P212121

We believe there is an opportunity to reduce the cost of invaluable research.

5 g Carbenicillin
You can pay 468 with USB, 320.50 with Fisher or 39 with P212121.

100 g IPTG
You can pay 2341.70 with Fisher or 99 with P212121.

Innovative products, you bet.
It’s coming, Monday.
Start hyperventilating now.

Last night, I was faced with a decision. I had 2 movies to choose from and, after some contemplation, decided to pass on Twilight: New Moon (apologies to the 14 year old girls in my readership). Instead, we went forExtraordinary Measures.


The movie, based on a true story, was about the Crownley family and an underfunded scientist. The family had 2 children born with Pompe, a progressive and (at the time) fatal disease. Their future was looking bleak with no drug currently available. The movie follows the research and scientists responsible for ultimately designing a drug for this disease.


I found the movie quite inspiring. If you feel like you’re losing your ‘oomph’ or passion for your work, this is a great movie to watch.


Have you seen the movie? What did you think?

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 doubled the number of resources in our tool page over the past couple of weeks. The focus has been on trying to bring together websites that are useful for macromolecular crystallography.

If there is a resource that we are missing and you’ve found it helpful, feel free to leave a comment.

RCSB PDB adds customized widgets to their homepage.

(if you are having trouble seeing this video try the full screen option)

What is the best method for setting up an expansion tray in protein crystallization?

After attaining an initial crystal hit it is often necessary to setup an expansion tray. The goal of the expansion tray is to optimize the hit condition, ideally producing diffraction quality crystals. Depending on the components of the solution one needs to decide which factors to vary such as temperature, pH, the precipitating concentration, etc… Let’s say your initial hit contains:
10 % (w/v) PEG 8000 with 0.1 M CHES at pH 9.5

You decide that would you like to setup an expansion from 5 % to 15 % PEG 8000.

Method 1:
Pipette each individual component into each well. If you unsure what I mean by ‘well’ here is a picture of a hanging drop setup keeping in mind that expanding upon other setups is possible. Pipetting by hand is the most tedious approach, error prone and is limited in its ability to produce very shallow (or fine) gradients.

Method 2:
A/B gradient is based on creating two stock solutions at either end of the range you would like to expand. This method is well suited for expanding upon a single parameter, which in our example is PEG 8000.

For example the two stock solutions:
Stock A: 5 % PEG 8000 with 0.1 M CHES at pH 9.5
Stock B: 15 % PEG 8000 with 0.1 M CHES at pH 9.5
The total volume of the stocks would depend on the well volume and number of trials.

In a 12 well expansion, stock A would be pipetted into well A1 followed by a 0.1 mL decrease in subsequent wells. Stock B would start B6 and proceed reverse of stock A. Note: A1 and B6 are referring to the common grid labeling on crystallization trays

Method 3:
Four corners method is essentially expanding upon the A/B gradient by including two more stock solutions. Instead of having a start and end point there is now 4 points that overlap within the middle of the tray. This results in the ability to screen two variables in the center of tray. The figure of the expansion is quite help although the zoom feature is not.

The authors state that this approach is so new that they have yet to extensively test the technique.

Do you see simultaneously adjusting two variables in the four corner setup as advantageous? Is the four corner setup the replacement of the A/B gradient? How are you optimizing your protein crystallization trials?

Neutron crystallography can be used to gain insight into hydrogen positions. This is extremely beneficial when trying to determine a mechanism. This was the case for endothiapepsin. If one is able to substitute hydrogen for deuterium, the scattering is significantly increased (see slide 14 of Roger Pynn’s presentation on Neutron Crystallography Theory). Deuterons scatter neutrons in a manner similar to that of carbon. In X-ray crystallography, however, we see that they are quite different.

Two methods are used to exchange hydrogens:
1) The crystal can be soaked in deuterated buffer or by placing deuterated water at the ends of the capillary to allow for vapor exchange.
2) The protein can be expressed by using perdueterated media, in which the carbon source for E. coli contains deuterium. Check with your favorite neutron beam line to find out if they offer perdeuteration services. Perdeuteration is the ideal method because nearly all hydrogens can be exchanged. The only downside here is that you may not be able to use the exact same crystallization conditions as the native protein.

If you are considering using neutron crystallography, I would suggest using these two general criteria based on previous published neutron structures.
1) Crystal size ~1 mm^3 or larger
2) Crystal has been solved to ~1 A or better with X-rays

Recently, a nice summary figure and table have been published. They show the current parameters of neutron structures that have been published up to 2007.

Data collection time can be greatly reduced if your crystal is in a high symmetry. High symmetry is a significant benefit in neutron crystallography since it may take 12 hours to collect a frame (mileage will vary depending on beam line).

Finally, it has been proposed that Oak Ridge be able to reduce crystal size to 0.1 mm^3 (pdf). If this is made possible, we may see neutron crystallography becoming a more routine crystallographic technique.

BRENDA is a gold mine for those studying enzymes! The database proclaims to be the comprehensive enzyme information system and with 5010 enzymes it looks to be the case. Here is a screenshot of the navigation bar. As you can see BRENDA brings together many different categories such as IC50 values, pH stability range and crystallization.

My only suggestion so far is to change ‘Recommended Name’ to ‘Enzyme name’. I think it would save some confusion in the search entry.

I have never seen another database bring together this much information about a class of proteins. If you have a colleague working in enzymology this is site is definitely worth passing along.

The way in which scientific publications appear online is going to become increasingly important. How literacy research is conducted is changing rapidly. People are turning to online resources rather than utilizing libraries. Publications that haven’t been made available on the internet may be overlooked. This could result in your paper not being referenced as often, or worse, your research may be repeated.

The number of citations a paper receives helps to determine the impact of the research within that paper. Even if your research is excellent, if no one finds the paper it won’t be cited.

The probability of your paper being read increases the closer it is to the number one search result.

So how do you become the #1 publication in search results?

SEO (search engine optimization) is a field that studies how search engines are influenced by content. The basis for this work is that it is believed that search engines are not perfectly efficient. Therefore with a little tweaking, you may be able to give your publication the boost it needs to be noticed and deemed an appropriate match by search engines. Just to be clear, papers should not be poorly written in an attempt to gain search ranking and in the end content will have a much great impact than SEO.

There are many factors a search engine considers when assembling a results page. The scientist in me cringes a bit at presenting this information since the search algorithms are kept secret making it very difficult to know exactly how search results can be influenced.

Today, we are going to focus on the two items that you can control.
1) Paper Title
2) Keyword Density

Paper Title:
The basics of SEO of titles is that your keywords (the search terms that you people may use to find your paper) should be contained within your title. Although opinions are mixed it is also thought that keywords placed near the beginning have a higher influence than those at the end.

Here’s a great example of how a title should be written for SEO from Acta D:
‘Eukaryotic expression: developments for structural proteomics’

An example of a disadvantageous title would be:
‘The 1.6 Å resolution crystal structure of a mutant plastocyanin bearing a 21-25 engineered disulfide bridge’

The problem here is the resolution of the structure is listed first in the title. More than likely, this paper is going to rank higher for the term ‘1.6 Å’ rather than, for example, ‘mutant plastocyanin’.
If you were the author of this paper and thought each word in that title was critical, it would be more beneficial to arrange the words as follows:

‘Mutant plastocyanin a 1.6 Angstrom crystal structure bearing a 21-25 engineered disulfide bridge’

This tweaked title would be ideal if you were looking to rank for ‘mutant plastocyanin’.

Keywords:
Of course, your paper should be written for humans, but it may help to keep search engines in mind. Although not as influential as in the past increasing the keyword percentage in your document should also help. For example, if you wanted to keyword for ‘protein crystallization’ think about if you can increase the number of times the term is being used.

Again, I don’t have direct proof of how much influence these changes make, but many top brands have excellent top keyword density (percent that term is used on their site) and my feeling is that it wasn’t by accident:
Zappos: shoe (8.33%), shoes (7.78%), zappos (5.00%)
Visa: visa (25.00%), card (10.61%), cards (4.55%), credit (3.03%)
JCrew: crew (6.77%), clothing (3.59%), dresses (3.19%)

Take some time and really think about the title of your paper and what keywords are important. You may find that the one thing standing between you and the first page of Google scholar……is a little tweaking.

A year ago today this site was created.

P212121 has been more successful than I would have ever imagined. My primary goal has been to help someone in the field of macromolecular crystallography. I hoped that by bring together resources, creating tutorials and answering questions that this website would be helpful.

I also need to say thank you. I have had many readers contribute so much time and effort answering my questions and sharing their experiences. My friends that have share with me more than I given with a comment here, thank you.

If you like numbers, the current RSS count here considering only Google is at 140 as a (probably bad) reference point Acta Crystallographica Section D is at 93.

The blogging community has been wonderful and for fear of leaving someone off of a list, I hope a general thanks will suffice.

A number of crystallographic companies have shown their link love:
Emerald Biosystems
Hampton Research
Microlytic
Rigaku

This site was also recently included in the science Alltop, which is cool resource for finding science information.

What’s next? I have a lot in the pipeline, but before we get there I would love to hear from you. Have an idea or suggestion to make this site better? If we have spent the last couple of months together and haven’t met just a Hi or Happy Birthday would be great.

Thanks again.