Single Particle Reconstruction with Reference Projections using SPIRE

This page describes how to do a 3D single particle reconstruction project using SPIRE.
Each step of a project is accomplished by running a series of SPIDER procedure files, described on the Single Particle Reconstruction web page.
SPIRE provides a graphical interface for running procedure files, and keeps track of all data files that are part of a project.

Starting a project in SPIRE
Project parameters
Input files needed for a reconstruction project
Running SPIDER procedure files
File numbers
Viewing procedure file outputs
The SPIRE manual

• Using your Laboratory database: If you have already entered project information into your laboratory's database system, you can type in the Project ID number and press the Get button. SPIRE will attempt to connect to the database and download information about the project.
  See how to configure SPIRE to connect to an external database.
• If you don't have the database connection, simply enter the information manually. No fields can be left blank.

  Project title:	give a descriptive title for your project.
  Project file:	a filename WITHOUT AN EXTENSION! e.g., "proj123", not "proj123.hrs"
  Data extension:	SPIDER data extensions must have 3 characters.
  Host machine:	the computer where the project physically resides.
  Project directory:	create a new directory for your project. By default, SPIRE suggests a directory with the same name as the data extension. Or, you can use the Browse button to select a pre-existing directory.
  Configuration file:	Select SingleParticle.xml or SingleParticleParms.xml
  Create directories and load procedure files:	make sure this box is checked.

  Click OK, and SPIRE will create the project directory and subdirectories, and load the procedure files into the appropriate locations.

Project parameters: the parameter file

Project-wide parameters are kept in a SPIDER document file, usually called params.ext. These are accessed by numerous procedure files. See the description of single particle reconstruction parameters.

When you start a new project, SPIRE opens up a Project Parameters window. (If it doesn't, then under the Parameters menu, select New.) Be sure to fill in values for:

zip flag:	whether or not the micrographs are zipped (i.e., have the extension .gz).
file format:	(of micrographs) May be SPIDER or output from a scanner.
pixel size:	Angstroms/pixel after scanning. Pixel size also sets the values for max.spatial frequency, window size, and particle size
electron energy:	(kV) Also sets lambda
spherical aberration:	Set appropriate value for Microscope
particle window size:	(in pixels) diameter of the small particle images
particle diameter:	(in pixels, approximate) default values are for Ribosome!!

Click OK, and SPIRE will save these values to the parameter doc file. Alternatively, you can read in values from a parameter file from a previous project.

Input files needed for a reconstruction project

1. The digitized micrographs should be in the Micrographs directory.
   Or, because they require so much disk space, it may be desirable to keep the micrographs in another location, and make Micrographs a symbolic link. In that case, cd to your project directory, remove Micrographs, and make a symbolic link:
        
   rmdir Micrographs
      ln -s /actual/location/of/the/micrographs Micrographs
   Your parameter file must reflect whether your micrographs are
        a) In SPIDER or some other format,
        b) Whether or not they are zipped (i.e., have the extension .gz).
2. A reference volume is required for reconstruction using reference projections. If the dimensions of your reference volume do not match the window size (key 17 in the parameter file), then you must resize the volume with resizevol.spi. This procedure file should be run in the top-level project directory, creating a new, resized reference volume called reference_volume.ext.
3. An image of background noise is needed by the particle picking procedure files lfc_pick.spi and pick.spi. Although a file can be generated by the procedure file noise.spi, ask your advisor if there is already a noise image being used to normalize the particle image set.

Some procedure files use a special parameter called [FILENUMS], which points to a document file that selects which input files are to be processed. The contents of the file numbers document file (usually called filenums.ext) can be controlled by the File numbers entry box in the lower left corner of the main SPIRE window. With the cursor in the File numbers entry box, hitting the Enter key writes the displayed values into the filenums file.

Sometimes it is useful to try out a procedure file with a single file. Once you've established that it runs correctly, set the file numbers to the rest of the input data files. If you run the same procedure file with different file numbers each time, those are considered distinct procedure runs.

SPIDER procedure files generate output data files, either text (document files), or binary (images, volumes, image stacks, etc). When a procedure file completes successfully, it is saved to the project file. You can see which procedure files you have run, as well as examine their output files, in the SPIRE Project Viewer. Under the Commands menu, select View project. This brings up the Project Viewer with completed procedure runs in the upper window. Clicking on a procedure run in the upper window displays the output files for that procedure in the lower window. Clicking on an output file displays it. Image files are displayed in Jweb; documents are displayed in a text editor.

• How to run SPIRE
• Starting a project in SPIRE
• Editing SPIDER procedure files
• Executing SPIDER procedure files
• Viewing the data files created

Source: mrspire.html     Page updated: 14/07/28