X-ray Crystallography: Practical 2 - Crystal Evaluation & Data Collection, Study notes of Biochemistry

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BCH 6744C: Macromolecular Structure

Determination by X-ray Crystallography

Practical 2

Diffraction Data Collection

Introduction Crystal usability is ultimately determined by the quality of the X-ray diffraction data obtainable, either ‘in-house’ at a home source or at synchrotron radiation sources. In the theory class crystal preparation prior to data collection has been discussed ( L4 ) in addition to the actual diffraction data collection process and the instrumentation requirements ( L6 ).

In this practical the aim is to provide you with ‘hands-on’ experience in crystal evaluation, preparation, manipulation and cryo X-ray diffraction data collection, using the lysozyme crystals that you grew in the first practical ( P1 ). You will be provided with nylon CryoLoops (loops), MicroTubes (copper pins) and a copper CrystalCap (caps) for staking (mounting) loops in which your crystals will be frozen prior to data collection. The copper pin and caps with its steel base are designed to prevent ice formation from the flow of liquid nitrogen. They also dissipate heat due to the X-ray source. The cap has a magnetic base that attaches it to the goniometer head. In the interest of time, cryo protectant solutions (in Tris or acetate buffer) have been prepared for you.

The data collection will be on an ‘in-house’ R-AXIS VII++ image plate system with a Rigaku generator, fitted with Osmic mirrors. The data collection equipment is operated using a PC. Due to time constraints, complete data sets cannot be collected for each student and thus a demonstration of the data collection procedure will be given after everyone has mastered “scooping” and aligning their crystal in the beam. Data will be collected on a selected crystal during the practical and made available for the next practical ( P3 ).

Step 2 Holding a copper pin with one hand, insert into the hole at the top of the copper cap held in the other hand. Put a drop of glue into the hole around the pin. Allow the glue to set.

Step 3 Holding the glued cap & pin in one hand, pick up the loop up with the forceps and insert the straight end into the hole at the top of the pin. Push the loop into the hole until only ~0.5mm of straight end is outside the pin

• ensures that it is held on straight and rigid and thus will not move too much in the cryo stream. Put a drop of glue into the hole to hold the loop. Allow to set.

Label the base with the size of the loop, screw on vial cover and label outside with loop size and your name. MOVE DETECTOR BACK to 300 mm TO AVOID DAMAGE.

Step 4 Take the mounted loop/pin/cap to the X-ray room. Just before you are ready to put your crystal in the beam, install the goniometer head onto the inverted
mount on the X-ray machine. Attach your cap onto the goniometer head using the magnetic base. Step 5 Align the loop so that it is co-axial with the position of the X-ray beam and cryo stream - X -marks the spot on the TV- using the translations on the goniometer head, which can be adjusted using the goniometer head key. I -Unlock the
mount. II -Turn the dial to 0º. III -Use the translations to bring the loop into the beam center. IV -Rotate the dial 180º. V -Repeat the translation operation from the opposite side. VI -Rotate the dial back to 0º. Check the loop to see if it is in the beam center. If not repeat III to VI until loop is centered. VII -Turn dial to 90º. VIII -Use the translations to bring the loop into the beam center. IX -Rotate to 270º (90º on other side). X -Repeat the translation operation from opposite direction. Check the loop to see if it is in the beam center by rotating it round. Repeat VII to X until loop stays in the same position during rotations - now co- axial. Lock
mount. Remove loop/pin/cap and protect with vial cover. You are now ready to put your crystal in the beam. Collect your crystal tray from its storage place and take it to the X-ray room.

Pin

loop

Cap

Steel Base

Crystal Preparation - In the X-ray room

Materials Supplied: Depression well plates P Yellow tips 30% glycerol in Sodium Acetate or Tris buffer Forceps Microscope

X-ray Instrument: The Rigaku generator current and voltage have been ramped up to their maximum, 50 kV and 100 mA. The detector is a dual image plate system, 300 mm in size.

Oxford CryoStream : The cryo cooling system has been filled with liquid nitrogen and the cooling program has been started prior to the lab since this takes several hours to reach the desired 100K. The procedure will be demonstrated. You need to check that the temperature is 100K before you freeze your crystal.

Step 1 Ensure that supplies and crystal tray are on a table top, conveniently located within easy reach of the optical bench and
mount. Put 50 l of the appropriate cryo-protectant (dependent in crystallization condition) in a depression well. Unscrew the cover from the loop/pin/cap and place within arm’s reach.

Step 2 Check your crystals under the microscope. Remove limbro plate lid from crystal tray setup after making a mental note of the drop with the selected crystal(s). Carefully remove the glass cover slip with crystals from the limbro plate with forceps and invert onto the lid of limbro plate lid or other desired clear surface, e.g. the depression well. Or If crystals in a sitting drop were selected for data collection, remove the glass cover slip above the well with the micro-bridge, with forceps.

X-ray Diffraction Data Collection using Lysozyme Crystals

Step 1 Log on to the PC outside the X-ray room. Check the disks to verify that there is sufficient space for the data to be collected. This is just good house keeping practice before you start data collection.

Step 2 Start the CrystalClear data collection package. Opens large window. You will only use the setup and collect images icons on the LHS for your data collection. Other windows will appear (images, status etc.) after data collection starts. Start data collection after editing in the appropriate settings.

Example of Settings: Crystal to detector distance Image scan size and image area Oscillation start position (
position), step size, oscillation angle and end position. Exposure time Temperature of data collection

Step 3 Make notes on the date, time, project for which data is being collected, crystal drop and crystallization conditions and your data collection settings in your note book and in the McKenna lab. data collection log book kept by the PC.

Step 4 Check the first image that is collected to ensure that the settings selected are appropriate for the crystals. Spot shape Spread Resolution Intensities What else?

Practical 2: Diffraction data collection - Assignment 2 - half a page of written text

Compare and contrast the image plate and charge coupled device (CCD) detector systems. For each system, give an example where its usage, for X-ray diffraction data collection, may be advantageous over the other system.

Xmm

Ymm

2 d sin =  d is your resolution and tan 2 = Y X

AND calculate the maximum resolution that you would be expecting for your data, given the figure below and Bragg’s equation.

crystal

detector