Single particle analysis
TSingle Particle Analysis(SPA) is, in Japanese, 単粒子解析法. Single Particle Analysis is the image analysis in two or three dimensions of a particle (or object), on the assumption that it has an identical shape in either two or three dimensions ( all images of a particle are assumed to present its own identical shape).
2D Single Particle Analysis
2D Single Particle Analysis does the following, 1) to classify the two dimensional electron microscopic images, which are essentially the projected images, 2) to average them, and 3) to discuss the obtained structural information.
3D single particle analysis
3D single particle analysis is the following method: by assuming that each particle has the same structure although the projection angle is different, we can reconstruct 3D structure of the particle from each projection. The protocol was the below.
1. Extraction of particle 2D images.
- Pre-processing of EM Images (CTF correction, noise reduction, etc.) (ctfDisplay, etc.])
- Extraction fo particles from pre-processed images (Display2 etc.)
2. Preparation of an initial reference model.
- Preparation of some 3D as an initial reference model (mrcImageModelCreate, pdb2mrc etc.)
3. 3D reconstruction
- Generation of a set of 2D images projected from the reference 3D (mrc3Dto2D)
- Estimation of the projection angle of each particle image as the projection angle of the 2D reference image which has the highest correlation coefficient or something like similarity between both (mrcImageAutoRotationCorrelation)
- 3D reconstruction (mrc3Dto2D)
- Check of the validity or quality of 3D (mrcImageFourierShellCorrelation, mrcImageFOMCalc, etc.)
- Refinement by repeated procedure.
-- In single particle analyses, the images of protein particles are collected, and images similar in the directions of three-dimensional projection are superimposed and averaged. The averaging results in low-noise two-dimensional images. These images are used again as references for positioning the original images to obtain new averaged images.
After repeating these procedures, three-dimensional angles (Euler angles) are determined to construct a three-dimensional structure. In addition, a re-projected image is created from the initial three-dimensional structure. The image is subjected to averaging through the superposition of the original image as a reference to reconstruct a three-dimensional structure. These procedures are repeated until the structure is stabilized, in order to complete a structural model. In this training, the principles of single particle analyses and single particle analyses using Eos will be learned.
