Welcome to my Cryo-EM 3D reconstruction Pages. Maybe you stumbled into this page, or you came to learn more about Cryo Electron Microscopy. Whatever the reason is, I hope you like what you see.
Why bother cryo-EM for the structure determination? Like Sir Aaron Klug said in his Nobel lecture" ... One studies a complex system by dissecting it out physically, chemically, or enzymatically, and then tries to obtain a detailed picture of its parts by x-ray analysis and chemical studies, and an overall picture of the intact assembly by electron microscopy." Because there is no method for directly imaging very large cellular complexes at atomic resolution, the current challenge for structural cell biology is to use X-ray Crystallography, NMR to obtain atomic models for domains structures, dock into the whole Macromolecular complex envelop from Cryo-EM and image reconstruction. Then use Molecular genetic information to test and refine the structures.
Negative stain, Frozen hydrated, Glucose, Tannic acid etc. When you cool down water, it become ice. There is a volume change during the phase transition, which cause freezing damage. If we can cool the water very quickly, water molecules will become immobilized before they have time to crystallize. This will prevent freezing damage. People were suspicious about freezing sample for structure determination. The breakthrough came in 1974, Taylor and Glaeser showed that, in the frozen state, the structure of catalase crystals can be preserved to atomic resolution. This shows different forms of ices in image and diffraction pattern. Vitreous ice forms when the water is frozen very rapidly. Cubic and hexagonal ice froms at relatively low cooling speed. There are distinct difference in the diffraction patterns: Vitreous ice ( fuzzy rings ), Cubic ice ( sharp rings ) and hexagonal ice ( spots ).
Some pictures of equipment from Scripps
Why bother with image analysis? DeRosier and Klug in 1968 first determine a three dimensional structure from electron images. In their work with T4 bacteriophage tail, they laid the foundation on how to apply image analysis. Then in 1975 Henderson and Unwin reached a major breakthrough in protein electron crystallography by obtaining the first membrane protein transmembrane 7 helicies from images of a 2D crystal of purple membrane. Projection Theorem The projection theorem The projection theorem show the 2D Fourier transform of a projection is identical with the corresponding central section of the 3Dtransform normal to the direction of view. So by tilting the sample, we can get different central sections. Combining those sections we can obtain the whole 3D Fourier transform of the original object, then Fourier inversion will generate the original structure. In EM the high energy electrons from the electron gun strike the sample, the energy depostied on the sample may cause ionization, charging effects, mass loss and contaimination etc.
Cryo-EM can be applied for Symmetrical or Asymmetrical samples.
Sample size ranges from 5 nm to 100 nm . Resolution from 0.35 nm to 3.5 nm. It can study native structure, conformational
changes and time-dependent events etc.
Applications can be described in several categories: 2D Crystals of protein, Helical polymers, Icosahedral virus and
Asymmetrical particles.
For 2D crystal, there is a Jaap Brink Homepages
for reference on lipid monolayer.
The high resolution structures includes Bacteriorhodopsin and
Light-harvesting Complex.
High resolution meets low is a comparative method where high resolution structures are
imaged at lower resolution to validate the interpretation of a low resolution structure.
Low resolution meets high , on the other hand, refers to the approach in which high
resolution models are fitted to low resolution density maps to derive pseudoatomic resolution models.
Another application of this combination is in Muscle contraction.
Some questions on CTF correction
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