If you are struggling with refolding your protein then you may want to take a look at REFOLD. The Refold database currently contains 759 protein entries.
The help pages are really nice in that they explain how to use the database and contain background information. For example, here is a pdf of ‘A practical guide to protein expression and refolding from inclusion bodies.’ Based on the current entries the most common method of refolding is by dilution.
Here is a shot of a couple of search options (note: all the panels are not open)
Graphs are updated nightly to reflect the current contents of the database:
A neat feature of this database is that it allows users to create their own free account which will track their past searches.
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.
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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
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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.
We have discussed the controversy regarding the 2009 Nobel Prize in Chemistry that was given for the structure and function of the ribosome. I was also thinking about what a great accomplishment it was and how we could pay tribute.
Here is the idea: Get together 200 of our closest friends of which many of whom would need to wear spandex and short shorts. We would make it a dance tribute and have people attach a balloon to their head for identification. We would then act out the function of the ribosome to music from the 1970s, you know – flute solos and a lot of cow bell.
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Well, I just found out from Molecule of the Day that this idea has already been done, dang – scooped again. Enjoy.
Fred points us to an eighteen minute introductory video on structural biology, but unfortunately the English version is not uploaded onto a video hosting site (the French version is here for my friend Julie). I lack the rights to the video so can’t post the English version myself.
I would recommend this video to any relatives that glaze over when you describe your job or perhaps to new graduate students. Enjoy.
I have had quite a bit of luck with this trick so thought it was worth passing on.
You may have noticed that you can lose quite a bit of protein when concentrating.
Using the following steps:
1) Remove the contents from the concentrator as normal
2) Refill the concentrator with your protein’s buffer
3) Centrifuge & remove remaining solvent (if you have any)
4) Check the solution for recovered protein
Why does this work?
Precipitated and "stuck" protein that you are unable to pipette becomes soluble due to the addition of the protein’s buffer.
The next step in science journals is online. In recent Nature news article, “the American Chemical Society (ACS) is taking steps towards turning most of its academic journals into online-only publications.”
I along with others have been expecting that science journals would move to an online format. I have been following the comments of other blogs that have discussed various aspects of the issue. The comments have been quite fascinating such as those from Farewell to Hard Copies at the blog In the Pipeline. A number of comments, from that post as well as the correspondence to Nature indicates that not everyone is going to happy with the change.
My guess is that the reasons mentioned are not going to convince the ACS to change its mind (assuming the rumor is true). If you are uncomfortable with the idea of having scientific journals online then here are some tools to hopefully make the experience less stressful.
1) How to find scientific articles
If you are simply using Google scholar or PubMed then you are missing out.
-I have already written about hubmed.org
These FireFox plugins are worth looking at Eccellio Science and i-cite
3) How to quickly and easily track multiple websites RSS feed reader
4) Difficulty reading a Computer Monitor
Firefox add on: NoSquint (version for Firefox 3.5 here)
This add on is helpful because it allows you to hold down the ‘ctrl’ key and then use the mouse to scroll and adjust image and text size.
6) Serendipitous discovery
I use Google Alert for certain key words that may relate to your research. For example, crystallography or phosphotransferase.
-If your word appears in any news stories then you will be sent an email with an excerpt and link to the article
2) Download the coordinates, followed by the map in CCP4 format
3) Extract the files that you downloaded
(7-zip will work for windows if you do not already have it installed) **Rename using the term map: example) 1c8u.ccp4 renamed to 1c8u.map.ccp4
4) Start PyMol and open the pdb and map files (File -> Open)
5) Display the map by clicking on the ‘A’ for actions then mesh followed by selecting your desired mesh level – as shown above (I did 2.0 in this example)
-Below: if you look at the upper right corner you can see what is displayed by if it is highlighted (light gray) in the PyMol window (in this case 1c8u.map is off)
6) Enter the information available on the PyMol Wiki into the command line (enter everything left of the # symbol)
select site, resi 39-50 # residues that are displayed for viewing
isomesh map, 1c8u.map, 2.0, site, carve=1.6 #display the map around the selected residues (change 1c8u.map to correspond to how you named your .map file)
color grey30, map # sets map to 30% gray
bg_color white #sets background to white
set ray_trace_fog, 0 #turns off raytrace fog–optional
set depth_cue, 0 # turns off depth cueing–optional
set ray_shadows, off #turns off ray-tracing shadows
I then zoomed in on one of the selections then showed ‘S’ ’sticks’ in the PyMol window.
7) Click on ‘ray’ for ray tracing then followed with ‘color grey30, map’ since the mesh looked black (note again what is toggle on and off in the upper right)
Finally, you can add labels using GIMP or Photoshop
Don’t forget to cite PyMol
If you would like to learn how to easily make a movie using PyMol check this post out.
I previously mentioned the Protein Data Bank (PDB). In brief, it is the world wide depository of macromolecular structures. Currently, 150,000 scientists from more than 150 countries visit the PDB each month. I am grateful that the PDB is freely available as it has served as an invaluable resource. A number of items have come to mind regarding the search feature in the PDB. I do not have the background to properly gauge the feasibility of these 10 search improvements, so please think of the following as a wish list rather than demands.
1) Eliminate the need for pop-up windows
-most browsers block pop-up windows, making these searches mildly annoying.
2) Have an option to simplify the results
-such as eliminating the images of the proteins (faster loading)
-reduced space would allow for more macromolecules to be displayed on each page (perhaps allowing 15 macromolecules to be shown, rather than 10)
3) List residues that are not within electron densities as a percentage
-residues at the termini or in flexible loops may not be within the electron density, however this may not be noticed by researchers who are unfamiliar with crystallographic models
-by seeing a percentage, a researcher may become aware that he or she should be examining the coordinate file with the electron density (like that which can be generated using the electron density server)
4) Eliminate separate ‘Evaluate Subquery’ toggles
-so it resembles a Google search
5) Save the original search
-it is frustrating having to toggle 5 ‘Evaluate Subquery’ entries and then, should you want to adjust one, have to completely re-enter all of them.
6) Provide an entry of when the structure factors will be available on the structure summary page
7) Ability to automatically load the structure factors with electron density
-similar to what can be done using the electron density server
Be able to search using metals
-instead of using a separate database
9) Be able to receive an email notification if/when a structure is deposited by a colleague
-this will allow you to monitor labs that are working on similar projects
-you’ll know when to send a colleague an ‘even a blind squirrel will eventually find a nut’ note
10) Have a ‘Find Related Articles’ button
-if applicable, auto enter the title of the article into Hubmed
Just don’t forget about attention to obvious.
