Heymann JB, Cardone G, Winkler DC, Steven AC. Computational resources for cryo-electron tomography in Bsoft. J Struct Biol. 2008, 161, 232-242.
The Bsoft package [Heymann, J.B., Belnap, D.M., 2007. Bsoft image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18] has been enhanced by adding utilities for processing electron tomographic (ET) data; in particular, cryo-ET data characterized by low contrast and high noise. To handle the high computational load efficiently, a workflow was developed, based on the database-like parameter handling in Bsoft aimed at minimizing user interaction and facilitating automation. To the same end, scripting elements distribute the processing among multiple processors on the same or different computers. The resolution of a tomogram depends on the precision of projection alignment, which is usually based on pinpointing fiducial markers (electron-dense gold particles). Alignment requires accurate specification of the tilt axis, and our protocol includes a procedure for determining it to adequate accuracy. Refinement of projection alignment provides information that allows assessment of its precision, as well as projection quality control. We implemented a reciprocal space algorithm that affords an alternative to back-projection or real space algorithms for calculating tomograms. Resources are also included that allow resolution assessment by cross-validation (NLOO2D); denoising and interpretation; and the extraction, mutual alignment, and averaging of tomographic sub-volumes.
Electron microscopy; Image processing workflow; Distributed processing; Micrograph alignment; Fiducial markers