PreprocessMicrographs

!Preprocess Micrographs

The following steps may be done in this protocol:

1. Convert each of the digitized micrographs (e.g., mic0001.tif) from TIFF to RAW format:
2. If the micrograph was digitized with a pixel size smaller than needed, perform a downsampling step (e.g. decrease the pixel size by a factor of 2):
3. Estimate the contrast transfer function (CTF) of the micrograph.

Analyze results offers to:

1. Visualize the CTF of each micrograph, and discard those micrographs that are of insufficient quality for further processing. If you have computed the CTF using Xmipp instead of CTFFIND, then you can recompute the CTF by clicking the mouse right button on the wrongly computed CTF, then choose "Recompute CTF". A graphical interface will be open in which you can help the algorithm by narrowing the region in which the CTF should be computed and by providing an initial solution.

How to recompute the CTF with Xmipp.

CRITICAL STEP: Micrograph selection may strongly affect the outcome of all subsequent data processing steps. The CTFs of good micrographs typically have multiple concentric rings, extending from the image centre towards its edges. Bad micrographs may lack any rings or only have very few rings that hardly extend from the image centre. Other reasons to discard micrographs may be the presence of strongly asymmetric rings (astigmatism) or rings that strongly fade in a particular direction (drift).

Example Data

• Input

  • micrograph pair in TIFF format: G40P_untilted.tif, G40P_tilted.tif. Tar file preprocess_micrographs.tar.gz contains all the data needed to execute the protocol: tiff files and the xmipp_protocol_preprocess_micrographs script. Files and directories created by this tar file follows the standard xmipp protocol conventions.

• Output

  • micrograph pair in XmippRaw format
  • down-sampled micrograph.
  • periodograms for CTF estimation

File output_preprocess_micrographs.tar.gz, contains all the files generated by thePreprocess Micrographs protocol. To examine the contents of this file, ungzip and untar it, change to the new created directory, lunch the scriptxmipp_protocol, click inpreprocess micrographs and then in theanalyze results button.

PSD of the tilted pair of negatively stainedG40P protein.

## USER's COMMENTS

--Main.RobertoMarabini - 08 Oct 2007