4 Comments

Crystallography for beginners: making the move into protein crystallography – Part 2

Lotus flower crystals of a Bacterial helicase grown in the Hampton Research Crystal Screen based reagent. By Naveen Vankadari institute of Molecular Biology, Academia Sinica, Taiwan (image hosted on Hampton Research website: http://hamptonresearch.com/)

In the previous post I have introduced non-crystallographers into evaluating whether their project is worthy of exploring in crystallography. Assuming you’re project is appropriate and you have the resources needed, read on for a primer on protein crystallography from protein to crystals.

Trial and error: Adapt your protein to crystallography world

You’re not the only one that needs to adapt to the protein crystallography world – your protein should too! You see, in protein crystallography the aim is to force billions of vibrating, moving and colliding protein molecules to pack into a crystal lattice in such a miraculous way that they will eventually form a solid creature, the crystal. For this miracle to happen the protein should be highly pure and contained within an environment which supports such delicate yet stable interactions.  A 0.5 change in the pH value (corresponding to five fold change in proton equilibrium) is enough to inhibit any crystal formation. So, how do you know which buffer will be appropriate for crystal growth? Well, you don’t. This is the name of the game: trial and error. However, there are several starting points which you should aim for:
•    Salt – for well behaving proteins (i.e. soluble at >5mg) you might want to limit the salt concentration in the buffer to 50-150mM (depending on the type of ion). Higher Salt concentrations can promote crystal formation in cases where it lead to dehydration of the protein which promotes protein-protein interactions. Of course, proteins should be kept at their solubility space and if higher salt concentration is needed so be it.
•    Buffer & pH – Use of low concentration buffer above or below isoelectric point (pI)– since you can’t determine the exact pI of your protein, and since it can have a critical effect on the protein-protein interactions it is wise to enable your protein to move across the whole range of pH in the presence of the crystallization cocktail (see below). Yeah, it for sure will precipitate in a “bad” manner but it can also precipitate in a “good” manner (i.e. form a crystal). Thus, use as low buffer as possible such as 10-30mM.
•    Additives – Remove any additives which are not required for the protein’s stability and solubility. This means, stabilizers, chelating agents, metal ions etc. as long as you KNOW these are a requirement.
•    Protein concentration – Another tough question to answer. Since it is easier to dilute a protein solution rather than concentrate it I would recommend that upon completion of your purification scheme you should concentrate the protein solution to a concentration of 20mg/ml or higher, divide your protein solution into aliquots of 20-30 microliters, flash freeze it in liquid nitrogen and store it in -80 degrees Celsius.  Why such small aliquots? That’s because once you’re thawed the protein solution you should not freeze it again (freeze-thaw cycles promote protein aggregation and denaturation).
Of course, all of the above are starting points; with time, you will familiarize with your protein in such a way that you will know which conditions are good for it and which are bad.

Next post I will discuss how to perform your first crystallization experiments!

Have any further questions? any further ideas?? Let me know!

Advertisements

4 comments on “Crystallography for beginners: making the move into protein crystallography – Part 2

  1. With havin so much content and articles do you ever run into any issues of plagorism or copyright infringement?
    My blog has a lot of exclusive content I’ve either written myself or outsourced but it appears a lot of it is popping it up all over the internet without my agreement. Do you know any methods to help prevent content from being ripped off? I’d genuinely appreciate it.

    • Hi Irish,
      Yes, I indeed had found one post being completely copied and posted by a south-east Asian blogger (thief is much more accurate) but I had little to do about it. Part of publishing your blog online is the exposure for possible plagiarism. I wish wordpress could enforce a copy-block function such that only search bots could scan and extract the text, while keeping the text copyright protected from copy-pasting by users.
      Unfortunantly, I am not aware of any function that can protect your text (unless you post it as an image but then the search bots can not scan that text so you’re loosing grounds here).
      Have a great day and thanks for stopping by,
      Chen

  2. I think the admin of this site is in fact working hard for his website, because here every stuff is quality
    based information.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: