Lifan Chen
- Dr. Lifan Chen
- Staff Engineer
- Read-Rite Corporation
- 44100 Osgood Road
- Fremont, CA 94539
Lattice constraints as well as differences in biochemical state of the independently acting myosin heads result in structural variations in situ that depart significantly from the uniform structure that is obtained by X-ray crystal structures. Our aim is to fit initially the atomic model for rigor acto-S1 complex into the envelope of 3-D reconstructions obtained by electron tomography. Adjustments are then made to the atomic model manually using the crystallography program "O" to correct for regions where model and envelope differ. Adjustments in the atomic model at this point are restricted to the light chain domain, which is pivoted about residue 770 of the myosin heavy chain. The motor domain and the actin monomers are kept constant. We have developed a procedure for correcting poor contacts that are produced by these manual adjustments using the RSref program. RSref is a real space refinement program that compares the electron density of the model with the density map. Movements of the model are determined using conjugate gradients. An independent scale factor is used to account for overall differences in density between model and map. The TNT refinement program is used in parallel to determine the corrections needed to improve poor geometry. A set of initial constraints is used to limit movements of the model. One of these involves maintaining the C-terminal residues of the two heavy chains to a minimum separation. Results obtained by modeling the acto-S1 structure into the envelope of the 2-headed crossbridge in rigor insect flight muscle show a range of axial movement in the light chain domain comparable to those hypothesized for muscle contraction. In addition, azimuthal movements are required to bring the two heavy chains into proximity.
Figure Legend. This picture shows an atomic model of an opposed pair
of 2-headed myosin crossbridges attached to an actin filament. The model has
been fit to an electron density map of insect flight muscle in the rigor state.
The 3-D map was obtained by electron tomography of a section of plastic embedded
tissue. The model was developed starting with the coordinates of rigor acto-S1
developed by Ivan Rayment and coworkers. We then moved the C-terminal residues
to within 12Å of each other by pivoting the light chain domains of both
S1s about residue 770. Then RSref was run to correct poor geometry and iverlaping
domains. The rsulting model fits the electron density much better than the
original starting model which had considerable density falling outside of
the 3-D envelope. This project done in collaboration with the laboratories
of Michael Chapman at Florida State University and Michael and Mary Reedy
at Duke University.
PUBLICATIONS
- Li Fan Chen, Eric Blanc, Michael S. Chapman and Kenneth A. Taylor.
Real
space refinement of acto-myosin structures from sectioned muscle. J.
Struct. Biol.¹ 133(2), 221-232 (2001)
- Kenneth A. Taylor, Holger Schmitz, Mary C. Reedy, Yale E. Goldman, Clara
Franzini-Armstrong, Hiro Sasaki, Richard T. Tregear, Kate Poole, Carmen
Lucaveche, Robert J. Edwards, Li Fan Chen, Hanspeter Winkler, and
Michael K. Reedy. Tomographic 3-D reconstruction of quick frozen, Ca2+-activated
contracting insect flight muscle. Cell 99, 421-431 (1999)
- Chen, Li Fan, Winkler, Hanspeter, Reedy, Michael K., Reedy, Mary C. & Taylor ,Kenneth A.. Molecular Modeling of Averaged Rigor Crossbridges from Tomograms of Insect Flight Muscle. J. Struct. Biol. (2002)