Crystallization and Heavy Atom Screening
Structural Molecular Biology Laboratory, ChemM230D, Room BH 269

A successful crystallization trial

Background Reading/Viewing 1) Pre Crystallization Lab Video. Michael Sawaya 1/5/2021 2) What a crystallographer should know about a protein before beginning crystallization trials by Michael Sawaya.  3) Heavy atom Safety, Selection & Screening by Michael Sawaya. 4) Screening for Phasing Atoms in Protein Crystallography by Boggon & Shapiro Structure 8, R143-R149 (2000). 5) Powerpoint presentation on crystallization and lab methods.

Laboratory Procedures

Part One: Crystallization Trials

Objective: To crystallize proteinase K and its complex with phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor.  You will optimize an initial set of crystallization conditions discovered earlier by sparse matrix screening kits.

Method: Hanging drop vapor diffusion.  Full matrix screening.

Caution: PMSF is extremely destructive to the mucous membranes of the respiratory tract, the eyes, and skin.  It may be fatal if inhaled, swallowed or absorbed through the skin.  In case of contact, immediately flush eyes or skin with copious amounts of water.  Solutions of PMSF can be inactivated by rendering it alkaline for several hours at room temperature.

Materials: 
1) 5 mg of Proteinase K (ProK) from Tritirachium album purchased from Sigma (cat. no. P2308).
2) 5 uL of 100 mM PMSF. 
3) 4M ammonium sulfate (NH₄)₂SO₄
4) Tris buffer pH 7.0 and pH 8.0
5) Distilled water
6) Greased VDX crystallization plates, 24-wells (Hampton Research (cat. no. HR3-140).
7) Siliconized glass cover slips (Fisherbrand Microscope Cover Glass 22x22-2 cat. no. 12-540B)

Proceedures:
1) Work in groups of 2-3 people.  Each group sets up 12 crystallization conditions for the native ProK (rows A & B of the tray) and 12 crystallization conditions for ProK-PMSF complex (Rows C & D).

2) Dissolve 5 mg of ProK (supplied in 0.5 mL eppendorf tube) in 100 uL of distilled water. Using pipet tip, stir gently to dissolve the protein. No bubbles! The final concentration of protein should be 50 mg/mL.

3) Prepare PMSF complex by pipeting 50 uL of the 50 mg/mL ProK solution into the tube labeled PMSF. This tube contains 5 uL of 100 mM PMSF dissolved in isopropanol.

4) Prepare reservoir solutions:  Pipet the indicated amount of reagent to each of the 24 wells.  [(NH₄)₂SO₄] concentration varies horizontally across a row.  The pH varies vertically down the column. The volume of each reservoir is fixed at 500 uL. Be sure to mix each reservoir thoroughly when you have finished pipetting.  You may do this by gently swirling the tray in a circular motion.

5) Prepare drops:  (a) Place a row of 6 coverslips on the top of the tray lid, at the edge of the lid.  (b)With the P-20 Pipetman, pipet 2.5 uL of the proteinase K solution onto the middle of each cover slip.  Use a steady hand to keep the drop in the form of a nice round bead. The coverslips have been siliconized in order to enhance bead formation.

How to avoid blowing bubbles in the drop: Normally, you expel liquids from the Pipetman by pushing the plunger to the second stop.  The distance between the first stop and the second stop blows air through the tip which ensures that all the liquid is expelled.  Unfortunately, this feature also blows bubbles in the drop you are forming on the coverslip. You can avoid blowing bubbles by pushing the plunger to the first stop only.  If you do get a bubble in your drop, you may remove the bubble by holding the pipet vertically and just touching the top of the drop. 

(c) Pipet 2.5 uL of reservoir A1 onto the first protein drop. The protein and reservoir will mix by convection; there is no need to mix with the Pipetman.  Mixing just increases the likelihood of smearing the drop.  (d) Add reservoir A2 to the second drop, etc. (e) Invert the coverslips onto their respective reservoirs.  If you haven't done this before, first try inverting a coverslip carrying a drop of water.  Keep the coverslip perpendicular to the acceleration, and banked a little.  Be careful to align the coverslips to the edges of the plastic tray.  Gently press the coverslips onto the grease seals around the edges using the point of a pipet tip.  It is best not to use your fingers because they get greasy and smear the glass, obscuring visibility of the crystals. Don't press directly in the center of the cover slip. (f) Repeat steps a-e for row B.  (g) Remember to use the ProK-PMSF complex for row C and D.

6) Label your tray with your names, the date, and the protein name using a Sharpie.  Crystals will appear in a few hours and reach full size overnight.

7) Next week: One group will collect data on the native crystal.  Another group will collect data on the PMSF complex.  All others will collect data on potential heavy atom derivatives. 

Interested students are invited to return on Monday to room BH269 for 10 minutes so that they can soak their crystals in heavy atom solution.  You can make an appointment with Mike for any time between 11:00AM-5PM by printing your name on the sheet posted on the lab door.

Part Two: Preparation of Heavy Atom Derivatives

Objective: Based on the results of the native gel band shift assay select a heavy atom and soak it into crystals for derivatization.

Procedures:
1) Look for a band shift in one of the lanes containing a heavy atom.  If the heavy atom complex is cationic, then the protein will have a higher mobility in the gel (remember, the gel is run with reverse polarity).  Lanes in which no protein appears are the result of aggretagion caused by the heavy atom, and should not be considered further for derivatization.

2) Select the heavy atom which you are most confident caused a shift.

3) Prepare a 1:1 and 1:5 dilution of the selected heavy atom.

4) Select three drops with nice crystals from the tray of proteinase K crystals that you grew. 

5) Add 1 uL of concentrated heavy atom to drop 1.  Add 1 uL of 1:1 diluted heavy atom to drop2.  Add 1 uL of 1:5 diluted heavy atom to drop 3.

Instructor's preparations

Back to CHEM M230D course syllabus 

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