Glycerol is the most common cryoprotectant used in macromolecular crystallography. If you are considering using glyercol as a cryoprotectant for your protein crystals it is worth looking into what has worked for others.
Obviously, your mileage will vary depending on your protein, however, there are insights that we can gain by examining the literature. I am fond of the notion that an hour in the library can save you a week in the lab.
Paper Selection:
1) Only papers that used the crystallization reservoir (mother liquor) with glycerol alone (not mixed cryoprotectants such as PEG 200 10 % with 20 % glycerol) were selected.
2) Papers that contained a range of minutes, such as 5-10 min, were not included.
3) Proteins were not listed more than once
In total, 27 papers met the above criteria and listed the soaking time in either minutes or a few seconds.
The % glycerol is presented along the top row while the 1st column is the soaking time:
.
5 %
10 %
15 %
20 %
25 %
30 %
50 %
.
few seconds
1
2
3
1
6
1
1
.
1 min
1
1
1
2
.
2 min
1
.
3 min
1
.
5 min
3
1
.
15 min
.
30 min
1
What percentage are crystallographers using?
The most popular percentage is 20-25 % glycerol.
How long are they soaking their crystals?
A soaking time of a few seconds is dominant as it was described in 15 out of the 27 papers.
Finally, I also noticed step soaking was used quite often (in 7 papers) which, in general, is performed in 5 or 10 % steps up to about 30 % glycerol with 1-2 minutes per step.
Whether you like online videos or not they are certainly gaining popularity. A year ago, Rigaku started their webinar series that touches on various aspects of crystallography.
Protein + Small Molecule Databases:
1) HIC-Up freely accessible resource for structural biologists who are dealing with small molecules
2) PRODRG2 a server for high throughput crystallography of protein-ligand complexes
3) ValLigURL protein ligand comparison and validation (post)
4) BindingDB experimentally determined protein-ligand binding affinities (Ki, IC50, ITC, post)
5) DBGET integrated database retrieval system for major biological databases
6) Ligand Expo provides chemical and structural information about small molecules within the structure entries of the PDB
7) LPDB selection of complexes based on various properties of receptors and ligands for the design and parametrization of new scoring functions or to assess and improve existing ones MSDChem provides web access to the “Chemical Component Dictionary” of the wwPDB as this is loaded in the PDBe database at EBI.
9) PISA tool for the exploration of macromolecular (protein, DNA/RNA and ligand) interfaces
10) Relibase search, retrieval and analysis system for protein-ligand complex structures (registration required)
11) RESID collection of annotations and structures for protein modifications including amino-terminal, carboxyl-terminal and peptide chain cross-link post-translational modifications
12) 15 Carbohydrate Databases
Small Molecule Databases:
1) CSD world repository of small molecule crystal structures
2) ChemDB nearly 5M commercially available small molecules
3) COD search over 88k small molecules for free
4) A La Mode building models of ligand & monomer molecular components (nucleic acids)
5) NCI Open Database 250k+ compounds searchable by name, CAS numbers, formula, name, etc.
6) ZINC free database of commercially-available compounds for virtual screening
7) ICSD inorganic crystal structure database (dead)
Phil Evans gives a 1 hr and 15 minute demonstration of the program Scala noting the capabilities (or lack thereof), commands and even opens up the closed tabs! This is the second part of a presentation on scaling and merging. The scrolling on the video doesn’t work so make sure to pay close attention. The screen is a bit hard to see as well as the questions to hear, but don’t get discouraged the presentation contains a wealth of information.
If you don’t have the 1:15 to spend right now here are some highlights:
15 – can have problems scaling data sets with large dynamic ranges (1 sec exposure vs. 10 sec)
26 – goes through log graphs
46:50 – frustration
48:40 – goes through log files
If you are also having trouble with the scroll working then you could run the talk in the background without sound until the right time.
Keep in mind that this video was made in 2001 and updates may have fixed some of the issues mentioned.
The BindingDB collects experimentally determined binding affinities of proteins and ligands (small molecules) that bind via non-covalent interactions. This database can be convenient in that it cuts through reading entire papers, if you are only look for experimental results.
What type of enzymatic assays have been run?
What type of concentrations have been used?
What type of affinities can I expect?
BindingDB includes the basics such as searching by name and sequence via BLASTP. You can also draw the chemical structure of the ligand, if needed. If you need the information in a particular format the database contains a monomer list in csv and Het list in SDF.
You can also use this database to find substrates and competitors that were used as binding substrates. Isothermal Titration Calorimetry (ITC) experiments can be searched by pH range in KJ/mole or kcal/mole. The ITC values can also be searched by each component in Gibb’s free energy (free energy, enthalpy and entropy). The Ki, IC50, Kd and EC50 values are available and can be searched in combination with molecular weight.
The database also has an author table, allows users to upload experimental data and contains PubChem BioAssay aid (identification input) information. Often crystallographers consider bringing together biophysical techniques to study their system of interest. This database can serve as an excellent resource in determining what has been done and gain an idea of what values may be expected. Great Stuff.
What is the best search engine for finding research papers?
I am pretty sure there are elaborate methods of comparing search engines. I decided to go with the ‘can you find the paper I am thinking of…method.’ The paper entitled, “Data-collection strategies” by Zbigniew Dauter is a classic. I would highly recommend it to anyone who sees themselves being involved in any sort of crystallographic data collection.
The comparison will involve 3 separate searches (from easy to hard), followed by counting from the 1st result until the paper is shown. The 3 search terms used are as follows:
(1) data collection strategies dauter
(2) data collection strategies crystallography
(3) data collection crystallography
The results are numbered with 1st result being given 1, second 2, etc…
Notes: Hubmed sorts by publication date and since the paper was published in 1999 resulted in a quite low ranking. ISI Web of Knowledge for the (1) attempt the entry had to be split apart with Dauter being placed as an author input (or not results appeared). Also with Web of Knowledge, I had to changed default from date to sort by relevance.
Google Scholar was the best search engine in this case. I still a big fan of Hubmed for its web-related terms functions despite its poor performance in this test. I included a number of search engines that I never use for finding publications such as Yahoo. The interesting part is that Yahoo ended up returning a better result than Pubmed!
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.
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
The