P212121

The phase diagram in protein crystallization is a schematic representation of how protein and precipitate concentration are related. Protein crystals are formed in supersaturated solutions. As shown below, low protein and/or precipitate concentrations will cause undersaturation that will not produce protein crystals.

The red line that separates undersaturated conditions from supersaturated is known as the solubility curve. A benefit of determining the solubility curve is that it can help guide you when analyzing your crystal growth conditions. A crystallization setup that is undersaturated or in the metastable zone will appear clear, however, the latter has the possibility of crystal growth if seeded.

The phase diagram is often broken down into 4 distinct zones one of which undersaturated we have already covered. Precipitation is when the protein comes out of solution as an aggregate and therefore is not useful for crystallographic studies. The labile zone (or nucleation zone) is important since this is where crystal nucleation and initial growth occur. As the crystal forms the protein concentration will be depleted causing one to move from the labile to metastable zone.

The journal Science has put together a forum that discusses careers in science. They have also created a forum primer that address a number of career questions:

• As an undergraduate, what can I do to position myself for a successful research career?
• Graduate school is not working out. I am having trouble getting along with my Principal Investigator (PI) and have not produced many publications. Is it worth it for me to finish my degree?
• How do I choose a postdoctoral adviser?
• What do I need to do at a postdoctoral interview? Who should pay my interview expenses?
• Should I do an industry postdoc?
• What do I need to know when interviewing for tenure-track positions?
• I am a senior scientist considering immigrating to the USA in order to take a job in industry. What tips would you have for me?
• Do I need to do a postdoc to work in industry?
• How do I make the transition from academia to industry? (related post)
• What is available if I have a PhD in the sciences but do not wish to work at the laboratory bench?

(Mary shares her journey as well as others)

• I would like to move to the business side of industry. Should I go to business school and get an MBA?

This is not an all inclusive list of questions so take a look if you see yourself going through a transition in the near future.

We traveled to the Advanced Photon Source this past weekend and rocked out on beamline 21 for 24 hours. The beamline had robotics for crystal mounting and although we did not use them for auto-mounting it was neat to see the set up. If you are interested in robotic mounting at a synchrotron near you then this page is worth exploring.

I picked a couple of bits of information about the robotics on this particular beamline.

1) Robots do not work well with certain pins. Although the 18 mm Hampton-style pin is recommended as the universal standard you need to be sure you select the right one. The Hampton Copper Magnetic HT was not allowed while the Hampton Copper Magnetic ALS HT was fine. The reason was due to the ‘upper lip’ that is present which differs between these two pins. ‘Upper lip’ meaning the ledge that is present before the pin tapers to meet the vertical copper pin. A picture can of these pins can be seen here (view full size – the left pin is not allowed while the center pin is fine). Molecular dimensions also has caps and pins that can be used.

2) Hampton caps (the plastic part that fits over the pin) were not allowed and instead had to use caps by Molecular Dimensions. The reason was being due to the consistency of length in Molecular Dimension caps. Personally, I have never noticed a difference in the lengths of the caps from Hampton.
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The crystal sits on a pin which is in a puck. The puck is placed in a container filled with liquid nitrogen. The robotic arm removes the pins from the puck, which is located inside the large container on the right side of the above picture.

I really enjoy reading and hearing stories about crystallography. A brief history of crystal mounting leading up to robotics is wonderfully covered in a paper (pdf) by Cele Abad-Zapataro.

Stephen also wrote a great post discussing his exciting times at a synchrotron awhile back.

I had the opportunity to see these dewars by Spearlab at the 2007 American Crystallography Association meeting in Salt Lake City.
They have become quite popular, but for those that have missed them they are WAY better than Glass Dewars. Foam dewars are virtually indestructible compared to Glass Dewars and run about half the price.

The dewars are also being used in ways that I would have never expected such as a transfer vessels of liquid nitrogen. Spearlab also just came out with a new 1400 mL dewar cryogenic dewar, which is shaped like the purple dewars shown above.

I still see quite a few people downloading a PDB and then opening it in Coot under File. Coot has the ability to open a PDB directly, if you know the 4 digit access code.


Note: You can also open maps from the Electronic Density Server (EDS) right beneath

I regret to inform you of the passing of Dr. Warren Lyford DeLano. Warren passed suddenly Tuesday morning on November 3rd, 2009.

Although the crystallographic community is small. I did not have the pleasure of knowing Warren personally. However, I wanted to express my sincere appreciation for his scientific contributions.

Warren was the developer behind the molecular graphics program called PyMol. I have written numerous posts about PyMol since it is truly one of the best molecular graphics programs.

I have had a love/hate relationship with PyMol, but one thing that I never questioned was Warren’s dedication in helping others. Warren set up his own forum just to help others with questions that they had about PyMol. Warren was also a leader in the open source movement within crystallography.

My wishes sincere condolences to Warren’s family.

A website has been setup by the Delano family. They have asked for all memories be posted at that location, thank you.

Written by Axel T. Brunger
Dear friends and colleagues:

It’s now been over a week since Warren has passed away. We are trying to move toward a permanent way to honor Warren’s memory and what he stood for: Open Source Computational Biosciences and molecular visualization. To do this, Jim Wells and I put together a mission statement with the approval of Warren’s family:

The Warren L. DeLano Memorial Award for Computational Biosciences

This award shall be given to a top computational bioscientist in recognition of the contributions made by Warren L. DeLano to creating powerful visualization tools for three dimensional structures and making them freely accessible. The award, accompanying lecture, and honorium will be given annually in the context of a national bioscience meeting or a Bay Area gathering of computational bioscientists at Stanford, UCSF or UC Berkeley. For the award special emphasis will be given for Open Source developments and service to the bioscience community.

The award selection committee, consisting of experts in the computational and biological sciences, will accept nominations from anyone..

To make something like this happen in perpetuity would take about ~100K for the endowment.

For donations, Warren’s family has set up a tax deductible fund:

Silicon Valley Community Foundation
memo: Warren L. DeLano Memorial Fund
2440 West El Camino Real, Suite 300
Mountain View, CA 94040
tel: 650.450.5400

We hope that you’ll consider making a contribution (not matter how small) in Warren’s honor. Also, please forward this message to anybody who might be able be willing to contribute.

Best regards,
Axel

**Update**
Fred notes:
For those of you who may worry about the future and future availability of PyMol, this is the information I have received from Elizabeth Pehrson (Warren’s wife, DeLano Scientific LLC):

“I would like to reassure all who fear for PyMOL’s future that DeLano Scientific still exists, we are still accepting (and receiving) subscriptions, we are still providing maintenance and support, and I am working closely with some of Warren’s most trusted developers to plan for the future of PyMOL”.

**Update**
Obituary by Axel Brunger and Jim Wells in Nature Structural & Molecular Biology

I will update this post as I hear more information.

The developers at Accelrys are behind Discovery Studio Visualizer 2.5 (DSV) are really pushing the molecular visualization field along. You can download DSV for FREE after a quick registration.

I actually tried to use this program a couple of months ago, but ran into problems because my computer did not meet the minimum system requirements.

I dropped Accelrys a quick note on their blog and they were able to connect with someone who was able to give me a hand (thanks again, Luke).

My reason for wanting to play around with this program is my need to find a simple and efficient way to create figures for presentations and publications. The program contains many other capabilities related to small molecule libraries and pharmacophore modeling, but those will have to wait.

A couple of items that I like right from the start:
1) I love that the program runs from one window and utilizes tabs

2) The layout can be customized by a simple drag and drop (ie. you can have your tools on the top, to the left or right, on the side bar, etc.)
3) You have the option for a full screen view that saves some squinting
4) I am fan of GUIs and not typing commands – DSV has really come through in that department
5) The Display Style window is really easy to use and check out the number of options, awesome.
One suggest is to add the ability to move the protein while the Display Style window is open
6) Check this one out called Line ribbonsI don’t think I would ever use it in a presentation or paper, but still think it looks cool.

The reason that I am such a fan of Pymol is its capability of producing high quality images. I selected the Ultra high resolution in DSV and exported it as a png file.

Here are two pictures side (left Pymol) by side (right DSV) both are 564×507 pixels.

I found it strange how the program displayed bonds such as the double bonds from carbon to oxygen. The reason being that this is usually not shown in other visual programs. I am guessing there is some way to turn it off, but by initial reaction was wow – look at all these dual conformers
Overall, definitely worth downloading. I would love hear your thoughts on how the DSV compares to your favorite visualization program.

How do you find structures related to a particular disease?

One method is to type the disease into HubMed, throw in a keyword like crystallography and start digging. Although this method is probably worth doing, you may want to check out the OCA browser-database for protein structure/function.

The disease input is listed on the main page in light purple.

The disease search is fairly forgiving and accepts symptoms such as cough or vomit.

Here is example of the search results:
Just as a heads up, the resolution is left as an integer.

This database would also be worth searching when starting up a collaboration with a colleague that is not in the structural field.

We have been running a poll here on P212121 for the past couple of weeks. Here is the distribution of the 88 votes:
An easy way to offend others is to give them advice – when they know more about the subject than yourself.

Paul comments:
“I guess with science becoming ever-more collaborative, this kind of stuff will become more and more common.”

I agree. The Hindu points use to a similar controversy in last year’s Nobel Prize selection in chemistry.

Abhishek comments:
“What you should be really doing is to dig more as crystallographer and come up with a technical comparison of their contributions.”

How about the percent of ribosome each investigator solved?
That would seem wrong since the difficulty of attaining (and importance of) a particular section can vary greatly. To complicate the matter, the award was also given for biochemical studies. We all know that a structure is worth a thousand gels, but are four assays more valuable than a structure? Depends.

Should I be coming up with a technical analysis to determine who wins a Nobel Prize by comparing contributions? Not interested.

The results of the poll reflect my feelings on the subject of ‘No idea’ and ‘They got it Right’. Unfortunately, I lack the background on the ribosome to determine if the Nobel Prize committee made a mistake. I would like to thank all those that gave their insight into this topic. I learned a lot, thanks.

Sparse matrix screening involves a combination of conditions (varying: pH, buffer, additive and precipitant) that have previously generated protein crystals.

This type of screening process is often recommended as the first method to attempt with a protein that has not been previously crystallized. Jancarik & Kim introduced this type of screening 1991.

Benefits:
Commercially available (see below)

Drawbacks:
Biased toward known crystallization conditions
Difficult to make a statistical conclusions due to ‘randomness’ of sampling

Here is a spreadsheet of the overlap between a number of commercially available screens that was adapted from UCLA. Sparse matrix screening is also utilized by Microlytic and Molecular Dimensions in their commercial products.