title: An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits.
abstract: 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 Å to 13 Å. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Shixin
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: John L
authors.initials: JL
authors.lastname: Woodhead
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Fa-Qing
authors.initials: FQ
authors.lastname: Zhao
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Guidenn
authors.initials: G
authors.lastname: Sulbarán
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address: roger.craig@umassmed.edu.
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D003599:Cytoskeleton / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009119:Muscle Contraction / Q000502:physiology; D009218:Myosins / Q000378:metabolism*; D013112:Spiders; D014961:X-Ray Diffraction
chemical_list: D009218:Myosins
keywords: 3D reconstruction; Cryo-EM; Muscle contraction; Single particle analysis; Thick filament