Growing Larger Protein Crystals: Some Insights From Fundamental Studies

Robert E. ThorneCornell University

Alexander MalkinLLNL

This work was supported by

Why Grow Larger Protein Crystals?

• Larger crystals reduce the radiation dose/volume required to obtain a structure.

• Flash cooling almost always degrades crystal order and diffraction resolution, and always degrades mosaicity.

• Viruses and large macromolecular complexes often freeze poorly (resolution MW1/3?).

• Flash cooled crystals are less suitable for time resolved and mechanistic studies.

• To collect the same amount of data at room T, the linear crystal dimension must be increased by 3 - 10

(5 - 30 if one dimension is fixed).

Obstacles to Obtaining Larger Crystals

1.Crystals develop cracks, twins, and other macroscopic defects as they grow larger.

2. Crystals stop growing.

3. Excessive nucleation depletes protein.

Problem 3 can be solved by careful control of growth conditions and by seeding. What about problems 1 and 2?

Q: Why Do Crystals Develop Cracks and Other Defects as they Grow Larger?

A: Because impurities incorporate nonuniformly within the crystal.

I. Sectorial Nonuniformity

• A crystal habit is defined by the growth faces, e.g., (101), (110), (111)

• A growth sector is the region of the crystal formed by adding molecules to a particular facet.

(101) facet

(110) growth sector

(110) facet (101) growth sector

Impurities preferentially stick to certain faces.

The impurity density and average lattice constant vary between growth sectors, creating stresses.

These sectorial stresses grow with crystal size.

Once these stresses reach a critical value, the crystal cracks and/or develops other defects to relieve stress.

II. Radial Nonuniformity

• Impurity incorporation increases with growth rate.

• Growth rates are largest just after nucleation.

Impurity Incorp-oration

rate

Growth rate

Growth rate

time

• Crystals tend to have impurity-rich cores.

Radial gradients in impurity density create stresses that drive crystal cracking and formation of polycrystals.

Impuritydensity

Radial distance from core

Q: Why Do Crystals Stop Growing?

A: Because impurities contaminate the crystal surface, and prevent molecular attachment and

growth.

• Impurities adsorb onto the growing crystal surface.

• Supersaturation and growth rate decrease as the mother liquor becomes depleted.

• Impurity coverage increases with decreasing growth rate.

• When the surface impurity density is large enough, growth step motion and growth cease.

• A minimum supersaturation required to sustain growth, determined by the impurity concentration.

• Due to macromolecule degradation, the effective impurity concentration increases with time during growth.

GrowthRate

supersaturation

no impurities

0supersaturation

GrowthRate

With impurities

0

time

Atomic force microscopy (AFM) images of impurity contaminated surfaces:

Removing the impurity layer allows growth to resume.

• Once growth has stopped, increasing the super-saturation usually will not cause growth to resume.

Conclusion

• Impurities present in growth solutions are the most important factor limiting the size of macromolecular crystals.

Conclusion

• Impurities present in growth solutions are the most important factor limiting the size of macromolecular crystals.

Your Response?

Conclusion

• Impurities present in growth solutions are the most important factor limiting the size of macromolecular crystals.

Your Response?

• Demand a bigger slice of the pie from your crystal-growing colleagues.

Conclusion

• Impurities present in growth solutions are the most important factor limiting the size of macromolecular crystals.

Your Response?

• Demand a bigger slice of the pie from your crystal-growing colleagues.

• Specific growth strategies: see Poster 45 or email ret6@cornell.edu

To reduce cracking and defects due to sectorial impurity concentration differences:

• Purify the growth solution.

• Refresh the growth solution to minimize degraded protein “impurities.”

• Reduce sectorial concentration differences by modifying solution chemistry, or by trying other crystal forms/habits for which the exposed facets are more nearly chemically equivalent.

To reduce cracking and defects due to radial concentration differences:

• Purify the growth solution, and use freshly purified solution.

• Use slower and more controlled supersaturation increases to allow sufficient time for nucleation to occur at lower supersaturations, reducing initial growth rates.

• Macroseed into solutions that produce slower growth rates.

Purify enough protein to make ~30 m seeds and then macroseed into less pure solutions.

To prevent impurity-induced growth cessation:

• Purify the growth solution, and use freshly purified solution.

• Reduce degradation by removing enzymes, lowering growth temperatures to 4°C, adding preservatives and reducing the time between drop setup and nucleation.

• Keep the supersaturation ahead of the knee in the growth rate curve. Add fresh solution, change the well solution or change the temperature.

• Since degradation products accumulate in the original solution, macroseed to a fresh solution. Only use seeds that are still growing.

If growth cessation has occurred:

• Try a relatively short period of undersaturation to remove adsorbed impurities and then seed into a fresh saturated solution. Periodic cycles of undersaturation followed by longer periods of supersaturation may revive and sustain growth.

• To reduce formation of inclusions and microcrystallites, after etching away impurities in an undersaturated solution, transfer the crystal to a modestly supersaturated solution that just sustains growth, and then increase the supersaturation to obtain a desired growth rate.