Random conical tilt reconstruction

As described in Nucleic Acids Res. (2011) 39: 2845-54.


Outline



Links to further information



General notes



Quick-start guide

This is a brief list of the steps comprised in this protocol. For simplicity, options have been limited. For more detailed information, see the full procedure below.

Data extension is assumed to be spi

  1. tar -xvf procedures.tar
  2. spider rct/spi @makeparams (or copy params.spi)
  3. (optional) tar -xvf data20060817.tar
  4. mkfilenums filenums.spi $MICROGRAPHS*
  5. (for non-SPIDER format micrographs) spider rct/spi @convertmics
  6. spider rct/spi @shrinkmics
  7. cd Micrographs/ ; montagefromdoc ../filenums.spi sm-mic*
  8. spider rct/spi @micpair
  9. (required for JWEB) spider rct/spi @padmics
  10. pick tilt pairs in JWEB or WEB
  11. spider rct/spi @makesomenoise (or copy noise.spi)
  12. spider rct/spi @windowparticles
  13. (for negative stain) Do both of:
    1. spider rct/spi @highpass-untilted
    2. spider rct/spi @highpass-tilted
  14. montagefromdoc listparticles.spi Zerodegr/stk2-winrot.spi
  15. (if necessary) spider rct/spi @removebad
  16. montagefromdoc listparticles.spi Tilted/stk2-winrot.spi
  17. (if necessary) spider rct/spi @removebad
  18. (slow) spider rct/spi @pairwise
  19. (optional) To reorient average, do both of:
    1. spider rct/spi @reorientavg
    2. spider rct/spi @sumalign
  20. spider rct/spi @filtershrink
  21. spider rct/spi @ca-pca
  22. Classify. (Ward's method shown here. For other options, see below.)
    1. spider rct/spi @hierarchical
    2. spider rct/spi @binarytree
    3. cd Zerodegr/Tree ; binarytree labeled001.spi 4 goodclasses.spi
  23. Iterate:
    1. spider rct/spi @multirefalign after the first iteration)
    2. spider rct/spi @filtershrink
    3. spider rct/spi @classifybyview
    4. verifybyview
  24. spider rct/spi @combinegoodclasses
  25. spider rct/spi @viewaverages
  26. spider rct/spi @centertilt
  27. spider rct/spi @storeangles
  28. (optional) spider rct/spi @d6symmetry
  29. spider rct/spi @bpclass
  30. spider rct/spi @volfilt
  31. (if multiple orientations) Do both of:
    1. spider rct/spi @volalignprj
    2. spider rct/spi @mergevols
  32. spider rct/spi @prepare-prjmatch


Getting started

There are two different choices for running SPIDER procedures: Using SPIRE or using SPIDER procedures directly.

  1. If you are using SPIRE, then read its documentation, starting with Starting a new project. In this case, the configuration file is called rct.xml, and is located in the directory in which you unpackaged the tarball.

    Even if you are not using SPIRE, its libraries are required for various Python scripts, such as mkfilenums.py and binarytree.py. These steps can be substituted with SPIRE-independent analogs.

    Installation instructions for SPIRE can be found here.

  2. If you are running SPIDER at the command-line prompt, the processing steps are carried out by procedure files, which run many SPIDER operations automatically. To use a procedure in this document, if you have not unpackaged the complete tarball, then right-click the procedure filename and save the procedure to your current working directory. At the beginning of each procedure, there is a list of parameters that can be adjusted according to the particular project. Some of the procedures will call additional procedure(s). You do not need to change anything in the procedures. Use the following format to run a SPIDER procedure:
    spider rct/spi @proc
    where rct is the procedure file extension, spi is the project data file extension, and proc.rct is the procedure file.


Procedure

This procedure comprises a complete series of steps required to compute a three-dimensional reconstrction using random conical tilt.


Miscellaneous procedure files


Modifications log


References

  1. Frank J. (2006) Three-Dimensional Electron Microscopy of Macromolecular Assemblies. (Oxford University Press, New York, NY).
  2. Marco S, Chagoyen M, de la Fraga LG, Carazo JM, Carrascosa JL. (1996) A variant to the random approximation of the reference-free algorithm. Ultramicroscopy 66: 5-10.
  3. Radermacher M, Wagenknecht T, Verschoor A, Frank J. (1987) Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J Microsc. 146:113-36.
  4. Shaikh TR, LeBarron JS, Trujillo R, Baxter WT, and Frank J. (2008a) Particle-verification for single-particle reconstruction using multivariate data analysis and classification. J Struct Biol 161: 41-48.
  5. Shaikh TR, Gao H, Baxter WT, Asturias F, Boisset N, Leith A, and Frank J. (2008b) SPIDER image-processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nature Protocols 3: 1941-74.
  6. Huang T, Shaikh TR, Gupta K, Contreras-Martin LM, Grassucci RA, Van Duyne GD, Frank J, Belfort M . (2011) The group II intron ribonucleoprotein precursor is a large, loosely packed structure. Nucleic Acids Res. 39: 2845-54.

Source: rct.htm     Page updated: 2015/07/29     Tapu Shaikh

Enquiries: spider@wadsworth.org