Firstly, I’d like to welcome you on our website and on my post. My name is Sema and I am coming from France. And my project within the Pathsense network is hosted by Newcastle University under the supervision of Dr Rick Lewis (Faculty of Medical Sciences, ICaMB) and Dr Jon Marles-Wright (School of Natural and Environmental Sciences).
In this first post, I would like to write about the main technique used in my project which is “Structure-function relationship of the sensory domain of Listeria monocytogenes’ Stressosome”. Why structure-function? Because the structure of a protein is highly related to its function. And how structure? Simply Structural Biology. But what is Structural Biology? How can we get a structure like the one below?
Structural biology is the study at the molecular level, the structure of proteins but also nucleic acid or even micro-organisms such as viruses. Studying the structural property of a protein will allow us to understand how it is acting within the organism.
There are 3 main techniques used for Structural biology:
- X-ray crystallography
- Cryo-Electron Microscopy
- Nuclear Magnetic Resonance
And in my case I am working on protein X-ray crystallography and later I’ll also use Cryo-EM method for my project. Proteins are very important for a living organism and there are thousands of different proteins, each with a specific activity. Structural analysis is required to better understand the mechanism of a protein.
To be able to use X-ray crystallography, we first need to have a highly pure and concentrated protein sample. Why? Because it all start with crystals. As we know crystals have a well-ordered atoms arrangement. Knowing this, the X-rays applied on the crystal, will diffract once it hits an atom (as they scatter the x-ray) and it will be seen as a diffraction spot. By this, really simply explained principle, we are able to have informations about the atomic structure of the protein. But it isn’t easy as it sounds like.
Protein sample preparation
So firstly, to have our protein of interest in high quantity we need some molecular biology, biochemistry techniques and knowledge to be able to clone, produce and purify the protein. Once the protein have been purify, its purity can be checked on a SDS-PAGE or with some biophysical study such as DLS (Dynamic Light Scattering). Having an almost pure (because 100% of purity is impossible) is one step, the second one is to concentrate the protein sample because during the purification process the proteins are diluted in the buffer. And some proteins which are not really stable tend to agglomerate, so when the sample is concentrated, a lot of protein can be lost during this process. But with determination and high hopes, we can obtain a concentrated protein sample. Finally, there isn’t a “right concentration” as it depends on the proteins as all aren’t the same. Some proteins may crystallize at 4mg/mL and some at 15mg/mL.
Secondly, to obtain a crystal of our protein we have to find the perfect crystallization condition. But what is a condition? Usually a condition is a mixture of a buffer, a salt and a precipitant and there are many many of them. So in order to increase the chance to get a crystal, different buffers, salt and precipitant are mixed at different concentrations and each forms a condition. Luckily, there are commercially available crystallization screening that are mixes of these different components.
The crystallization will then start with a crystallization screening which are generally composed of 96 different conditions that can be tested with our protein.
You also need to know that there are 2 types of setting up crystallisation essays: the sitting drop and the hanging drop as shown on the following diagram:
Diagram of the crystallisation drops methods commonly used
Usually the sitting drop is used for the first crystallisation screening and the second one is used for the crystallisation optimisation that we’ll see later.
In order to set up crystallization tray, in my lab, we use the Mosquito robot. This robot is able to set up crystallization drops of 200nl which is extremely small to do it by hand. As I’ve said for the crystallisation screenings we use the sitting drop technique. Here you can find one of the many different plate used for this method which is the MRC 2 well plate as shown bellow:
Picture of the MRC 2 plate with 96 wells for each conditions that are stored in the reservoir
(rectangle well) with the 2 different drop on its side (round wells)
It allows the screening of up to 96 conditions that are stocked in the reservoir (represented by the rectangle-shape well) and 2 sitting drop place next to the reservoir (the round shape well). This plate will allow to have 2 drops for each conditions, one at a 1:1 ratio and the the second one at 2:1 which means that the drop will be composed of 100nl of the protein sample and 100nl of the condition for the 1:1 ration, and 200nl of the protein sample and 100nl of the condition for the 2:1 ratio.
Each condition are loaded into the 96 wells and then the Mosquito will set up the drops. Here you can find a video that explains and shows how the Mosquito works: https://youtu.be/_iP-PclaY38. Finally the plate is sealed and the crystal forming method generally used is the vapour diffusion, so the drop will get at a hyper-saturation state by equilibrating its solution with its reservoir.
Some crystals can be formed within the hour, some may take days. This is why, after setting the plate, a regular plate check is made during the first 2 week and then less frequently after the 2 weeks.
I’d like to share also the first crystals ever that I’ve got for my project and in my life, it is such a nice feeling to see nice crystals like this (even if they didn’t diffract which is another story that I’ll try to cover later)
Now that we have crystals, it is time to harvest them.
To be continued…
Have a nice day/night/weekend!
Sema Ejder, ESR 13 from Newcastle University.