Tilt-Pair Reconstruction

Access: CP::3D Model::Classification and CP::3D Model::Reconstruction (Tab)

The Random Conical Tilt (RCT) method uses tilted particle-pairs for 3D model reconstruction.  Because the tilt geometry comes from experimental observation, this method can reduce error in particle angular alignment and produce a quick initial model reconstruction.  In addition, the known tilt-geometry between particle pairs can be utilized to validate a model reconstruction.  PARTICLE sets up an internal data structure for the "book-keeping" during a tilt-pair data analysis, so that the RCT reconstruction can be accomplished in a few simple steps.  The program strives to simplify and expedite the RCT data processing at the application level, thus enabling more and more molecular structures to be analyzed properly and efficiently from tilt-pair data.  In the implementation, the RCT module interacts closely with the particle-pair selection and particle classification modules.

In PARTICLE, to use the RCT method for model reconstruction,

1. Collect tilt-pair micrograph data and record the nominal reading of the tilting geometry;
2. Enter the pair data into the project space, interleaving the master-image (from the first exposure) and the pair-image (from the second exposure);
3. For each image pair, refine the tilt-geometry and select particle pairs (see Particle-Pair Selection);
4. Build two particle stacks: the master-stack and the pair-stack.
5. Classify one of the stacks (the pair-stack is recommended, especially for cryo-EM data), and load the class averages via Class Editor::Classes.  At least one class should be selected (CTRL+LeftClick) for the RCT model reconstruction.
6. In 3D Model::Classification::Data for Reconstruction group, select the Random Conical Tilt method and activate the Modeling button, which will automatically switch the system to the CP::3D Model::Reconstruction module;
7. In the Reconstruction::R.C.T. Reconstruction group, open the file (with the extension "rct") that specifies the pair stacks;
8. Upon activating the Model button, and RCT reconstruction will proceed for each selected class.  When the Merge box is checked, multiple RCT models from the selected classes will be merged into one density map at the end.

Once a RCT model has been derived, it can be used as the reference for the subsequent model refinement.  A variety of model refinement functions can be accessed from CP::3D Model::Reconstruction.