期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 193, 期 1, 页码 45-54出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2015.11.007
关键词
Single particle analysis; Cryo-EM; Thick filament; 3D reconstruction; Muscle contraction
资金
- NIH [R01 AR034711, P01 HL059408, R01 AR067279, R01 AR062279]
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 angstrom to 13 angstrom. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits. (C) 2015 Elsevier Inc. All rights reserved.
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