Molecular crystal: all atoms can be described in terms of regular lattice. Number of possible space groups is 230

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1 Crystallization Primary difference between molecular crystal and protein crystal is solvent content. Protein crystals much less ordered than molecular crystals. Thus, diffraction data cann not be measured to resolution normally obtainable for molecular crystals. Molecular crystal: all atoms can be described in terms of regular lattice. Number of possible space groups is 230 Protein crystal: crystalline array coexists with high percentage (30 to 75) of liquid. Number of allowed space groups is 65. Growing molecular (inorganic) crystals Prepare hot, saturated solution and slowly cool. Polar organic cpds similar or slow precipitation from aq. solution with organic solvent. Does not work with proteins heat and organic solvent usually denature them.

2 protein crystals ~1mm subtilisin cellulase The color you see is birefringence, the wavelengthdependent rotation of polarized light.

3 Crystallization robot High-throughput crystallography labs use pipeting robots to explore thousnds of conditions. Each condition is a formulation of the crystal drop and the reservoir solution. Conditions can have different: protein concentration ph precipitant, precipitant concentration detergents organic co-solvents metal ions ligands concentration gradient

4 Protein crystal growth protein concentration precipitant concentration Arrows indicate different diffusion experients. A,B,D,F,G. Vapor diffusion. E. Bulk C. Microdialysis L=liquid S=solid m=metastable state (supersaturated) blue line = saturation of protein red line = supersaturation limit Crystal growth occurs between these two limits. Above the supersaturation limit, proteins form only disordered precipitate.

5 vapor diffusion setup a Linbro plate Volatiles (i.e. water) evaporate from one surface and condence on the other. Drop has higher water concentration than reservoir, so drop slowly evaporates.

6 Other ways to supersaturate slowly Sitting drops Microdialysis Gel filtration

7 precipitants A precipitant (r) causes proteins (p) to stick to each other by competing for solvent. p p p p r r r r r r r = EtOH, (NH 4 ) 2 SO 4, methylpentanediol, polyethylene glycol, etc

8 50 of the most successful crystallization conditions:

9 Crystallization theory Nucleation takes higher concentration than crystal growth. slow slow fast After nucleation, the large size of a face makes the weak bond more likely. not so slow fast

10 Periodic Bond Chain theory Bonds A,B are stronger than P,Q. Dimensions of crystal at equilibrium are proportional. More on Periodic Bond Chain theory:

11 Periodic Bond Chain theory Growth is unfavorable directions increases as the crystal grows. Weak bonds in Z favor growth in XY, forming plate xtal. Growing crosssection in XY favors growth in Z. atio of cross sections is inverse to ratio of bond strength.

12 diffusion depletion Crystal growth depletes the surrounding solution of protein, while concentrating impurities. Local depletion......prevents nucleations close to a growing crystal...slows and eventually stops crystal growth...concentrates impurities on the surface of the crystal...causes convection currents. Cobalt impurities in SiO 2 (amethyst) are concentrated in the part of the crystal that formed last (the tip).

13 Better crystals in micro gravity? Higher concentration of protein = higher density Differences in densty cause convection currents, which might cause crystal defects. Microgravity eliminates convection currents. More at:

14 Thin-walled glass capillaries (<1mm in diameter) are filled with mother liquor (the fluid in which the crystal was grown) and a crystal is carefully dropped in. The mother liquor is removed using filter paper cut to fine strips. The crystal sticks to the glass, immobilized. The xtal remains in vapor diffusion contact with the mother liquor. If not it will dryout and crack. mounting crystals Protein crystals are extremely fragile!!! They may break upon sudden contact with a solid object. Tiny pipets are used to pull crystals from drops.

15 Crystal mounting If not freezing If freezing (preferred) Xtal is mounted in a thin-walled glass capillary tube Xtal is mounted on a thin film of water in a wire loop. The loop is fixed to a metal or glass rod. Mounted xtal is attached to a goniometer head for precise adjustment. Low-melting hard wax is used to glue the rod or capillary here. wax Crystal must be kept at proper humidity and temperature!! Very fragile! Must freeze immediately or film will dry out.! Small wrenches fit here, here, here and here. eucentric goniometer head (made by Nonius)

16 Why freeze? Essentially eliminates X-ray damage to crystal. Crystals do not decay during data collection. Why not? wire-loop crystal catcher Cryo equipment is expensive. Ice crystals may form if freezing is not done properly, ruining data.

17 Crystals must be flash frozen...to prevent glass->ice transition Water must be frozen to < 70 C very fast to prevent the formation of hexagonal ice. Water glass forms. How? Crystals, mounted on loops, are flash frozen by dipping in liquid propane or freon at 70, or by instant exposure to N 2 gas at 70 C. hexagonal ice

18 Centering the crystal in the whoops it s off center. Fix it! beam xrays machine center is the intersection of the beam and the two goniostat rotation axes. Must be set by manufacturer! To place crystal at machine center, rotate ω and κ and watch the crystal. If it moves from side to side, it is off center. If it is off-center, we adjust the screws on the goniometer head.

19 Aligning crystal lattice with the beam. This is where the a* axis is pointed otate the crystal until the zero-layer disappears and the 1-layer is centered on the beam. misaligned aligned h=1 h=0 h=-1 h=1 beam is here Concentric circles around beam means axis is aligned with beam.