Protein crystallography services provide a one-stop solution for determining 3D structure of proteins, protein-protein complexes, protein-small molecule complexes, antibody-antigen complexes at high resolution. Protein crystallography projects typically carried out in gene-to-structure format encompassing gene cloning, protein expression, purification, crystallization, screening, synchrotron x-ray diffraction, structure solution and refinement.
Our structural biology expertise also includes protein engineering to enhance crystallizable of difficult proteins.
We seamlessly integrate structural biology with drug discovery programs for end-to-end SBDD and FBDD offerings
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The quality of protein crystal diffraction can be directly correlated with its internal order. Better the internal order of a protein crystal, more accurately to which atomic positions in the structure can be determined. Protein crystals generally have limited extent of diffraction due to its solvent channels. Although the solvent content of protein may be a major contributor in poor diffraction quality, it provides valuable insights into the protein biochemistry. The protein crystals are frozen in liquid nitrogen and data collection is usually performed under a cryo stream of nitrogen (100 K or -173 °C) to preserve the crystallized protein from X-ray-induced damages.
The diffraction quality of protein crystals mainly depends on their solvent content. Reducing the solvent content improves diffraction in general. Once a protein crystal is identified as poorly diffracting due to high solvent content, steps to improve the diffraction by progressively removing excess solvent may be undertaken. For example, the crystal may be incubated in solution containing progressively higher concentrations of precipitant solution. Alternatively, in situ “annealing” in which the frozen crystal is thawed and refrozen immediately can be employed for improving the diffraction pattern of a protein crystal.
We use the vapor diffusion technique for crystallization by setting up sitting or hanging drops. In some cases, we also apply micro-batch under oil. The purpose is to achieve a supersaturation of the macromolecule usually present in aqueous buffers. Supersaturation is defined as “a non-equilibrium condition in which some quantity of the macromolecule in excess of the solubility limit, under specific chemical and physical conditions, is nonetheless present in solution”*. The supersaturation causes energy equilibrium to tilt towards the formation of solids from the aqueous phase causing proteins to form crystals.
* Source of quoted text- McPherson, Alexander, and Jose A. Gavira. "Introduction to protein crystallization." Acta Crystallographica Section F: Structural Biology Communications 70, no. 1 (2014): 2-20.
Proteins that have hydrophobic surfaces or are membrane associated tend to have exposed hydrophobic region causing them to be “sticky”. This would bind to membrane filters during processing. At APSL, non ionic or zwitterionic detergents, and other additives and excipients are added to protein during the purification and concentration process. The use of detergents during purification helps in keeping the protein from sticking to tubes and filtration devices.
Transporter proteins are integral membrane proteins that help transport ions, small molecules and macromolecules across the membrane. G protein coupled receptors or GPCRs are a type of integral membrane proteins that are targeted by various drugs.
Since, they are membrane associated they pose unique challenges to crystallization. There are several ways to purify and crystallize membrane proteins. For example, use of detergents during purification and crystallization is a tried and tested method. Often, a membrane like environment is created using artificial lipids like monoolein, monopalmitolein etc.,. These lipids are mixed in certain ways with the membrane protein in detergents to form what is called as “Lipidic cubic phase” (LCP). Protein in LCP are set up for crystallization using special crystallization plates designed to handle LCPs. High through put LCP crystallization set up is also used in several cases.
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