Advances in Diffraction Rastering Technology for Protein Crystals

Abstract

Diffraction rastering technology has become indispensable in protein crystallography, especially when dealing with heterogeneous or non-homogeneous protein crystals.Through the accurate determination of the best diffraction areas within a given crystal, diffraction rastering enhances the quality of the data collected and the resolution of protein structures. This paper explains diffraction rastering, the uses of the technique/tool and its impact on structural biology.

Introduction

Protein crystallography has been and is still to this day one of the most trusted and applied techniques in the determination of the three-dimensional structures of proteins. Despite its usefulness, the method suffers from one major drawback which is the dependence on the quality of the protein crystals. Many times, protein crystals are anisotropic and, therefore, have different diffraction characteristics, which makes the diffraction data messy and hard for computational programs, such as XDS or DIALS, to analyze correctly. This heterogeneity can, therefore, hinder both the resolution and the completeness of the structural information one can gain, sometimes even preventing successful structure determination. Enter diffraction rastering technology (otherwise known as X-ray centering). Diffraction rastering allows for the best possible centering on the crystal by providing an entire grid map of diffraction for theentire crystal at any chosen rotation. The user is then able to manually or automatically pick the best spot for a data set to be taken.

Diffraction Rastering in Detail

Diffraction rastering therefore works by placing a protein crystal in an X-ray beam and then taking diffraction photographs at different positions. While the older methods for data collection presuppose that the diffraction quality is homogeneous within a given crystal, this method acknowledges the fact that diffraction within a given crystal may not be homogeneous. Thus, one is able to determine where the diffraction is best and where it is not by taking samples from various parts of the crystal.

 Typical steps include:

1.    CrystalMounting: The sample is set on a goniometer which is used to maneuver and locate the sample in space.

2.    RasterScanning: The crystal is transported through an X-ray beam in a fix grid pattern and diffraction patterns are exposed at each point.

3.    ImageAnalysis: The diffraction images which are obtained from the scan are evaluated based on the intensity and sharpness of the image. The parts of the object that give the most distinct peaks are highlighted as requiring further analysis.

4.    FocusedData Collection:Scientists then confine data acquisition to these high-quality areas, which enhances the obtainable structural resolution.

Applications of Diffraction Rastering

Clearly, diffraction rastering has been of immense benefit in the analysis of protein crystals that are difficult to work with or those that are hard to crystallize.Here are just a few notable applications of the technology:

1.    MembraneProteins: Crystals of membrane proteins, especially GPCRs, are notoriously difficult to grow and work with. Many times the crystals have to be grown under Lipidic Cubic Phase(LCP), are radiation sensitive, and very difficult to reproduce and/or optimize. Rastering helps identify the areas of good-quality diffraction within these crystals and enables one to capture data that could not have been obtained using older methods such as visual centering.

2.    LargeMacromolecular Complexes:The crystals of large proteins, such as ribosomes and enzyme complexes, may have different diffraction qualities on some parts of the crystal versus other parts. There are many instances where diffraction is usable in only a tiny area of a crystal (i.e. facets/points). Rastering allows scientists to collect data only from the areas that have the best resolution and that are single lattices, enhancing the overall resolution and successful solving of these structures.

3.    Difficult-to-CrystallizeProteins: Proteins that are hard to crystallize due to their flexibility, large size, or any other reason may produce poor-quality diffraction. However, using raster scanning, it is possible to identify the zones with the highest diffracting quality, which allows scientists to obtain high-quality data.

Impact on Structural Biology

The advancement in diffraction rastering is now a must-have to the protein crystallography community since it provides a key technology that helps identify protein diffraction from small crystals, finds the highest resolution diffraction on a crystal, and also the ability to localize the data set on an area of the crystal that will successfully process. This has opened up new possibilities to study the protein interactions and functions, and has a prospective effect in pharmaceutical research and development.

Additionally, diffraction rastering has the potential to broaden the number of proteins that can be targeted for crystallographic analysis, thus allowing the study of complex and clinically important proteins. Its role in revealing new biological knowledge shows that it is still an important tool in modern structural biology.

Conclusion

Diffraction rastering is a revolutionary development in protein crystallography which provides ways of dealing with the complications that are brought about by non-homogeneous or hard to crystallize samples. It helps to distinguish the best areas of a crystal to get accurate data and enhance the resolution of protein structures. In the future, as the method goes on to improve, it is set to open new fronts in the field of structural biology and bring about new findings in the area of protein architecture and function.