P212121

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.

Protein crystallization is influenced by a number of variables. Initially, one must decide what type of setup will be used when attempting to grow a protein crystal.

A common method is hanging drop, which utilizes a process called vapor diffusion. The benefit of vapor diffusion is that it allows for the increase of protein and precipitant concentrations. The concentration is increased due to vapor leaving hanging drop to reach equilibrium with its surroundings.

Hanging Drop Setup:

Note: This setup does not allow for evaporation to take place although it may be used to encourage crystal growth. The cover slip is often sealed in place using vacuum grease.

The contents of the hanging drop includes your protein with its buffer, well solution and sometimes water. A common setup is to use 2 µl of protein solution + 2 µl of well solution, suspended over 998 µl of well solution (the 2 µl are added from the well solution to the hanging drop).

10 questions to consider before setting up protein crystallization trials:

1) How pure is your protein sample?

2) What chemicals are present in the protein buffer?

3) Is the protein modified such as phosphorylated, glycosylated or methylated?

4) What temperature and pH is the protein stable?

5) What concentration causes the protein to precipitate out of solution?

6) Does the protein have any known substrates, metals, inhibitors or ligands?

7) Have related proteins been crystallized?

Is the protein sensitive to proteolysis?

9) Does the protein have cysteines?

10) How has the protein been stored?

Feel free to add any questions that you consider in the comments.

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.

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.

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.

Crystal! Sweet.

The next step is to determine whether the crystal is comprised of salt or protein. Here is a list of methods to help you determine whether you are dealing with a salt or protein crystal.

1) Prediction Tools - can be used to determine if the drop components have the possibility of forming a salt crystal.

2) Birefringence – is a sign of salt crystals although it is not visible in protein crystals that are cubic. A set of polarized lens are needed to check for to see if the crystals are birefringent.
Look for salt crystals to have strong birefringence while proteins are weaker

3) Dyes - such as methylene blue are able to enter large solvent channels of proteins that are not present in salt crystals
Look for dye to be absorbed into the protein crystals and not salt

4) Dehydrate - by removing the crystal from the drop.
Look for salt crystal to maintain its structure while protein to turn to mush

5) Crush - the crystals using a small tool. Protein crystals are softer than salt crystals due to their crystal packing. It is helpful to try this technique with a number of known samples to gain experience. Tip: Wick away excess solvent to save time
Look for protein crystals to collapse when compared to that of salt

6) Glutaraldehyde - can be added to your drops (~1 %), if protein is present the drops will turn a yellowish color. Since glutaraldehyde is quite volatile it may also be added to the reservoir, if you decide to go this route then use ~2 % concentration. Any free amines will turn yellowish so watch out for the presence of other protein or components in your buffer such as TRIS.
Look for a protein crystal to turn a yellowish color

7) SDS-PAGE - can be used by dissolving your crystal (or the entire drop)
Look for: the correct electrophoretic pattern

Control experiment - can be done (this is science) by setting up the exact same condition without your protein.
Look for motivation

9) Crystal shape - alone can be very misleading between salt and protein crystals. However, I have noticed the wings of death crystals (magnesium phosphate, in this case) appear quite distinct.
Look for wings of death

10) X-ray diffraction - although obvious, it is not possible for everyone. If you have easy access then this is the only definitive method out of all the methods mentioned.
Look for salt crystals have fewer reflections (larger lunes) compared to protein crystals

The Marseille Protein Crystallization Database (MPCD) combines the Biological Macromolecule Crystallization Database (BMCD) (post) and the IBS Conditions Yielding to Crystallization Of Proteins Database (CYCLOP) (no longer online) databases (ref).

CYCLOP contains 3,157 crystallization conditions of 1,726 different proteins while the BMCD (v.2.0) contains 2,220 crystallization conditions of 1,485 different proteins.

Unfortunately, that means the MPCD does not include the most recent version of the BMCD, which contains 14,372 crystal entries. According to the 2006 publication, the MPCD contains 5376 crystal entries. Despite being smaller, the MPCD contains a large array of unique search options for crystallization information that are quite useful.

The database includes information about the macromolecules themselves such as name, pI, molecular weight and number of subunits. In addition to crystallization conditions, methods and additives, which are presented in an easy-to-compare table form.

To use the database click on ‘Consult’.

Example of Results: Here are a couple of the 104 conditions that contain PEG 3350:

The MPCD also allows users to upload their own crystallization information by clicking on the Connect option.