I am spoiled.
I don’t have to collect images using film or kill whales for my haemoglobin samples.
So how do you treat the information from those that were not so lucky? I am referring to older structures that were deposited in the PDB.
For example, here is a link to the electron density server for the structure 1DCL.
The structure was solved to 2.3 A and has a Rvalue of 0.140 (wow!).
According to the EDS server the completeness is at 34.7 %. I have yet to go through the CCP4 uniqueify script and determine if the EDS calculates to 2.0 (highest resolution that the data was deposited) or 2.3 (highest resolution the data was solved), but at first glance the completeness looks to be very low.
The structure contains 9 waters which are over 70 angstroms from the protein. My guess is that these waters were incorrectly placed based on a symmetry neighbour.
The Ramachandran plot indicates nearly 11 % of the structure is comprised of outliers.
I have a knack for crashing iMosflm, but love its user interface. Here are some tips that should save you time and help you attain the right space group.
Masking the beam stop:
1) Select the green box in the image GUI
2) Zoom (using the magnifying glass) left click and drag to desired view
3) Select the arrow and drag the rim of the green circle that appears to your desired size
4) Right click to zoom back out
Note: I often see people using the masking tool (the icon that looks like a mask) for the beam center. The masking tool is best used for the shadow that extends from the beam center.
Selecting Images to Index:
The Grey boxes cannot be selected under the ‘Use’ column
1) Double click on the red circle (under the Search column) and that image will be used in indexing
Multiple Lattices:
If you suspect multiple lattices this may eliminate a lattice, if it is comprised of weaker reflections.
1) Click on Indexing on the main GUI
2) Adjust the I/sigma cut-off from the default 20 to 40 (or higher): see image for where to adjust
Note: Keep an eye on the number of reflections (want ~400)
Cell Refinement:
Instead of typing in images manually, you can automatically select them
1) Select by clicking on the repeating blue circles
2) Select the images that you wish to be included during the cell refinement
Note: You can adjust the view of the selected images are displayed
The very basics of using Coot for those that have not used this program. I am a big fan of Coot and think if you give it a try then maybe you will be too. This post will cover three basics: 1) opening a PDB file, 2) loading maps, and 3) how to move about using your mouse.
1) Opening a PDB file with Coot
Find your desired PDB file (coordinates) and select it
Tip: Using the Filter option can save a lot of time
2) Loading a 2Fo-Fc map
Simply hit ‘Ok’ and you did it!
Here is how to load a Fo-Fc map:
Select (its below the default option) and open with the ‘DEL’ for the phases and amplitudes
3) Moving the macromolecule with your mouse
Left click (hold) allows you to rotate the coordinates
Right click (hold) allows you to zoom in (move toward you or right) and out (move away or left)
Center click allows you to center (focus) on an atom
A couple of years ago, I read the book, The E-Myth Revisited
and have felt that certain aspects of small business could be applied to a crystallography lab. The ideas in this book could be applied in a variety of settings, but this post will focus on a professor in academics.
The vision of McDonald’s and your lab maybe different. Well, maybe not, if that cure for malaria ever pans out, you too can add “Billions Served” and your office door.
What is the vision of your lab?
What do you want your lab to accomplish?
Think about it. Be succinct.
What do you need to do to achieve it?
All too often, the clear focus (the project that landed you the position) becomes muddled. In an effort to combat the “I need a paper/funding to get tenure,” you end up trying to do everything yourself. This feeling is justified in that you have the technical abilities to run every aspect of your lab and as the saying goes, “if you want it done right then do it yourself.”
The reason you have an academic position is largely due to your technical skills: purifying protein, running gels, data processing, publishing papers and presenting research.
However, now you have more responsibility than ever before and doing everything becomes overwhelming.
Maintain all your instruments, order all your supplies, purify all your protein, set up all those crystallization experiments, be on department committees, prepare for and teach classes, stay current on literature, review and write papers and of course write grants. You don’t scale. There are 24 hours in a day and if you want a life (ie. family, vacation, volunteer, hobby, etc.) you are going to need help from others.
Lucky, there are two groups to sell your vision too, graduate students and post docs.
So you teach them all the skills that you know and after a couple of years, the older students pass it on to the new students. If you can afford a post-doc then they too can answer questions that would otherwise come to you.
It works, but not well.
The technical skills will only take you so far, two additional components should be added: managing the system and a vision for the future.
The reason that it does not work well is it is like playing the telephone game. Instead a system should be put in place that helps to serve the vision of the lab. Research is largely about doing something novel, but many aspects are repeated over and over again, which led themselves to becoming a system.
For example, the operation of a HPLC can be largely systematized: what column should you use? what is the maximum pressure that the column can withstand? how do you clean the HPLC? what solutions are needed for each column? how to connect the column? what is the appropriate flow rate? what to do if air bubbles get on the column? who gets to use the column when? etc…
If a system were in place that addressed these questions then all of sudden, you have time to focus on your vision of the lab.
What is the role of each person in your lab/system? Here is one possibility:
New students (1-2 years of grad school)
Technical skills:
Basic lab protocol (PPE, cleaning)
Able to understand manuals and protocols
Where everything is
How to make solutions
How to operate equipment (centrifuge, HPLC, set up crystallization trays, etc.)
Limited by how much work he or she can do
Older Students (3-12 years of grad school)
Manager skills:
Schedule equipment usage (such as HPLC, autoclaving tips or incubators)
Learn interpretation of results
Assign tasks such as areas to be cleaned
Orders supplies
Update technical manuals (very detailed, every aspect that you can think of including trouble shooting)
Post Doc
Manager skills:
Learn new techniques, protocols, software and instrumentation
Assist in initial writing of papers and grants
Help review papers
Write papers, new technical and proof read manuals
Limited by how many people they are able to manage
Professor
Visionary skills:
Final editing of papers
Write grants
Hire post docs – bring in graduate students
Departmental obligations
Committees and meetings
How do you develop a better system?
What is the vision of the future?
Where are the opportunities? (collaborations, funding)
Limited by how many people they are able to buy into their vision
This is just an example, if your vision includes you working in the lab because that is your passion then do it!
McDonald’s (Walmart, Starbucks, etc.) have developed a system that scales. I don’t believe that a crystallography lab can be run as hands off as McDonald’s, but I do believe there is room for improvement.
What do you think? How do you feel about the organization of your lab? Could it be improved?
The web-based tool ValLigURL, which is totally pronounced Valley Girl (serious, check the pdf) allows a crystallographer to compare and validate ligands in the PDB.
This server provides a way to examine whether the ligand in a structure has been seen in a particular conformation. The great part is that this server allows you to upload your own structure for a comparison against existing structures. The server takes about a minute to run.
This could be helpful if you are trying to determine whether you have kissing waters, crystallization remnant or your ligand of interest.
The following is a JMOL output (one option) of a search ligand (thick lines) verse the ligand (thin lines) from MSDchem.
Finally, the server can be used by structural bioinformaticians to examine conformational diversity and quality for one or more ligands in the PDB. In other words, this server is hella sweet.
The Abysis database brings together Kabat, IMGT and structural data from the PDB. Users have many search options available to them. The database even allows you to select the format of your search results.
I previously mentioned the IMGT/3Dstructure database which allows for searching related to antibodies. However, if you are just looking for information related to antibodies then this is worth checking out.
The database has three main search functions: basic, sequence, and structural.
1) Basic
2) Sequence
3) Structural
A topic, linked to a crystallographer that works on it:
1) cancer
2) malaria
3) bioenergy
4) drug resistance
5) materials
6) solar cells
7) enzymology
viruses
9) bacteria
10) vaccines
Thanks for the work that you do.
Quick answer: hell no.
Here is the equation:
Did that help you?
Did you feel that light bulb go on, like YES! Now I know if this structure is good
Thank goodness they mentioned that equation in 6 point font
Yeah, I get that Rfree came out only 17 years ago and some people still haven’t figured out how to use a seat belt on a plane, but seriously, why is this going on?
(the classic)
