Stack Creation » History » Revision 4
Revision 3 (Anke Mulder, 05/18/2010 07:25 PM) → Revision 4/15 (Anke Mulder, 05/18/2010 07:39 PM)
h1. Stack Creation This procedure boxes out particles and is also able to apply CTF and astigmatism correction. 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. # Select the [[Particle_Selection|particle selection]] run to use for boxing from the drop down menu. # Enter a description of your run description. # Enter a box size into the description box. # Select the appropriate preset of images stack to search (default is en if you collected with leginon). # From align from the drop down menu, select whether you want to box particles on tilted images as well (for RCT and OTR, make two separate menu. Note that stacks for tilted and untilted particles!) # Check these boxes if you are creating can be identified in this menu by stack concurrent with data collection, particle selection, name, stack ID, and CTF estimation. Appion will wait that the number of particles, pixel and box sizes are listed for more picms to roll in. each. # You can pre-filter particles in accordance with your rejection (hide) or exemplar decisions in the image viewer and/or using the [[Image_Assessment|image assessment tool]]. # 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). # Check the "invert image density" box if you collected ice data and wish to perform 2D alignment and classification. Also, if you wish to normalize your stack to STDEV of 1.0, select the "Normalize Stack Particles" box. # To apply CTF correctio, select the "Ctf Correct Particle Images" box, and choose the CTF correction method from the drop down menu. We like the ACE2 WeinerFilter Whole Image corrector. # Click on "Make Stack" "Run Maxlike 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/212/Picture_119.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 "Stack creation" "Run Alignment" submenu in the appion sidebar. h2. Notes, Comments, and Suggestions: # Default In the parameters work well generally. 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. # Remember that, if 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 low pass 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 high pass filter your stack no particles, then any filters applied during subsequent processing will be in addition as well as a few classes that appear to this original filter. have two particles where one of them is on the edge. In our hands, such classes are eliminated using the sub-stack tools. # We tend have not noticed significant advantage to filter during decreasing the angular increment, so that we generally use maximum likelihood with a 5 degree angular increment to create templates for subsequent finer alignment instead of during stack creation. via reference-based tools. # Clicking on "Show Composite Page" in the Alignment Stack List page (accessible from the "completed" link under "Run Alignment" in the Appion sidebar) will expand the page to show the relationships between alignment, feature analysis, and clustering runs. ______ [[Stacks|<Stacks]] [[Run_Alignment|<Run Alignment]] | [[Particle Alignment|Particle Alignment [[Run Feature Analysis|Run Feature Analysis >]] ______