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Overexpression, Crystallization and Preliminary X-ray Crystallographic Analysis of the C-terminal Cytosolic Domain of Mouse Anoctamin 1

Published: Tuesday, March 27, 2012
Last Updated: Tuesday, March 27, 2012
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This article describes how researchers initiated structural study of the C-terminal cytosolic domain of mouse ANO1.

Summary

Transmembrane protein 16A (TMEM16A, also known as anoctamin 1; ANO1) is a bona fide Ca2+-activated chloride channel that is activated by intracellular Ca2+- and Ca2+-mobilizing stimuli and plays important roles in a variety of physiological functions. To elucidate the structural features of ANO1, structural analysis of the C-terminal cytosolic domain of mouse ANO1 (mANO1-CTD) was initiated. mANO1-CTD was overexpressed in Escherichia coli and was crystallized at 297 K using a reservoir solution consisting of 0.2 M sodium acetate trihydrate, 0.1 M Tris–HCl pH 8.5 and 30%(w/v) PEG 4000. X-ray diffraction data were collected to 2.3 A ° resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 73.96, b = 103.73, c = 114.71 A ° . If it is assumed that eight copies of a monomer molecule are present in the crystallographic asymmetric unit, the crystal volume per protein mass (VM) is 2.38 A ° 3 Da_1 and the solvent content is 48.38%. Attempts to solve the structure of mANO1-CTD by the MAD method using selenomethionine-labelled mANO1-CTD or heavy-atom-derivatized crystals are in progress.

This article was published online in Structural Biology and Crystallization Communications and is free to access.


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