This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
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This article is dedicated to the memory of our good friend and colleague Nicolas Boisset, who passed away on January 4, 2008. The authors would like to thank Jesse Brown for batch files on the common-lines approach and helpful discussions. We also thank Michael Watters for assistance with the preparation of the illustrations. Supported by HHMI and NIH grants P41 RR01219 and R37 GM29169 (to J.F.).
The authors declare no competing financial interests.
Supplementary Fig. 3. JWEB window after clicking Fit Angles. The tilt angle theta, and tilt-axis directions phi and gamma have been calculated. (PDF 217 kb)
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Shaikh, T., Gao, H., Baxter, W. et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc 3, 1941–1974 (2008). https://doi.org/10.1038/nprot.2008.156
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