I have come across quite a few papers and books about protein crystallization. Unfortunately, I have not yet found a resource that answers the more practical questions about crystallization:
Typical Questions:
1) What should I read to understand the basic theory of protein crystallization?
2) How much protein do I need for crystallization?
(Note: Peter did a good post on this topic: Risky business or, how much protein sample do you need?)
3) How much will it cost to attain a protein crystal?
4) What is the ideal buffer for my protein?
5) Which method should I use for crystallization?
6) What concentration should I use when setting up my crystallization trails?
7) What size should my drops be during expansion set ups?
When should I expect to see crystals?
9) How can I tell that I have salt crystals and not protein crystals?
10) What screen(s) should you use and in what order?
Have I left any questions out?
Please feel free to add more questions below in the comments section or send me an email: sean@p212121.com
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This is not listed in any manual or wiki, but if you watch an experienced crystallographer you will see that after clicking Run.
The very next thing you should do is to see if the process failed.
I attended the Mid-Atlantic Graduate Student Symposium (MAGSS) in Medicinal Chemistry this week.
My goal was to see how structures from the Protein Data Bank (PDB) are used by the non-crystallographic community.
I had a brief discussion with a colleague who was doing a number of modeling experiments. She was, essentially, modeling how the protein would move over the course of 35 nanoseconds.
I asked her how she determines which model to use from the PDB. The two criteria were:
1) the PDB entry had been used in other publications
2) the entry had the highest resolution
The most useful piece of information in deciding how to determine the proper position of the coordinates is the electron density map. However, I believe there is currently no resource that displays the final electron density map generated by the crystallographer.
We are currently making a huge effort to generate and store the coordinates of proteins, but are we doing enough to make them useful?
What criteria should the modeling community be using to judge the quality of coordinates from the PDB?
I always get a kick out of listening to colleagues describe what they do for a living. I came across this video and thought it was a nice ‘elevator pitch’.
When explaining what we do I normally use hand gestures. With one hand (the inhibitor) over the other (the protein) I’ll say, “If you want to stop a reaction you need to know the shape so the inhibitor can bind in the right spot.” I think this video could be helpful for a friend, spouse or parent that finds his/herself in an inquisitive mood.
1) Go to the electron density server
2) Save the map in CCP4 format.
Note: if you save the map as an ‘XPLOR’ or ‘CNS’ file, you will need to rename the extension ‘.xplor’. This will enable you to extract the files you have just downloaded.
Example: 1w5x.cns -> 1w5x_2fo-fc.xplor
(I like to enter in the type of map (2fo-fc) as soon as I download it)
-If you do not change the extension, pymol will not recognize/see the file when you are searching/browsing.
3) Load pdb that corresponds to the map you downloaded into pymol
Example: 1w5x.pdb
4) Diplay -> Background -> white
5) H-> everything S-> sticks
6) Load the map that was downloaded from the electron density server into pymol.
Example: File -> Open -> 1w5x.ccp4
You will still NOT see electron density, you need to use the mesh command next (see image below).
7) Mesh Displayed:
If you don’t want your structure looking like clown vomit, enter the following into the command line:
isomesh mesh1, 1w5x_2fofc
9) mesh1 -> C -> grays -> gray70
(see how to use the electron density server post for a publication)
The fourier duck began in the book Optical transforms, their preparation and application to X-ray diffraction problems by C. A. Taylor and H. Lipson. The old school, 1966 book review contains a brief history on the subject. The first time that I met the duck online was in Kevin Cowtan’s Book of Fourier.
I came across the site Diffraction and Fourier Transform via twitter (@olchemist) and in Web 2.0 style decided to draw my own.
The site loads and you are given a brush to draw on the left panel then click FFT (center). Once you have the basic idea then try adjusting various components such as cell parameters, masking and the draw function. In addition, you can display how the components of complex, magnitude and phase contribute to the resulting pattern.
Enjoy.
If you have ever wanted to generate space group diagrams that are found in the International Tables of Crystallography (vol A). There is a simple free program called Space Group (Mac, Windows, Linux) created by George Lisensky. The program operates in the orthorhombic and monoclinic crystal systems and can generate equivalent positions, centering, glides, screws, mirrors, etc…
The program could be helpful in preparing diagrams and as a teaching tool.
I have been unable to find out whether photography of presentations will be banned at the 2009 American Crystallography Association conference. I am doubtful there will be any information on the issue, but felt it was important to bring up nevertheless.
I have never felt comfortable when someone in the back of the room photographs each slide of a presentation. I also have not had the guts to ask them to stop.
A conference that promotes speakers to share new ideas and information is exciting. However, if the speakers are worried about being scooped then they will hold back and simply regurgitate their most recent papers.
The same reasoning applies during the poster sessions. I do see some people asking permission to take photos of posters. I have also seen thieves attendees taking pictures while the presentations were going on with the owner of the poster not present. Plan on your poster being photographed.
I was just asked to speak at the 2009 American Crystallography Association meeting this year. I will be presenting during the Educational Outreach in Crystallography session at 9:30 am. This was a last minute switch, as the original speaker was unable to attend, so I may not be listed in the conference information/flyer/brochure/pamphlet.
If you will be attending the conference, please stop by and say ‘hi’! I would love to be able to put faces to names. Finally, here are a couple tips I have picked up over the years from attending ACA (or any conference really):
Make a Plan: Presentations are usually given simultaneously, so take some time to figure out which ones you want to attend the next day. You would think this would be obvious, but I can’t tell you how many people I see trying to figure which talk they want to see next.
Take some Snacks: I usually end up waiting on others when lunch or dinner time rolls around. I have found that having a granola bar on hand can be a life saver.
Call your own Room: As soon as you get your room key, ask the front desk for the phone number to your room. Use your cell phone to call your room. You will then have your room phone number in your call log to give to others if the need arises.
Get to Know Someone New: Reach out, ask questions, be helpful, be yourself.
If you have any more conference tips, feel free to leave a comment.
I previously mentioned the Quick Lookup tool and had suggested having the condition results appear on the same page. I ended up having a number of great email exchanges with Paul, the developer of the site. Not only did he implement this change but Paul also added a more user friendly ‘tray reference’ instead of the original drop down menu.
If you are checking a large amount of crystal trays this can be a real time saver. As an encouraging side note, Paul says he is open to new ideas and suggestions regarding the Crystal Screen Wizard site. If you have any ideas for improvements or would like to simply say, “Thanks Paul. You’re a swell guy”, please feel free to leave a comment.