P212121

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.