Structural Biology, part 2
Hello, I am back to talk about my passion about the X-ray protein crystallography. In the first blog, I’ve explained the set up of crystallisation screening, optimization and the visualisation of the crystal. Now that we have crystals, we have to harvest or to mount them in a loop. The loops used are in nylon and they come with different sizes from 0.025 to 1mm.
The first step is to harvest one crystal per loop in order to avoid the multiple crystal diffraction that wont’ give a good dataset. Before harvesting, the size of the crystal should be determined and the loop associated to the approximate size should be used. Then the seal of the drop is taken off, so now we have access to the reservoir and the drop. And then the crystal is harvested into the loop, but it isn’t as easy as it sound. The crystal is a tiny entity and so the movement of the hand has to be coordinated. In order to do so, a lot of dexterity and concentration is required to harvest a crystal.
The second step is to cryoprotect the crystal before flash-freezing it. But why? Because the crystal contains water molecule that has to be rearranged to avoid forming ice when the crystal is flashed-freezed in liquid Nitrogen. The cryoprotectant solution can be many components such as PEG400, 6M Sodium Formate, oil or Ethylene Glycol… the mostly used is the 20% of PEG400 mixed with the reservoir solution of the crystal drop. Once the crystal has been harvested from its drop, it is then transferred into the cryoproectant solution drop for few seconds (or more if the crystal is too big), which is deposited next to the drop to harvest.
Then the crystal mounted on the loop is dipped quickly in clean liquid nitrogen to avoid condensation water to form surface ice on the crystal as we can see on the picture below. And now that the crystal is flashed-freezed, it can be stored in liquid nitrogen dewars until it is tested for diffraction.
Good vs bad loop: On the left a nicely harvested crystal on the right a fluffy loop
The next part of this story is the crystal testing for diffraction, impact of bad cryoprotection, fluffy loop and data collection that I will write more in the next post.
Have a good day!!!
Sema Ejder, ESR13 from Newcastle University