Cryocrystallography is the collection of crystallographic data at cryogenic temperatures primarily with the use of liquid nitrogen. The following advantages and disadvantages are comparing cryogenic vs. room temperature data collection.
Advantages
1) Reduced Radiation Damage: when compared to room temperature collection
2) Fewer Crystals: as to be expected if the radiation damage is reduced the crystal lifetime is increased therefore reducing number of crystals
3) Better Data: increased resolution, I/sigma, redundancy, reduced B values and stronger anomalous signal
4) Better Crystals: protein crystals can be selected at opportune times during the crystallization process (possibly avoid dissolving or cracking) as well as be easily transported within cryogenic dewars
Disadvantages
1) Icing: even if icing occurs it may be possible to salvage the collection during data processing
2) Cryoprotectants: many protein crystals require the use of a cryoprotectant (the most popular is the use of 20-25 % glycerol) prior to being exposed to the cold stream
3) Increased mosaicity: this can be reduced with the proper cryoprotectant as well as mounting technique (ref pg. 32)
4) Non-isomorphism: unit cell size may vary (ref)
5) Cost of equipment: include in grant applications or have at least two bake sales
We have had a couple of instances when the beam center was incorrect in the header of our images. The header of an image is written out when you collect a diffraction image. The header may contain the wavelength, collection time, beam center and oscillation step size, which are read by data processing software (in this case Ipmosflm).
Unfortunately, the information contained within the header may not be correct.
How can you look for to tell if your beam center is incorrect?
One way is by looking at where the Bragg reflections are predicted vs. where they actually are located. It is important to check if the orientation of the lunes and the spacing between Bragg reflections looks reasonable. If they are not then you may an issue with your unit cell and/or space group and not a beam center problem.
If you are having trouble seeing the offset in the video here is a screen shot:
The blue arrow is pointing toward the Bragg reflections while the red arrow indicates the predicted reflection locations. Also you can see that there are fewer Bragg reflections (dark spots) than what is predicted (yellow squares) which is a sign that there is a problem.
CNS (Crystallography and NMR systems) is able to perform simulated annealing to get started, one must first create a generate file.
1) Input then scroll down to Refinement, refine.inp and Edit
2) amy.pdb needs to be replaced with your pdb file
3) The space group, unit cell, angles and amy.cv need to be updated
4) Adjust the resolution to your desired range. The overall B-factor correction should be set to isotropic unless you are dealing with very high resolution data (~1 Angstrom). Set Bulk solvent correction needs to be set to False
5) Change annealing schedule to slowcool
Note: Not shown, but I usually set the map grid to 0.25 for better viewing
Save an updated file
In your terminal:
type: cns < refine.inp > refine.out &
Note: if you renamed your generate files then use them as your .inp
The ‘&’ symbol allows your cursor to be free
type: tail -f refine.out
This will allow to see the progress of the processing in your terminal
This allows you to quickly see if the inputs have generate an error
Tableau allows for the searching of protein folding patterns of substructures in the PDB structural database. This type of searching can be helpful in understanding protein structure, function and evolution.
The server searches using secondary structure elements and is capable of finding either an entire structure or a substructure of a larger structure (ref).
Tableau has a number of simple inputs (title, email, desired structure, output), however, you want to read the suggested tips. You can submit structures that contain multiple chains or domains, but it is not recommended. I submitted a number of structures and had a response time of about 3 minutes.
I may have been putting off this post perhaps because it is not as fun reporting as good news.
I went to APS a couple of months ago to collect data on the miracle crystals (see link above). Cory from Emerald Biosystems was able to swing by (at nearly midnight!) and mount then plunge the crystals. Despite the channels being narrow, the mounting process is easy and by adding the appropriate buffer after opening the card there is no need to rush. I then transfered and screened the crystals. The result…
Salt crystals…
The crystals had a number of different morphologies, but unfortunately they were all salt crystals.
Although I have seen many others have the fun of collecting on salt crystals this was my first time. If you haven’t had the experience it is like watching a unicorn die.
The CCP4bb is the most popular electronic mailing list that is related to macromolecular crystallography. The idea would go even further and bring together all bulletin boards (such as Phenix, Coot, PyMol) into one location. This would benefit developers and users in that they would only need to check one location to ask and answer questions.
It is time to move on when:
1) Members have a drawn out (here, here, here, here, here, here, here, here, here, here, here) discussion on attachment size and html formatting in emails which is fine.
Does everyone need to receive each of these emails? No.
Both problems would be fixed by using a forum instead of an email based system.
2) Members are being used as targets for scams (yeah, I don’t know who falls for these either).
3) Have your work email address spammed due to being on the bulletin board.
4) You would not have to depend on others to summarize answers to the question they asked. This would not only save time of those asking questions, but allow others to have access to all the answers.
5) A forum could be organized by topic instead of the entire list receiving every email.
Members of the email bulletin board have already expressed their dissatisfaction in receiving every email.
—
What would the topics look like for this new forum?
Protein Cloning, Expression and Purification
Protein Crystallization
Data Collection
Data Processing
Data Refinement
Program Installation Problems
Program Execution Problems
Employment
—
The topics will need to be adjusted. It won’t be perfect at first, but will be much, much better.
It is time to make an old system fit the next generation.
Please drop me a line in the comments on what you think about this and if you would join.
The process in macromolecular crystallography for generating heavy atom derivatives can be tedious. Problems may arise from heavy atoms not being incorporated into your protein to difficulty in producing crystals for derivatization trials therefore making each attempt critical.
The Heavy-Atom Database System: HATODAS II has been created to address these problems. The database uses 93 known heavy atom binding motifs (derived from 3103 heavy atom binding sites) and can take into account the amino acid sequence as well as the crystallization condition (ref).
Here is an example of a prediction that HASTODAS generates for potential heavy-atom reagents:
The following is a list of the suggested motifs that are present in the submitted sequence:
If your protein does not contain a His, Cys or Met then you maybe forced to mutate a residue for derivatization, but which one do you choose? HASTODAS addresses this question by suggesting a point mutation(s) based on multiple sequence alignments of homologous proteins.
The catalytic site atlas (CSA) is a database that displays active sites and catalytic residues of enzymes (ref). The database is regularly maintained, but has not been updated since August of this year. The database currently contains 25,537 entries based on 968 literature references.
The site has a number of search options (located at the top of their page) that include PDB, Swiss-Prot code and EC number.
Below is an example of some of the results that are produced by the CSA. The catalytic residue in this case is an aspartate located at position 93. I would like to see the catalytic residues highlighted differently so that they are can be identified quickly.
The CSA also performs a homology search using Psi-BLAST. This information is very helpful if you are looking for evolutionary relationships between proteins (ref).
The CSA help page is really good (although a number of links to it are dead) if you would like more information or are having trouble interpreting the results.
I have been to the APS Synchrotron quite a few times and realized that there are a couple tricks of where to sleep, if you don’t stay at the Guest House. I often see newbies trying to do the head on the table approach, but in a rolling chair this can be tricky.
So what to do?
1) Sleep on the floor
Pro: plenty of space
Con: the floor is concrete
If you are going to go this route then at least sleep on a coat or put your head on a bag (bring a pillow!).
2) Conference room (located along outer rim of the building)
Pro: decent space, very thin carpet, darker and less noise than being near the beam lines
Con: group members may think you have disappeared, if you don’t let them where you are sleeping
3) Couch
Pro: located near the beam line and a lot better than the floor
Con: bright, few around, maybe on the shorter side if you are tall
The process in macromolecular crystallography for generating heavy atom derivatives can be tedious. Problems may arise from heavy atoms not being incorporated into your protein to difficulty in producing crystals for derivatization trials therefore making each attempt critical.
The 
group members may think you have disappeared, if you don’t let them where you are sleeping