Protein Crystallography Course
The most important thing you will ever put in your crystallization trials is your protein.
Some basic things to check before you start are:
Is it pure?
- Purity is the most important prerequisite for crystallizability
- Run an overloaded gel to see contaminants present at 1% of the total protein
- Purity also means that there is no heterogeneity in posttranslational modifications
- If you are crystallising a protein-protein complex that will stay together at concentrations of 1mg/ml, purify the complex from uncomplexed protein before setting up trials
Is it folded?
- Check the activity of your protein if you have an assay
- Check the CD spectrum of your protein to see if it has secondary structure
Is it fresh?
- Proteins break down with time and the mixture becomes heterogeneous
- Always try to set up your trials as soon as possible, preferably the same day as the last step of the purification
Is it monodispersed?
- Monodispersity means that the protein exists in solution as a single oligomeric species eg. monomer or dimer
- It means the protein is free of non-specific oligomers and aggregates
- Use a size-exclusion column as the last step in the purification
- Use the dynamic light scatterer to see if your protein is monodispersed
(Set up trials anyway)
- Does your protein need to be kept reduced?
- Does your protein need the addition of something (eg salt) to stay in solution?
- Is your protein stable at room temperature?
- Does your protein break down rapidly?
- Has anything similar been crystallized before?
And now for something completely different...
If you are getting nowhere with your protein, try crystallizing something else.
Does your protein bind a ligand? If it does, it can help because the binding of a ligand
- is likely to order the region of the protein that binds ligand
- may bring two subdomains together and reduce flexibility>
- will change the surface properties of the protein
- may cause a conformational change in your protein
Heterogeneous tertiary/quaternary structure can hinder crystallization
- Does your protein break down to a stable proteolytic fragment (either "spontaneously" or with the help of an added protease)?
- Does the homology of your protein to others in the same family drop off at the N- and C-termini?
- Does your protein have a domain structure?
- Does your protein have low complexity regions?
Sometimes, a point mutation is all that is required to stop/start a protein crystallizing.
Working on a different species is the easiest way to get a collection of
point mutations that do not affect function.
If your protein is glycosylated the floppy and heterogeneous carbohydrates may be interfering
with the crystallisation. Try enzymatic deglycoylation.
Know your protein
© 1999-2005 Airlie J McCoy,
University of Cambridge. All rights reserved.
7 June, 2005