Three months in a structural biology laboratory.


As part of our final year, Biological Sciences students at the University of Leicester have the option to either do a experimental project or a literature based project. We are then expected to write a final dissertation based on this project.

Owing to my dreams of one day doing my own research, I was really keen on being chosen to do an experimental project. The process for being dealt projects involves filling in a form with your options, and based partly on your grades and other factors, the Biological Sciences School Office sorts out who does what. It’s quite a nerve-racking process, baring in mind that the resulting project is going to be a major part of your final year grade (40 credits) and will probably have a great impact on your research career choices.

Luckily, I got one of the options highest on my list. I was chosen to do a project in the same lab that I did my summer project in. Even though I am technically a medical genetics student, I am constantly crossing between both genetics and biochemistry modules. The description of the project I was given was mainly biochemistry based as it involved working in one of the structural biology labs. The project description said I would be studying HDACs (histone deactylase enzymes), which are enzymes involved in the removal of acetyl groups from lysine residues found at the n-terminal of histone tails. This allows histones to bind DNA more tightly and aids heterochromatinisation.

However, during one of my initial meetings with my project supervisor he said he would like me to work on ‘a slightly different project’ as it would allow me to achieve a more complete project. Slightly different project seems to mean completely different project, because the project I ended up doing was based on solving the structure of the low density lipoprotein receptor (LDLR) bound to the FERM domain of IDOL (inducible degrader of the LDL receptor). Anyhow, I felt this was a really interesting project and was excited to get stuck in.

What was interesting to me is that in structural biology, the methods used are very similar in every project. The aim is to express and purify a stable protein or compound that you wish to deduce the structure of, set up crystal trails using generic screens, and if you manage to get crystals, optimise your screen to achieve better crystals, use x ray crystallography to get a diffraction pattern and use this to deduce the structure of your protein. If you think that sounds simple, you would not believe the number of road blocks you can hit in the middle.

Rather than go into the specifics of my project, I thought it would be interesting to highlight a few key methods that are commonly used in structural biology.

1) Designing Constructs

So, before you can express any protein, it is really important to think carefully about the constructs you wish to purify. In order to increase your chance of achieving nicely diffracting protein crystals, the constructs can’t be too long. Floppy chains and unstructured regions do not crystallise well.

If the tertiary structure of the protein you wish to purify is available, it is more simple to predict which regions are more likely to fold on their own. However, when this is not available, secondary structure prediction tools such as PHYRE2 and JPRED can be used to predict which regions are likely to be structured.

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Programmes like PHYRE are really simple to use. Simple put in the amino acid sequence of your protein and it will predict the secondary structure based on known sequences.

It is also important to put a tag of some sort on your protein so that you can purify it later – an example of a tag is an MBP tag that can be purified on amylose resin or a FLAG tag that can be purified on FLAG resin. The tag must also be cleavable, for example by having a TEV cleavage site.

2) Express your protein

Before you can express your protein, it is important to think about which expression vector you will use. Some proteins express much better in mammalian cells, and our lab is very lucky to have mammalian cell culture equipment. However if your protein is happy, it can be better to express your protein in bacteria such as E.coli as it is much cheaper, faster and generally produces a lot more protein.

Once you have chosen your vector, you must then transfect and induce your cells to produce protein.

3) Purify your protein

Now that you have your protein expressed, it is possible to purify your protein by binding it to an appropriate resin, washing it to make sure only your resin/protein remains, and then eluting it from the resin. If you are using an MBP tagged protein bound to amylose resin, for example, it is possible to elute your protein using maltose.

Once your protein has been eluted, you can cleave off the tag using TEV if you have inserted a TEV site in the construct.

Using gel filtration it is possible to purify your protein further. It is important to select the right column for your protein. If you are using an ion exchange column, you must first find out the isoelectric point (pI) of your protein. Programmes such as protparam can predict your protein’s pI. When selecting an anion exchanger, such as Q or DEAE, select one with a buffer pH above the pI. When selecting a cation exchanger, such as S or SP, select one with a buffer pH below the pI.

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The net charge of a protein can vary with pH. Every protein has its own charge/pH relationship.

Gel filtration columns, such as those that employ columns of beads of gel that have small pores, separate proteins depending on size.  Small proteins would be able to fit through these pores and spend more time in the medium than larger proteins which are eluted first. A S200 column for example can separate proteins up to 200kDa in size, and a S75 can separate proteins up to 75kDa in size.

The AKTA is a machine with which you can load columns. The machine measures the absorbance of each fraction at different wavelengths so that you can work out which fraction contains your protein.

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A typical AKTA machine with gel filtration and ion exchange columns attached. It is usually attached to a computer that gives you a readout.

Once you have the protein in the fractions, you can collect the fractions and concentrate them, if required.

4) Set up crystal trails

Choose a generic screen with which to set up your crystal trails. Examples of crystal screens are PROPLEX and JCSG.

In our laboratory we are lucky to have robots able to set up the crystallisation screen for us. This allows for very precise measurements in terms of how much protein is dispensed and allows the protein to go further. The robot our lab has is called the Mosquito.

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This is the mosquito crystallisation robot.

Next you must record in which conditions potential crystals are seen, and it is possible to optimise the conditions.

So far in my lab work I have got to the stage of setting up crystal trails, but haven’t managed to grow crystals. Keep your fingers crossed though.

That’s all from me. Merry Christmas, everybody!

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