Transmission Electron Microscopy (TEM) is an analytical technique that is complementary to the other analytical instrumentation we have at Analytical Answers. It uses a high energy incident electron beam that passes through a specially-prepared, thin sample to produce high resolution images of the sample.
CryoEM (coupled with single particle analysis) has become a popular method for high resolution 3D protein structure determination. An aqueous biological sample is frozen rapidly and irradiated with a beam of electrons from a transmission electron microscope. A detector senses how the electrons are scattered and computerized image processing techniques are used to reconstruct the 3D-shape of the molecule. Single particle analysis (SPA) is the most cutting-edge technology of cryo-electron microscope. There is no need to crystallize the sample but only purified. The three-dimensional structure of the macromolecular protein is resolved by 2D image projection and 3D image modeling analysis.
Microcrystal electron diffraction (MicroED) is a structural technique that leverages the strong interactions of matter with electrons. MicroED requires crystalline material, though material unsuitable for X-ray analysis is often permissible. A transmission electron microscope is used to collect diffraction data, which can be processed by standard crystallographic programs, allowing for rapid structure determination.
X-ray crystallography is currently the most robust technology for structural determination of proteins and other macromolecules. The requisite for a successful X-ray crystallographic application is to obtain single crystals of the target protein. Data is then collected by diffracting X-ray from the single crystal that has an ordered pattern of atomic orientation. The assembly of atoms and molecules in the crystal can be deduced from the measurement of X-ray scattering.
Negative staining TEM is a technique for preparing electron microscope sample images to show negative contrast. It is used to observe particulate matter or biological macromolecules in samples. It is applicable to most samples even when their concentrations are low (typically below 0.1 mg/ml). 2D Images or 3D tomograms of the complex of interest can be obtained. Classification and averaging of the particles, both in 2D (Single Particles Analysis – SPA) as well as in 3D (volumetric averaging), can be performed within few hours depending on the nature of the sample.
Protein Production and Purification
Molecular biology techniques are used to connect the target gene to the protein expression vector to construct a foreign protein expression vector. Use a suitable expression system to express the target protein and purify it to provide qualified samples for the analysis of the three-dimensional structure of the protein cryo-electron microscope.
Structure Based Drug Discovery
Virtual screening is a computational technique used in drug discovery to search libraries of small molecules in order to identify those structures which are most likely to bind to a drug target, including a protein receptor or enzyme.
Molecular Dynamics is a computer simulation method for analyzing the physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a fixed period of time, giving a view of the dynamic "evolution" of the system. It is useful for addressing the flexibility issues in computational drug design. Molecular dynamics (MD) has become a particularly important tool in drug design and discovery.
Molecular docking is one of the most applied virtual screening methods, especially, when the 3D structure of target protein is available. This method could predict both the binding affinity between ligand and protein and the structure of protein–ligand complex, which is useful information for lead optimization.
Lead optimization is a critical process that culminates in the identification of a preclinical candidate. The most promising hit series, once they are identified through hit-to-lead efforts, advance into the lead optimization stage of drug discovery. The goal of this stage is to extensively optimize, in parallel, both the biological activity and the properties of the lead series, through a dedicated screening funnel of both in vitro and in vivo assays.