So, you've got something that looks like it might be a crystal, or could become one; what's next?
SCALING UP - FEWER DROPS
When optimizing you will probably use fewer conditions as you are focusing on a particular part of phase space. In addition you will probably be scaling up to larger drops to try and grow bigger crystals.
When scaling-up from a 96-well plate, be aware that the dynamics of the experiment will change. Scientists at the MRC in Cambridge came up with the MRC Maxi plate to help combat this problem. Scale up your experiments by hand, or using a robot.
You might want to set a few drops at a time, then develop your strategy by screening additional conditions. With QuickTear ClearVue™ perforated strips you can seal just 2, 4, 6 or 8 rows at a time, leaving the others free for later use and refined conditions.
WHAT TO TRY
Start by checking the formulation of your hit. Then you can work out a strategy for what to vary to try and improve your crystal. The most common things to try changing are:
- Protein concentration
- Precipitant concentration
- Salt or other additive concentration
- Try switching the anion or cation by moving along the Hofmeister series
- Vary the sample to reagent ratio. Eg. If you started with 1:1, try 2:1, or 1:2
Molecular dimensions stock solutions provide piece of mind that you are working with the same solutions that produced your initial hit. If you are optimizing from an initial Morpheus® hit, you can get the constituent mixes for optimization here for Morpheus® I and here for Morpheus® II.
There are many small molecules that can be added to a crystallization conditions to try and increase the size and quality of your crystals. At molecular dimensions we sell a number of screening kits that make it easy to find out if any of these improve your crystals. Simply pippette your hit condition across the whole plate and then introduce a different additive to each well. Molecular Dimensions Single Reagents make replicating the initial condition easy. Our additive screening kits include:
- The ANGSTROM Additive Screen™ - Contains a range of polyols to improve crystal quality and simultaneously cryoprotect. Designed by Fabrice Gourrec at the LMB, Cambridge, this screen has been exclusively licensed to Molecular Dimensions.
- The Morpheus® Additive Screen - Developed by Fabrice Gourrec, contains precipitants, stabilizers, nucleants, cryoprotectants, surfactants and even heavy atoms for phasing in one screen.
- CryoProtX™- From Enrico Stura and Laura Vera at SEA, Saclay. These mixes balance protein preciptating and solubilizing agents to allow cryoprotection without affecting crystal quality.
- CryoSol™ - Ideal for fragment-based drug design projects. These solutions balance protein stability with the ability to dissolve hydrophobic ligands. They allow you to carry out soaking or co-crystallization experiments without degrading X-ray diffraction quality.
- The CALIXAR™ v2.0 Additive Kit - Calixar's can form supra-molecular micellar type structures that facilitate protein-protein interactions. They can improve crystal quality or stabilize membrane or flexible multidomain proteins.
- MemAdvantage™ - An additive screen developed specifically for membrane proteins and including detergents, polyols, organics and amphiphiles.
- RUBIC Additive kit - A protein stability screen to identify small molecules including salts, chaotropic reagents, amphiphiles, polyols, amino-acids and carbohydrates that may stabilize your protein and enhance its crystallizability
- The Molecular Dimensions Additive Screen - An ideal starting point. Sampling examples from different additive types, this screen can give an indication of which factors are most likely to improve your crystal quality.
THE 3 'F' SOLUTION
Polyvalan's Crystallophore No1(patent pending) - A luminescent Terbium complex that is an effective nucleant and provides significant isomorphous and anomalous phasing power. The only single compound that solves three problems at once.
Forming: Growing a crystal capable of producing high quality diffraction data
Finding: Identifying crystals in drops quickly and accurately
Phasing: The f"~30 electrons for the Tb3+ LIII edge at 1.6 Å or 7.5 KeV
One of the most common problems with initial hits and optimization is that there are either clear drops or drops with a showers of microcrystals, but no drops with just a few crystals that are suitable for data collection. Seeding can help in this situation. There are many different ways to seed - as described in these tips from Terese Bergfors. But simply put, to seed you reduce the precipitant or protein concentration to a metastable point where the drops are all clear, after allowing some time for equilibration, then add the seeds.
Seeds can be a few microcrystals or a pieces from a large crushed crystal. Alternatively, hetero-seeding involves introducing a nucleant other than the protein. Try Microseed Matrix Seeding (MMS) as described in D'Arcy et al (2003) with our kit. Alternatively, we supply Naomi's Nucleant - which has promoted the crystallization of at least 14 different proteins and is described in Saridakis and Chayen (2009). Fine tweezers and microprobes are useful for manipulating seeds.
An alternative approach to the problem of too many crystals in a drop is reduce the number of nucleation events. This can be done by using an additive to 'poison' nucleation: ethanol and dioxane are often used as additives for this purpose. Alternatively, the precipitant concentration can be reduced after a time, so that there is initial nucleation period and then a growth period in the metastable zone. The 3D Structure Screen provides the necessary solutions to do this. Finally, the number of sites for nucleation can be reduced using a 'containerless' crystallisation method, such as that provided in our Gelled Surface Crystallization™ kit. In this case the protein drop sits between gel and oil removing the possibility of nucleation on the vessel wall.
TROUBLE REPLICATING THE INITIAL HIT?
How did the manufacturer pH the buffer? This can vary from screen to screen, and in some cases within the screen. PEG stocks can oxidise rapidly and significantly alter not only the chemical composition but also the pH of the solution. The molecular weight distribution for each PEG solution can also vary by supplier. Always check the data sheet. If you have any questions about our screens, please contact us on firstname.lastname@example.org