Growing Protein Crystals

Contents

• Pre-lab Exercise
• Introduction
• Materials
• Hazards
• Procedures
• Study Questions
• Report

This experiment is based on "A Novel and Innovative Biochemistry Laboratory: Crystal Growth of Hen Egg White Lysozyme,", Elizabeth Garrett, Audrey Wehr, Rebecca Hedge, David Roberts, and Jacqueline R. Roberts, Journal of Chemical Education, 79, 366-368, 2002. Thanks to the authors for permission to provide the procedures, suggestions, figures, and questions on this page.

Background Reading

Look up "crystallography", "x-ray crystallography", and "x-ray diffraction" in the index of your biochemistry textbook, and read all entries.

Important Note About Pre-lab Exercises

You MUST turn in pre-lab assignments at the beginning of the pre-lab meeting in order to receive any credit.
The point of each exercise is for you to invest some thought in the upcoming experiment BEFORE you come, and thus come to lab better prepared. You will get a chance to correct your pre-lab exercises only if you hand them in on time.

Pre-lab Exercise (Due at first lab meeting on this project)

BEFORE COMING TO LAB, write a procedure for preparing 100 mL of 1.00 M sodium acetate buffer, pH 4.50 from glacial acetic acid (density = 1.049 g/mL) and sodium acetate. Use 4.80 as the pK_a of acetic acid. Assume that the user of your procedure has a pH meter for checking or adjusting the pH. Best marks for the simplest and most reliable procedures. Copy the procedure into your lab notebook before coming to lab, and

PREPARE A NEAT, HANDWRITTEN OR TYPED COPY TO HAND IN AT THE BEGINNING OF THE PRE-LAB PERIOD.

During the first lab session, you will use your procedure to prepare a buffer for use in growing crystals.

Introduction

The first step in determining the structure of protein molecules by x-ray crystallography is to grow crystals of the protein. For most proteins, if you cannot grow crystals, you will never know the structure of the protein. Crystal growth is part science, part art. The researcher finds optimal growth conditions by systematic trials.

A widely used means of growing crystals is called the sitting-drop vapor-diffusion method. A drop of protein solution containing a low concentration of a precipitant (such as a salt) sits in a well above a reservoir containing a higher concentration of the precipitant (see Figure 1 in Procedure). The exchange of water vapor between drop and reservoir (vapor diffusion) results in shrinkage of the drop. This shrinkages raises the protein concentration, and also raises the concentration of precipitant in the drop toward the concentration of precipitant in the reservoir. When protein concentration exceeds the solubility of the protein in the drop, protein precipitates. If the protein is pure and precipitation is slow, crystals of the protein may form and grow. Under less optimal conditions, the protein may precipitate as an amorphous solid or gel, which is useless for structure determination.

For this project, you will receive the materials for growing crystals of lysozyme, along with suggested ranges of conditions. Your goal is to optimize the growth conditions for the materials provided to your team. Your finished products will include lysozyme crystals, photos of the crystals, and a written procedure that gave your best results.

• At the first lab meeting, prepare the acetate buffer according to the procedure you devised in the Pre-lab Exercise. Then set up your first trials.
• Check your trays for crystals as often as possible. Sometimes crystals form in a few days, and then conditions deteriorate and crystals redissolve, or additional small crystals form.
• At the second lab meeting, make crystal-quality assessments and set up the second trials.
• In the following weeks, work on this project at your convenience, either during lab meetings or at other times approved by your instructor.
• Check the course calendar to find out when your final report is due.

Materials 

Reagents

Lysozyme, 100 mg/mL in water (a fresh batch will be available each Monday)
Precipitant, 100 g (each teams gets one of the following salts: NaCl, NaBr, NaI, or NaNO₃)
Glacial acetic acid (See Hazards, below.)
Sodium acetate

Supplies

Crystallization trays (24-well plates)
Clear tape for covering trays

Equipment

Microscope
pH meter and electrode
Magnetic stirrer and stir bar
Digital camera (borrow from instructor when needed)

Hazards

Glacial acetic acid causes severe burns. Avoid contact with skin. Wash spills with lots of water.

Procedures

Preparation of the Crystal Tray

Figure 1 illustrates the crystallization plate and the contents of a well. The reservoir surrounding the pedestal contains the buffer/precipitant solution (0.500 mL). In the depression at the top of the pedestal is the "sitting drop", which contains equal amounts of protein (3 µl of lysozyme solution) and the precipitant solution (3 µl) from surrounding reservoir.

 Figure 1

Wells are sealed with clear plastic tape, making each well and its pedestal a closed system where vapor diffusion can occur. Two lengths of tape running lengthwise are sufficient to seal and isolate all wells.

Initially, your group will receive three crystal trays to complete the project. This number is limited to encourage you to make decisions rationally and systematically, as opposed to just attempting every possible combination. Drawing a diagram in your notebook with a representation as given in Figure 2 is an excellent means of recording results.

 Figure 2

Each student group will be assigned one precipitant. Table 1 provides a list of concentrations ranges for each precipitant. All conditions employ an acetate buffer system at pH 4.5. To somewhat limit the variability for this experiment, the pH of the buffer system is held constant. In broader searches for crystallization conditions, you could vary pH also, as well as temperature.

You are responsible for making all of the appropriate stock solutions. A good plan is to make a supply of your buffer at its maximum concentration, and then plan to dilute with water in each well to achieve lower buffer concentrations. One way to achieve proper salt and buffer concentration is to weigh quantities of salt for each well, transfer the solid to the well, then add buffer, and if needed, water to obtain the correct buffer concentration and make up the final reservoir volume. Assume that the salt does not increase the final volume.

NEW FOR 2007: Here's an Excel spread sheet that computes suggested conditions for your first trials, along with quantities needed to make up solutions. These conditions are suggestions only, but they conveniently provide rough coverage of the full range of your conditions. I recommend that you first calculate a set of conditions and quantities by hand, and then use the spreadsheet to check your work.To use the spreadsheet for your assigned precipitant, replace the zeros in the appropriate labeled spaces with the minimum and maximum concentrations of each component.

After you have added all the buffers to the correct reservoirs, you are ready to prepare each drop. Into the depression on the top of a pedestal, pipette 3 µl of the appropriately diluted lysozyme solution. With the same pipette tip, dip into the reservoir that is being set up, remove 3 µl of precipitating buffer, and add this to the protein. Shake the tip to clear it completely before moving to the next well. Repeat this procedure until all of the drops have been set up. Move quickly so that early drops do not have time to evaporate before all wells are sealed. Small drops evaporate rapidly.

IMPORTANT NOTE ABOUT CLEARSEAL TAPE: When cutting the tape, don't let the roll end fall back against the roll! If you do, you will spend a long time getting it unstuck. (But you knew that.)

When you have set up all wells in the first two rows of the plate (rows A and B), cover these two rows with one piece of clear tape, making sure to get a good seal around every well. Continue setting up rows C and D, and seal them in the same way.

When finished, look at each well under the microscope to see (and record!) if anything has happened (Note if any precipitate has formed already). Set the tray in a location where there will be minimal disturbance due to vibration or temperature changes (ask your instructor). Check on the tray as often as you can, daily if possible. Even if good crystals appear and then disappear or deteriorate, this is an accomplishment, because if you were growing crystals for structure determination, you could harvest them when they are at their best. (Removal of crystals to sealed capillary tubes with their mother liquor usually stabilizes them for a time.)

Getting Better Crystals

Remember that the goal of this project is to grow the "best" crystals you can. Because there are so many different possibilities for protein and reagent crystallization, we limit your choices in order to help increase your chances of success. Each team should vary the concentration of precipitating agent, protein, and buffer. Even with these three variables, the possibilities are numerous. Remember that varying these conditions will affect crystal size, shape, definition, and the number of crystals produced. The best condition will yield the fewest crystals (one crystal is best) of good size and good definition (not a shapeless blob, but having with a very defined shape, like a cut gem). Once you find the best condition, repeat the condition to see if it is reproducible (it takes more than one crystal to solve a structure, so one must be able to make more).

General Hints and Suggestions

• Choose your conditions wisely. Changing too many variables at once may lead to incorrect assumptions and will not allow you to successfully interpret results.
• Start in the middle of the suggested ranges of all variables. Try going both above and below the midranges in your first trials. Start off with larger increments, and when you find a good condition, try smaller increments.
• Check trays frequently to catch transient crystal formation that may be useful or may signal conditions worth exploring.
• Look for trends in the results. Does something happen consistently when the concentration of one of the components is increased? How does the number of crystals and their size change? What are the different shapes? Putting the results in table form to help sort through the data is a good idea.
• Because there are so many possible conditions, teams should compare results and discuss different things to try. Also, make sure to keep good notes so that you do not have to repeat trials until you are retesting your best conditions.
• If you get no good results (for example, all trials make precipitate, but no crystals), recheck calculations on reagent preparation, and recheck buffer pH. Did you prepare your reagents correctly?

Study Questions

1. What condition yielded the "best" crystals of lysozyme?

2. What changes did your group try to make, and what effect did they have on the crystals?

3. How many of your conditions yielded suitable crystals (nice ones, and only one to a few per well)? How many did the entire class get (use the email discussion to share and obtain this information)? Give the success fraction (number of conditions that gave crystals/number of different conditions that were set up) for your attempts, as well as for the entire class. What does this tell you about protein crystallization?

4. What methods can you think of to confirm that the crystals are lysozyme and not salt (many salt crystals are colorless as well)? Hint: think of protein properties and ways to assay or detect proteins, and focus on methods available in this lab.

5. Many protein crystals are formed at or near the pI of the protein being studied. Explain why the pI might be a good pH for crystal formation.

6. Why does vapor diffusion decrease the volume of the drop, rather than increasing it?

Report

NOTE: Be sure to use our digital camera to photograph your best crystals, and any others you want as illustrations in your report!!

I'll transfer them from the camera to my computer. Then I'll email copies to you.

Turn in the following materials by 1:00 PM on the Monday specified on the lab calendar. Follow the guidelines for Laboratory Reports, being sure to include all specific materials listed here:

1. Table: Using the format of the sample Excel table I distributed in lab, provide a complete listing of results of all crystallization trials. Construct your own compact labeling to show, in each box representing a set of conditions you tried, whether conditions produced no crystals, unsuitable crystals (fuzzballs, needles, and so forth), and suitable crystals (one or a very small number of well-formed, clear, sharp-edged crystals). Leave boxes blank if you did not try those conditions at all.
2. Brief Interpretation (be sure that your interpretation fits the guidelines): The main thing you need to do to show your understanding of the molecular basis of protein crystallization is to explain how salts precipitate proteins, and why salt concentrations both higher and lower than ideal can fail to produce crystals. Then briefly describe how you chose your first trial conditions and how you decided on the second and subsequent trial conditions. Finally, tell about your attempts to reproduce crystal growth from your most promising conditions. Illustrate your interpretation with your digital photos (color printing not required).
3. Study questions: Answer the study questions above.

Turn in separately: Lab-Notebook Copy Sheets.

CHY 462 Schedule