X-ray crystallography is now a prevalent but still very powerful experimental technique which can reveal the atomic structure of proteins. Basic principle for this is the analysis of the diffraction pattern such as the angles and intensities of the diffracted beams from protein crystal. Overall procedures of X-ray crystallography are as followings, 1) Protein Purification, 2) Protein Crystallization, 3) Diffraction Data Collection, 4) Calculating Structure Factor – Amplitude and Phase (by Multiple Isomorphous Replacement(MIR), Multiwavelength Anomalous Dispersion (MAD), or Molecular Replacement(MR), 5) Model building and Refinement.
Transmission Electron Microscopy
Transmission Electron Microscopy (TEM) has long been utilized in the various research fields including material science, nanotechnology and semiconductor research as well as biology and medical science. Biological researches with TEM have provided the morphological information of subcellular organelles, cells and tissue. Thereby, it could give insight into diverse biological and pathological processes. Moreover, TEM is of high value in the diagnosis of clinical specimens related to renal diseases, tumor processes (especially for questions concerning the grade of differentiation of tumor cells), storage disorders and the identification of infectious agents.
Recently, TEM becomes a major tool for the structural research of protein complex as well. Based on the type of samples, the specimen preparation and the analyzing method, the technique for structure determination with TEM can be classified into three categories – (1) Electron Crystallography, (2) Electron Tomography, (3) Single Particle Electron Microscopy. Due to the fact that biological samples are intrinsically very sensitive to the radiation, all three techniques prefer to take an image under cryogenic condition (-180℃) for obtaining higher resolution structure, which are called cryo-electron microscopy (CryoEM).
Electron Crystallography studies the structure of two-dimensional crystals of membrane proteins or other crystalline arrays. Two types of data are collected in electron crystallography, images and electron diffraction patterns. Images contain both amplitude and phase information, and structures can be determined exclusively from image data, but electron diffraction patterns provide more accurate amplitude information.
Electron Tomography developed for the structural analysis of thin cells or section of tissues. Electron tomography collects images of specimen at different orientations by tilting it, after which a 3D reconstruction can be computationally generated by aligning and merging the images.
Single particle Cryo-Electron Microscopy (CryoEM) is a method that obtains the structural information from many copies of identical protein complex with different orientations embedded in vitrified ice. This method prevents protein complex from the deformation and dehydration at the high vacuum conditions in TEM column. However, in order to reduce the radiation damage to proteins, very limited electron doses that can be transmitted through the specimen are allowed, leading to low contrast images. Thus, thousands to millions of images of individual assemblies must be collected, computationally aligned and merged to arrive at a three-dimensional structure. The great advantage of Single Particle CryoEM is that it allows direct visualization of protein complex without crystallizations which are usually the rate-limiting step and can sometimes cause the artificial packing-interaction in X-ray crystallography. In addition, the protein quantity required for single particle EM, is significantly less than the amount for conventional structural techniques such as X-ray crystallography and NMR. Low resolution structure of macromolecular complex from Single particle CryoEM (10~30Å) has been used for determining the location of each component, characterizing the multi-component interactions and identifying the conformational change in different states by fitting individual or partial protein’s crystal structures into CryoEM map of large assemblies. However, recent advances in microscope design, cryogenic sample preparation, image processing software, high sensitivity detector and automatic data acquisition etc., make it possible to determine 3D structure of macromolecular protein complexes at atomic or near-atomic resolution. For example, the Single particle CryoEM structure of membrane protein, TRPV1 (~300kDa) at 3.4Å resolution solved by Liao M and Cheng Y (UCSF), are good enough to recognize amino-acid side chains and β-sheets, and to trace the polypeptide backbone of the protein (Liao M et al, Nature, 2013 Dec 5, 504(7478), 107-12). This outstanding success is the milestone in Single particle CryoEM field and herald the dawn of CryoEM as a technique to understand molecular mechanism of various macromolecular protein complexes that are currently challenging to structurally characterize with any technique.
Tecnai G2 spirit TWIN (120kV, FEI)
Eagle 4K High Sensitivity CCD
Vitrobot for Cryo-sample
Ultramicrotome for Ultrathin Section