1. One 24-well plate requires ~0.5 mg of PTP1B, which crystallizes at a concentration of 10 mg/mL. More protein may be needed depending on the number of crystallization conditions that will be tested.

2. EDTA is insoluble in solution until the pH reaches approximately 8.

3. Hydrogen peroxide is light sensitive and will degrade over time. It should be stored in the dark and purchased in small quantities. For these procedures it is recommended that the H2O2 is not more than 6 months old. The reagent can be tested following the procedures in Section

4. DTT is a reducing agent. It will react with cysteine residues within a protein and keep the cysteine residues in a thiolate anion state (see Table 8.1). It is more susceptible to oxidation once it is dissolved in aqueous solutions and should therefore either be prepared freshly on the day of use or stored in aliquots at -20°C.

5. Even within a single crystallization well there is variability in the quality of the crystals. Preparing several crystals at a time improves the probability of finding a crystal that will diffract well in the crystallography experiment. In addition, the crystals will be transferred to several different buffers and can be damaged or lost as they are transferred. Preparing several crystals at once requires minimal extra time and again improves the probability of having a high quality crystal for the x-ray diffraction experiment.

6. The concentration of PEG in the crystal wash buffer, the crystal oxidation buffer or the crystal reduction buffer should match or slightly exceed the concentration of PEG that was present in the well solution while the crystal was growing. For example, crystals that grew in 12% PEG 8K should have at least 12% PEG 8K in the various buffers.

7. Glutathione can become oxidized rapidly once it is dissolved in solution. This oxidation also occurs slowly in the solid state. Liquid solutions should be made up freshly on the day of use. It is recommended that the bottles of glutathione not be more than 1 yr old.

8. The appropriate cryoprotectant buffer is dependent upon the conditions that were used to grow the crystal. For PTP1B crystals that grow in relatively high PEG concentrations (16-17%) a cryoprotectant buffer with 20% PEG is more suitable. For those that grow in lower PEG concentrations (12-15%) a cryoprotectant buffer with 18% PEG is more suitable.

9. While freezing crystals, it is convenient to be able to check that the crystal is in the cryoloop and to check how well it diffracts. However, if no cryostream is available, the crystals can be frozen and stored directly in the liquid nitrogen without the need for a cryostream or goniometer head. If the crystals are being frozen immediately prior to data collection they can be frozen in the cryostream and data collection can be initiated immediately.

10. This assay can also be used to test different concentrations of hydrogen peroxide or different times of oxidation.

11. The concentration of p-NPP can be adjusted such that the absorbance at 405 nM is detectable and linear for most of the 5-min assay.

12. If the spectrophotometer is not set up to continuously record the absorbance versus time then the absorbance can be recorded at 30-s intervals.

13. If a change in absorbance at 405 nm is observed for the sample with H2O2 (i.e., the protein is still active even though H2O2 was added), then it may be necessary to use a longer oxidation time or to use a fresh supply of H2O2. If no change in absorbance at 405 nm is observed for the sample without H2O2, then it may mean that the protein sample is not properly reduced. The assay could be repeated after treating the sample with freshly made 5 mM DTT. If there is still no observable change in absorbance for the sample without H2O2, then a fresh protein sample may need to be prepared as the catalytic cysteine may have become oxidized to a sulfonic acid.

14. Proteins are usually purified with reducing agents present, but the reducing agents need to be removed before the addition of hydrogen peroxide. This dialysis step removes the reducing agents. A buffer exchange column could also be used for this step.

15. The selection of a ratio of 1:1.25 for the ratio of PTP1B to H2O2 is to ensure that there is enough H2 O2 so complete oxidation can occur, but to prevent the further oxidation of the PTP1B catalytic cysteine to a sulfinic or sulfonic acid, or of non-catalytic cysteine and methionine residues.

16. Oxidized PTP1B forms precipitates in solution more rapidly than reduced PTP1B. At 8.5 mg/ml there was a small amount of protein precipitation, but the majority of the protein was still soluble. This precipitate was filtered and the protein was immediately placed into crystallization trials. Reduced PTP1B can be crystallized at a higher concentration (10 mg/mL).

17. Bradford protein assays require a smaller volume than measurements of the protein concentration at 280 nm and are convenient for checking protein concentrations if only a small amount of protein is available.

18. Some proteins such as PTP1B crystallize at 4°C whereas others crystallize better at greater temperatures. For proteins that crystallize at room temperature this step can be omitted.

19. The timing of crystallization and data collection is important. Sufficient oxygen from the air can enter crystallization wells and cause the reversible sulfenamide bond to become oxidized to a sulfonic acid. It is therefore important to freeze the crystals and collect data in a timely fashion to trap the readily reducible oxidized intermediate.

20. To trap PTP1B in the sulfenamide bond state, the crystals were soaked in a small volume of 4 pL with 20 pM of H2O2. This was done to limit the availability of H2O2 to prevent oxidation of the catalytic cysteine of PTP1B to sulfonic acid. The selection of 20 pM was determined based on estimating the total amount of PTP1B in the crystallization drop. After diluting the protein 1:2 in crystallization well buffer the final PTP1B concentration was 5 mg/ml. The molecular weight of the PTP1B catalytic domain is (37 kDa) which made the concentration of PTP1B in the crystallization drop approximately 130 pM.

21. Glass coverslips can crack easily as they are removed. This can be minimized by gently rotating the coverslip to loosen the grease seal before prying it off. Also, although the external appearance of a protein crystal does not always correlate to how well it will diffract, larger regular shaped crystals tend to give a better diffraction pattern and were chosen at this step over smaller less regularly shaped crystals.

22. Four crystals were found to be suitable for this procedure. Preparing more crystals at this step may enhance chances for finding a crystal that diffracts well. However, adding too many crystals at this step may leave insufficient hydrogen peroxide for complete oxidation to the sulfenamide state. A third microbridge with an additional 4-pL drop of crystal oxidation buffer could be used to oxidize four additional crystals.

23. Cryoprotectant buffers help to minimize ice formation and crystal damage during the freezing process. For more information on selection and optimization of cryoprotectant buffers, see refs. (16-18).

24. Mounting and dismounting crystals is a user- and facility-dependent procedure and detailed protocols are not presented here. Common procedures are the cryotong method (Hampton Research) and the detachable extended arc method (Molecular Dimensions Limited).

25. Handling of the crystals should be minimized as much as possible. To prevent the presence of excess cryoprotectant buffer surrounding the crystal, it is advantageous to pull the cry-oloop from the cryoprotectant buffer such that the cryoloop is perpendicular to the meniscus of the solution. Also, the crystal should ideally be placed in the center of the cryoloop.

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