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    Overview of Key Protein Structure Analysis Techniques

      The function of proteins directly depends on their structure, their interactions with other proteins, and their location within the cell, tissue, and organs. Large-scale studies of protein structure and function in proteomics make it possible to identify protein biomarkers associated with specific disease states and provide potential targets for treatment. Understanding the structure of proteins, as well as mapping protein locations, expression levels, and interactions, can generate valuable information that can be used to infer protein function. The main techniques for protein structure analysis include X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), cryo-electron microscopy (Cryo-EM), and atomic force microscopy (AFM). Here is a brief introduction in the following.


      X-Ray Crystallography

      1. Principle

      Protein X-ray crystallography can obtain the three-dimensional structure of proteins by crystallizing proteins' X-ray diffraction. By seeding a highly concentrated protein in a solution that promotes precipitation, orderly protein crystals are formed under appropriate conditions. The protein crystal scatters X-rays to an electronic detector or film when X-rays are aimed at it. Rotating the crystal to capture three-dimensional diffraction allows for the calculation of the position of each atom in the crystalline molecule through Fourier transformation.


      2. Characteristics

      It provides atomic-level resolution structural information; requires high-quality protein crystals; not applicable to proteins that are difficult to crystallize or cannot maintain functional structure in a crystal state.


      Nuclear Magnetic Resonance Spectroscopy (NMR)

      1. Principle

      Nuclear magnetic resonance (NMR) spectroscopy is used to obtain information about protein structure and dynamics. In NMR, the spatial position of atoms is determined by their chemical shifts. For protein NMR, proteins are usually labeled with stable isotopes (15 N, 13 C, 2 H) to enhance sensitivity and facilitate structure deconvolution. Isotopic labels are typically introduced by providing isotopically labeled nutrients in the growth medium during protein expression.


      2. Characteristics

      NMR can provide dynamic structural information and is suitable for proteins in solution. It requires relatively low sample amounts. However, its spatial resolution is lower compared to X-ray crystallography, and there are limitations in studying large molecular systems.


      Cryo-Electron Microscopy (Cryo-EM)

      1. Principle

      Electron microscopy imaging of protein samples at extremely low temperatures, by collecting a large number of images and combining them to construct a three-dimensional structure.


      2. Characteristics

      Cryo-EM is suitable for large molecular complexes and proteins that are difficult to crystallize. In recent years, the resolution has significantly improved, reaching near-atomic levels. It does not require protein crystals.


      Atomic Force Microscopy (AFM)

      1. Principle

      The probe scans the sample surface, recording minor force changes on the surface, thereby drawing a three-dimensional image of the surface.


      2. Characteristics

      AFM is suitable for imaging proteins and protein complexes under physiological conditions, providing single-molecule-level information. It does not require special sample preparation. However, its spatial resolution is relatively low, generally insufficient for detailing atomic-level structural features.


      Circular Dichroism (CD)

      1. Principle

      Circular dichroism is based on the principle that different wavelengths of light are absorbed to different extents when polarized light passes through optically active molecules (such as proteins). The difference in absorption of left and right circularly polarized light through the sample produces a circular dichroism signal, which can reflect the secondary structure characteristics of proteins, such as α-helices, β-folds, and random coils.


      2. Characteristics

      CD is particularly useful for analyzing the secondary structure of proteins. It provides information about the relative content of structures like alpha-helices, beta-sheets, and random coils. CD can also be used to monitor changes in protein structure in real time, such as during folding and denaturation processes.


      Determining the three-dimensional protein structure at atomic resolution can be used to elucidate protein function, structure-based drug design, and molecular docking. Each method has its advantages and limitations, so researchers may use multiple methods in combination to obtain complete and detailed structural information about proteins in actual research.

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