Appion

h1. Spider Reference-free Alignment

This method uses the Spider AP SR  command to align your particles.  WARNING: this method is very quick (~few minutes), but also very sloppy and does not always do a great job. The only way to obtain decent results is to run several times and compare the results.

h2. General Workflow:

# Make sure that appropriate run names and directory trees are specified.  Appion increments names automatically, but users are free to specify proprietary names and directories.

# Enter a description of your run into the description box.

# Select the stack to align from the drop down menu.  Note that stacks can be identified in this menu by stack name, stack ID, and that the number of particles, pixel and box sizes are listed for each.

# Select a method for initializing reference-free alignment by activating one of the radio buttons.  If you wish to use a template image to initialize alignment, you can locate the template ID number by clicking on "1 available" under "Upload template" in the "Import tools" submenu on the appion sidebar.

# Make sure that "Commit to Database" box is checked.  (For test runs in which you do not wish to store results in the database this box can be unchecked).

# Click on "Run Spider NoRef Alignment" to submit your job to the cluster.  Alternatively, click on "Just Show Command" to obtain a command that can be pasted into a UNIX shell.

!http://emg.nysbc.org/attachments/161/Picture_14.png!

# If your job has been submitted to the cluster, a page will appear with a link "Check status of job", which allows tracking of the job via its log-file.  This link is also accessible from the "1 running" option under the "Run Alignment" submenu in the appion sidebar.

# Once the job is finished, an additional link entitled "1 complete" will appear under the "Run Alignment" tab in the appion sidebar.  Clicking on this link opens a summary of all alignments that have been done on this project.    

# Click on the link next to "reference stack" to open a window that shows the class averages and that contains tools for exploring the result.  Such tools include the ability to browse through particles in a given class, create templates for reference based alignment, substack creation, etc.

# To perform a feature analysis, click on that button.

!http://emg.nysbc.org/attachments/157/Picture_8.png!

h2. Notes, Comments, and Suggestions:

# In the parameters box on the right, the most important one to play around with is "Number of References" under "Job parameters".  This specifies the number of "seeds" (classes) that will be used for reference free alignment of your particles.  A good starting place is 10 references.

# For the rest of the parameters, default values are a good place to start, but can be adjusted in accordance with the demands of your project.  

# If you place your mouse cursor over a parameter name, a help box appears with a thorough description of it.

# Maximum likelihood usually generates a few "empty" classes that contain junk or no particles, as well as a few classes that appear to have two particles where one of them is on the edge.  In our hands, such classes are eliminated using the sub-stack tools.

# We have not noticed significant advantage to decreasing the angular increment, so that we generally use maximum likelihood with a 5 degree angular increment to create templates for subsequent finer alignment via reference-based tools.

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[[Run_Alignment|<Run Alignment]] | [[Run Feature Analysis|Run Feature Analysis >]]

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