anastasia A Chernyatina et al. Methods Enzymol. .

Abstract

Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the "divide-and-conquer" approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer-dimer contacts.

Keywords: Amino-acid sequence analysis; Coiled coil; Crystallization; Electron paramagnetic resonance; Filament assembly; Site-directed spin labeling; Three-dimensional structure; X-ray crystallography.

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