Open AccessPurification, crystallization and preliminary X‐ray crystallographic analysis of squid heavy meromyosin
Author(s) -
O'NeallHennessey Elizabeth,
Reshetnikova Ludmila,
Senthil Kumar V. S.,
Robinson Howard,
SzentGyörgyi Andrew G.,
Cohen Carolyn
Publication year - 2013
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112049925
Subject(s) - heavy meromyosin , myosin , meromyosin , crystallization , actin , crystallography , myosin head , squid , chemistry , biophysics , dimer , electron microscope , biology , biochemistry , myosin light chain kinase , physics , optics , ecology , organic chemistry
All muscle‐based movement is dependent upon carefully choreographed interactions between the two major muscle components, myosin and actin. Regulation of vertebrate smooth and molluscan muscle contraction is myosin based (both are in the myosin II class), and requires the double‐headed form of myosin. Removal of Ca 2+ from these muscles promotes a relatively compact conformation of the myosin dimer, which inhibits its interaction with actin. Although atomic structures of single myosin heads are available, the structure of any double‐headed portion of myosin, including the ∼375 kDa heavy meromyosin (HMM), has only been visualized at low (∼20 Å) resolution by electron microscopy. Here, the growth of three‐dimensional crystals of HMM with near‐atomic resolution (up to ∼5 Å) and their X‐ray diffraction are reported for the first time. These crystals were grown in off‐state conditions, that is in the absence of Ca 2+ and the presence of nucleotide analogs, using HMM from the funnel retractor muscle of squid. In addition to the crystallization conditions, the techniques used to isolate and purify this HMM are also described. Efforts at phasing and improving the resolution of the data in order to determine the structure are ongoing.