Dr. Yong Tang, a senior researcher at the Wistar institute, is going to give us a talk on protein crystallography on July 2, 2008.
Practical approaches of protein crystallography
– your beloved protein! your own structure?!
In this talk yong is going to cover various aspects of a "typical" protein crystallography project with an aim to stimulate your own enthusiasm of obtaining crystals of proteins you may be working on.
Q1: Is my protein a potential (or exciting) structural target?
A1: There are ways to help you evaluate whether your protein is
workable or not, and if workable, how big the impact may be. Secondary structure prediction, structural homology to known structures, expected structural novelty.
Q2: How do I get started and what are the secrets of getting proteins
"suitable" for crystallization?
A2: Examine your gene first with a bacterial-expression perspective
and engineer your protein expression vector to be crystallization-friendly. These includes rare codons, cleavable tags (like GST or 6His), minimum vector-derived sequence, co-expression. What if all these fail? – I would follow up with this.
Q3: All right, I got my protein as you have recommended, but it just
won't crystallize!
A3: These are tricks to modify proteins at both the gene and protein
levels. Subcloning to make truncated or internal-deletion constructs
(and what are the guild lines?), limited proteolysis to get rid of
"unstructured" tails or internal fragments.
Q4: Well, I still have no clue how to crystallize a protein!
A4: I would bring in a simple but comprehensive set of crystallization
tools and reagents for demonstration. It is easy – you would graduate
the course in 10 minutes, but indeed it takes years for you to claim
yourself a "crystallization expert".
Q5: Hooray, I got my protein crystallized! But how to proceed then?
A5: Improving protein crystals takes a combination of fine-tuning your
crystallization conditions on one hand, and improving your protein
itself on the other. I would introduce some guild lines.
Q6: How to know if my protein crystals are good enough for structural determination?
A6: Congratulations, you are very close to getting a protein structure
– at this point, I would suggest that you consult a professional
crystallographer with collaboration perspectives.
Q7: How to determine and refine a structure?
A7: Save your time and effort for what you are really good at and let
a professional crystallographer take it over from here.
Q8: Hooray again! My collaborator determined the structure for me
perfectly. But, sorry, how to understand all these chicken wires,
cartoons, surfaces, and save me!, the table that seemed to be written in another language?
A8: Good point. Even if you don't work on proteins, it is indeed
essential to understand how structural biologists present protein
structures – truly protein structures have been in the spotlights now
and then. I would guild you through these various bare-bone
experimental data (like coordinates and electron density map) and all
these "artistic" renditions.
Q9: Hey, my crystallographer collaborator stole!!! my leading
co-first-authorship in CELL. How do I survive collaboration like this?
A9: We mostly think that crystallographers are service person for
pioneering cell biologists or biochemists, but believe it or not,
crystallographers indeed have their own advantage to identify novel
and dominating aspects of a collaborative project. I would explain
with examples.
Q10: So, am I ready to move on as a crystallographer after my first
success with protein crystallization?
A10: There are indeed well-known crystallographers coming from
"nowhere" and I would share with you what I know about the pros and cons.
Sounds very interesting? Come and join us at CRB 302, 12:00 - 1:30 PM on Wednesday (July 2, 2008)!
