X-Ray diffraction experiments with protein crystals at cryogenic temperatures

Author: Prof. Mariusz Jaskólski

Aims

Purpose of this practical

In this practical you will carry out real-life X-ray diffraction experiments on protein crystals. You will learn how to mount protein crystals in cryo-loops and how to cryoprotect them before vitrification at 100 K. You will be able to collect X-ray diffraction data using a rotating anode X-ray generator and an Image Plate (IP) detector.

In this exercise you will be working with real X-rays. The working environement is completely safe, provided that you exercise all necessary safety measures and strictly obey all directions from your instructor. X-rays are invisible to the eye but can be very dangerous because of their ionizing character. Other hazards connected with this practical include high electrical voltage (50 000 Volts), vacuum equipment (0.000001 Torr), and very low temperatures (work with liquid nitrogen). In addition, because of the use of liquid nitrogen, the room you will be working in can very quickly fill with gaseous nitrogen, creating a suffocating atmosphere. Make sure that the room is well ventilated!

Things you should know or prepare...

• The rotating anode X-ray generator is made by Siemens
• It will operate at 50 kV and 112 mA. Calculate the power of the generator
• The anode of this X-ray generator is made of copper (Cu), therefore the characteristic radiation has the wavelength corresponding to CuKalpha, i.e. 1.54178 Å
• This characteristic (or monochromatic) radiation is selected from the polychromatic spectrum of this generator using a monochromator made from a single crystal of graphite. (Graphite, space group P6₃mc, is an allotropic form of crystalline carbon, the other forms being diamond, fullerene, and lonsdaleite). The monochromator acts as a diffracting crystal, selecting from the polychromatic spectrum just one wavelength through a particularly powerful reflection (002) from one particular set of lattice planes.
• Knowing that the c parameter of graphite is 6.708 Å, calculate the Bragg theta angle of the 002 reflection for CuKalpha radiation (lambda=1.54178 Å) and for MoKalpha radiation (lambda=0.71073 Å)
• The low-temperature attachment is a gas-blowing device using nitrogen evaporated from the liquid. The cooling gas stream has the temperature of about 100 K (specify the temperature in degrees Celsius). The liquid nitrogen tanks and all the tubing is made of Dewar vessels (what is a Dewar container?). This apparatus was made by Oxford Cryosystems
• The Image Plate scanner has a diameter of 300 mm. It was made by MarResearch.
• What is the principle of operation of an Image Plate equipped with a laser scanner?
• The IP scanner can be moved closer or farther away from the crystal, within a distance range of about 70 - 700 mm. If you want to achieve the highest resolution, in which direction would you move the detector? What would be the maximum resolution attainable with this detector and CuKalpha radiation?
• You will be working with single crystals of the enzyme lysozyme from hen egg white
• Ask your tutor about the crystallization conditions of this particular form of lysozyme crystals
• Using available resources (handbooks, scientific literature, the Internet), find the basic facts about lysozyme
• Search the PDB for crystal structures of lysozyme. Are there any? If yes, try to find out which crystalline (polymorphic) form of hen egg white lysozyme (HEL) you've been working with
• What buffer did you use to cryo-protect your crystal?
• What were the physical dimensions of your crystal?
• Note the experimental parameters for the diffraction image(s) that you have recorded (name, date, distance, resolution, rotation, exposure time, temperature, crystal size)

Your Report

• For your report, please compile your answers to the questions posed above, plus include any other observations that you might have. As usual, please email your report to your tutor (szymon@amu.edu.pl) within one week. Please indicate in the Subject field of your message: SERP or ERASMUS Practical-2 your name.

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Mariusz Jaskolski, 30.03.2009