X-ray crystallography is currently the most accurate technology for determining 3D structure of protein. It uses the ability of proteins to form crystals under specific conditions.
Molecules in the crystal form a very regular three-dimensional pattern, which upon being exposed to radiation of X-rays allows the registration of the diffraction pattern on a special detector. Such data is usually collected by synchrotron X-ray sources (available for example in Berlin – synchrotron BESSY). Analysis of the resulting images allows the researchers to determine the position of each of the thousands of atoms of the test substance with great precision, which makes it possible to create a three-dimensional model.
This level of observation of either the proteins or protein-ligand complexes makes crystallography one of the most advanced and promising approaches to new drugs discovery. Medications are often designed to block the activity of the protein (inhibitors), if the excess is the cause of the disease.
High resolution of the protein structure, which is a potential goal for therapy, allows for the design of specifically tailored chemical molecules that can interact with it.
This technique is also necessary to verify that the designed molecule combines with the protein as expected and that it has the potential to be used as a drug.
The use of crystallography is becoming a standard in the new drug creation. It often eliminates the cumbersome need of searching libraries of thousands, or millions of chemicals and tailoring them to a particular protein. This significantly limits the number of required experiments and greatly improves the final result of the project.Crystallographic methods are the last and crucial stage of the cycle of work necessary to know the atomic structure of protein. Before starting the crystallization it is necessary to produce a very pure sample of a protein that has the appropriate concentration level.
First the gene of the target protein is optimized, usually for expression in bacterial cells (ie. the preparation of expression constructs – gene cloning), and then the gene is introduced into the cells which are then grown in bacterial cultures. During their growth the modified cells produce protein encoded by the introduced genetic information. A purified product is obtained through the extraction of cell biomass (ie. production and purification of recombinant proteins).
PROBIOSTRUCTURES UNIQUE TECHNOLOGY IN X-RAY CRYSTALLOGRAPHY
X-ray crystallography is a well-established and routine method ProBiostructures team has applied in numerous R&D and scientific projects. A remarkable advantage is unique expertise in the crystallization of protein-nucleic acid complexes. CSO, dr. Marcin Nowotny has determined complex crystal structures of important nucleic acid enzymes – UvrA, RNase H1, RNase H2 and Rad2/XPG to name just a few. This expertise can be used to understand the mode of action of nucleic acid-based therapeutic agents and to understand the mechanism of nucleic acid enzymes to devise better strategies of their inhibition. ProBiostructures cooperates closely with IONIS Pharmaceuticals, the leader in RNA-targeted drug discovery.
The unique technology that we offer allows us to specifically tether the proteins with their nucleic acid substrates. This can be used to stabilize transient and unstable substrate complexes of these proteins.
Single-particle cryo-electron microscopy (cryo-EM)
In 2017, the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution”. This novel approach enables the determination of high-resolution structures of proteins under near native condition at close to atomic resolution and complements existing atomic-resolution approaches, such as X-ray crystallography. Recent publications show that cryo-EM can be used to investigate a broad spectrum of drug-target interactions with routine resolution of ~4 Å, possibly up to 2 Å. Cryo-EM enables the analysis of proteins or protein complexes with a molecular weight ≥ 150 kDa (e.g., antibodies and membrane proteins) that are otherwise difficult or impossible to analyze using other structural biology methods. ProBiostructures is the first in Poland to offer a commercial service of that emerging technology.
ProBiostructures guarantee rapid access to in-house 120 kV electron microscope equipped with all the accessory devices for cryo-EM experiments what enables affordable sample preparation and primary characterization. Afterward for the cost optimization purpose the first runs are on a 200kV microscope followed with the final high resolution data collection on a large 300kV Titan Krios microscopes.