The Screening Techniques:

Footprint Screen #1

The footprint screens are based on the concept of screening the protein precipitant solubility curve . In the screen below screening is done at acidic, neutral and basic pH's for polyethylene glycols and salts:

Footprint #1
-

1

2

3

4

5

6
- PEG 600 PEG 4K PEG 10K A.S. PO4 Citrate

A

 15% 10% 7.5% 0.75M 0.8M 0.75M

B

 24% 15% 12.5% 1.0M 1.32M 1.0M

C

 33% 20% 17.5% 1.5M 1.6M 1.2M

D

 42% 25% 22.5% 2.0M 2.0M 1.5M
- 0.2M imidazole
malate
pH 5.5		 0.2M imidazole
malate
pH 7.0		 0.2M imidazole
malate
pH 8.5		 0.15M sodium
citrate
pH 5.5		 NaH2PO4
K2HPO4 pH 7.0		 10mM sodium
borate
pH 8.5

PEG Screen Footprint type screen #3.1

Footprint #3.1
-

1

2

3

4

5

6
- MPEG 550 PEG 600 MPEG 2K PEG 4K MPEG 5K PEG 10K

A

 30% 18% 18% 8% 12% 9%

B

 40% 27% 27% 15% 18% 15%

C

 50% 36% 36% 20% 24% 22.5%

D

 60% 45% 45% 30% 36% 27%
- 0.1M HEPES
pH 8.2		 0.1M HEPES
pH 7.5		 0.1M sodium
cacodylate
pH 6.5		 0.2M imidazole
malate
pH 6.0		 0.1M sodium
acetate
pH 5.5		 0.1M ammonium
acetate
pH 4.5

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Hampton Research Screens

Reverse Screening:

Stura E.A, Satterthwait, A.C, Calvo, J.C, Kaslow, D.C, Wilson, I.A. (1994) Reverse Screening. Acta Cryst. D50 : 448-455.

Abstract: A major emphasis has been placed in recent years on ``kits'' for screening crystallization conditions of macromolecules. Such approaches have undoubtedly speeded up the initial screening and to a certain extent helped in reducing the protein required for the initial survey. Factorial screening techniques, either full factorial or sparse matrix approaches, have proved successful in the crystallization of many proteins. In cases where the amount of protein is limited a systematic approach based on a priori choice of precipitant may be preferable to an extensive search. The approach described here targets such situations.The approach consists of an initial search to determine the solubility characteristics of the macromolecule under study as a function of precipitant and macromolecule concentrations. This information is used to define a working range for the precipitant and protein concentrations. Changes in solubility as a function of pH and as a result of the introduction of additives are evaluated at a later stage. This analysis is important for establishing conditions under which the protein is highly supersaturated and hence more conducive to nucleation, one of the dominant problems that hinders successful crystallization of proteins. When ligands are available for the formation of macromolecular complexes, the screening of the different complexes of a particular macromolecule is advocated as a means to increasing the probability of nucleation. Solubility information derived from one complex that crystallizes may be used to aid in the crystallization of other complexes. Cross-seeding between complexes is an intrinsic part of the method and provides an efficient way of obtaining crystals where spontaneous nucleation is hard to achieve.

Refining Crystallization Conditions

Updated/Created on Oct-8-97 by EAS, TSRI