Structure determination is a procedure to solve the three-dimensional atomic coordinates of a molecule or biomolecule through analytical techniques. A variety of strategies are utilized in antibody structure determination, such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, Cryo-electron microscopy density maps, and so on. With years of experience in the antibody engineering, Creative Biolabs offers the first class antibody structure determination services for customers to promote your antibody design and drug discovery progress.
X ray crystallography is a technique to get a three dimensional molecular structure from a crystal. Firstly, the purified sample at high concentration should be crystallised and the crystals are exposed to an x ray beam. The resulting diffraction patterns are then processed, to produce information about the crystal packing symmetry and the size of the repeating unit that forms the crystal. This is gained from the pattern of the diffraction spots. Then, the intensities of the spots are able to be used to measure the “structure factors” from which a map of the electron density enable to be calculated. A number of approaches are able to be used to promote the quality of this map until it is of sufficient clarity to permit the building of the molecular structure through the protein sequence. Finally, the resulting structure is refined to fit the map more accurately and adopt a thermodynamically favoured conformation.
Fig 1. Structure determination by X-ray crystallography.
Nuclear magnetic resonance (NMR) spectroscopy is a technique that determines the absorbance of radio frequency (RF) radiation which occurs when certain kinds of atomic nuclei (1H, 13C, 15N) are kept under strong magnetic fields. The positions of the absorption frequencies or “resonances” can be measured are called chemical shifts. Chemical shifts are highly sensitive to the electronic environment surrounding each nucleus and enable to offer a number of information about a molecule’s covalent and non-covalent structure. During the past years, NMR spectroscopy has become a crucial alternative to X-ray crystallography, because it enables protein structures to be determined in conditions that are very close to the physiological state (i.e. in solution). In addition, NMR spectroscopy offers structural biologists the opportunity to determine events or processes which cannot readily be seen or quantified by X-ray crystallography, for example, protein kinetics, dynamics or thermodynamics.
Fig 2. Structure determination by NMR.
Single-particle cryo-electron microscopy (cryo-EM) is becoming a more and more popular method for structure determination, especially for macromolecular complexes which are not suitable for crystallographic studies. Thanks to the great progress in direct electron detectors and image-processing techniques, now cryo-EM reaches near-atomic resolution (3–5 Å) from small particles with low or even no symmetry is available. Formerly, high resolution EM imaging is mainly based on photographic film, which is very slow, cumbersome, and limited in sensitivity, or it relied on charge-coupled devices (CCDs), which is a more convenient but indirect detection. Currently, the production of direct detectors records the incident electrons in a thin, sensitive layer so that the signal is not scattered into surrounding pixels. The sensitivity of the detectors has extremely increased, and was determined as detective quantum efficiency (DQE). Besides, the readout is much faster, enabling images to be acquired as a set of movie frames at 17–400 frames per second.
Fig 3. Cryo-EM structure of Ad5F35. (Cao, C., 2012)
With our comprehensive structure determination services, designing and engineering novel antibodies with desired therapeutic properties is available. We customize the service according to the specific requirements from the customers. Please contact us for more information and a detailed quote.
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