Difference between revisions of "Single particle analysis"
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1. Extraction of particle 2D images. | 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. | + | 2. Preparation of an initial reference model. |
| − | * | + | * Preparation of some 3D as an initial reference model ([[mrcImageModelCreate]], [[pdb2mrc]] etc.) |
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| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
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| + | 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. | 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. | 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. | ||
Revision as of 03:13, 19 June 2013
The overview of single particle analysis is shown.
Protein particles are ice-embedded in random directions, thus allowing the three-dimensional electron microscopic imaging of protein particles from various directions of 2D images/projections. The images allow the structural analysis of proteins using single particle analyses.
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.
