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Friday, 14 March 2014 10:49

A customer analyzes In Situ 1 crystallization plates

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Dr. Ivan Campeotto

MRC Centre for Molecular Bacteriology and Infection
Imperial College London
United Kingdom

In my current project at Imperial College London, I am focusing on several proteins from different human pathogens. One of these proteins forms crystals, which tend to be looped with the unique c axis of ~500Å in length and oriented in parallel with the X–ray beam. This led to a severe spot–overlapping problem, which was resolved with the aid of a kappa goniometer.
Gentaur products came to the rescue in different steps of this project.
The first issue to solve was the fact that crystals grew on layers of microcrystals mixed with protein skin, which, when disturbed with a traditional loop, would break into many pieces, enter the loop and thereby abolish diffraction. The Microloops™ allowed for precise selection of the desired crystals without perturbation of the drop, so that skin-free crystals inside the loop could be obtained.

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Figure 1. Micro–crystals were used for in situ at Diamond beamline i04–1. X–ray beam and ruler are represented in red.Another problem was the reproducibility of the crystals, when crystallization trials were set–up in 96, 48 or 24 well plate formats.  I decided to try the new in–situ plates and to compare them with traditional 96 well, 48 well and 24 well plates. This experiment was performed on the same day and using the same protein batch. Whilst the other plates gave either only micro–crystals (<1μm) or failed completely to produce crystals within a given crystallization condition range, the Gentaur plates gave crystals of about 30μm in size (Fig.1)!

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Figure 2. In situ diffraction at the Diamond Light Source synchrotron from Gentaur plate (beam line i04–1).In addition the Gentaur in situ plate was easily transported to the synchrotron (Diamond, beamline i04–1), as it could be packed vertically. Diffraction experiments were performed in situ and crystals diffracted approximately to 3.8Å (Fig.2) with a very low background diffraction compared to competitors.
In my opinion the greatest benefits were: crystal reproducibility, crystal–friendly plate transport and low background scattering. There are a few others benefits, which are not less important, such as the possibility of shooting the crystals with better orientations for data collection or the possibility of testing lattice disorders from data collected at RT vs. cryo data. These are all applications, which I am currently exploring at the i24 beamline at the Diamond Light Source (Oxford, UK), where complete data collection is currently achievable in situ.
We all know that obtaining crystals and manipulating crystals represent a continuous challenge; therefore I would like to thank Gentaur for producing innovative products, which assist us crystallographers not only in delivering better results but also in overcoming experimental obstacles, which otherwise could severely delay or hamper our work.

Gentaur’s In Situ 1 crystallization plates

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