An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits
Department of Cell and Developmental Biology
Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 A to 13 A. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.
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Citation: J Struct Biol. 2016 Jan;193(1):45-54. doi: 10.1016/j.jsb.2015.11.007. Epub 2015 Nov 22. Link to article on publisher's site
3D reconstruction, Cryo-EM, Muscle contraction, Single particle analysis, Thick filament
Journal of structural biology
Wang, Shixia; Woodhead, John L.; Zhao, Fa-Qing; Sulbaran, Guidenn; and Craig, Roger, "An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits" (2016). Cell and Developmental Biology Publications. 188.