Before a data set can be collected—from which the final structure of the protein can be deduced—it is necessary to mount a crystal in the X-ray beam of a diffractometer and determine if it diffracts to sufficient resolution to justify collecting a full data set. The initial crystal screen can often be used to determine the space group of the crystal, an important piece of information is planning data collection. The exact instructions for collecting data will vary depending on the type of equipment used. Instructions for several facilities are given here.

Oxford Diffraction Gemini R Ultra (Colgate)

The following instructions are for collecting protein data using a Gemini R instrument located in the Department of Chemistry (Wynn Hall) on the Colgate University campus.

Figure 1. The Colgate University Department of Chemistry Oxford Diffraction Gemini R system.

Startup

• Verify that the external water cooling supply is on. Valves near the computer workstation and near the filtration units on the opposite side of the room should be in the open positions. Water should be seen exiting the outlet in the drain closet.
• Verify that the instrument has been left configured in the (default) Mo X-ray source. (This configuration will require no helium gas flow.)
• Turn on the cryojet and autofill controllers, if not already on.
• Verify there is sufficient liquid nitrogen and helium gas to complete the experiment.

Note: Current recommended settings for the cyrojet are 6.5 L/min sample and 4.5 L/min shield flow for 110 K and good frost suppresssion. At this setting, the cryojet will consume about 40% of the cryojet dewar capacity per day of operation. It is programmed to auto-fill at 25% capacity to a final fill of 90%. Therefore, it should be expected to autofill about every 36-40 hours. A full 160 L dewar of LN2 can be reliably expected to perform two full autofills. Do not start an experiment unless there is sufficient LN2 to complete the run. Long runs (more than 2 days) may require attachment of a new 160 L LN2 dewar to the autofill. Helium consumption for the Cu source will deplete about 100 psi per day of operation. Do not start an experiment unless there is sufficient helium pressure to complete the run.

• Turn on the helium flow by opening the main valve on the helium tank. Verify that the helium flow in the Gemini electronics cabinet to 30-40 cm³/min. The indicator LEDs in the flow meter should be green if there sufficient helium flow. If there is insufficient flow, the ineicators LEDs will be red. Adjust the flow rate if necessary.
• Start CrysalisPro on the computer workstation. Check the status panel for the instrument in the upper right hand corner of the display.
• Click on the Cyro button in the status panel of CrysalisPro. Verify that the set temperature of the cryojet is set to 100-110 K. Set the sample flow to 6.0-7.0 L/min, and the shield flow to 4.0-5.0 L/min. Turn off the heater until the cryojet temperature approaches the final temperature, then switch it on. The cryojet will stabilize in about 30-45 minutes.
• Click on the XRAY-Mo button on the status panel to switch to the Cu source. After accepting a warning dialog box, the instrument should switch to the Cu X-ray source.
• Click on any status light to get additional info about the CCD, cryojet, X-ray source, and nitrogen level. Note: The Peltier cooler in the CCD should be working at less than 70% capacity for nominal operation. (Above this, and the vacuum should be replenished in the CCD.)
• Set the Cu X-ray source to 40 kV x 40 mA (1.6 kW) for protein data collection. The source will take several minutes to ramp up to the desired power setting.
  • Note: recent CrysalisPro software updates have limited the PX Ultra Cu source to 1.6 kW operation. Previously the source was operable at 2.0 kW.
• The instrument is now ready for data collection.

Configuring the autofill

The default settings for the autofill are

• Fill level = 90%
• Autofill trigger = 25%
• Low (alarm) trigger = 15%

This settings will result in an autofill about every 36 hours during normal cryojet operation. I may be desirable to change these settings slightly to maintain cryojet temperature stability, and schedule autofills more frequently during data collections. The following settings will schedule an autofill when the LN2 level drops to 50% in the cryojet dewar, and will result in an autofill approximately every 24 hours so the filling can be easily monitored during working hours:

• On the autofill controller, press and hold down the RAISE, LOWER, and SILENCE buttons at the same time. The LCD should briefly display "tSt" follwed by "t.00".
• Press RAISE or LOWER repeatedly until "t.11" appears.
• Press SILENCE until "FIL" appears in the LCD display. (This should require two presses of the SILENCE button.)
• Hold the SILENCE button down and press RAISE until the fill trigger value is 50%.
• Press SILENCE to exit the manual configuration mode. The LCD display should flash "5.06" then "n2" before reverting to the display of the current fill level.
• To return the autofill to the default state after data collection, simply power off the autofill controller and then power it back on.

Performing a manual nitrogen fill

When the supply LN2 dewar is low (i.e., insufficient LN2 to complete a full fill when the next autofill is triggered) it can be advantageous to perform a manual fill of the 75L cryojet dewar. Use the following steps to do a manual fill:

• On the autofill controller, press and hold down the RAISE, LOWER, and SILENCE buttons at the same time. The LCD should briefly display "tSt" follwed by "t.00".
• Press RAISE or LOWER repeatedly until "t.11" appears.
• Press SILENCE until "FIL" appears in the LCD display. (This should require two presses of the SILENCE button.)
• Hold the SILENCE button down and press RAISE until the fill trigger value is larger than the current fill level, but not more than 85%.
• Press SILENCE to exit the manual configuration mode. The LCD display should flash "5.06" then "n2" before reverting to the display of the current fill level. If the fill trigger level was properly set, an autofill should commence immediately.
• Do not abandon a manual fill. If the LN2 runs out before the fill can be completed, the autofill solenoid could be damaged from overheating.

Loading, aligning, and removing crystals

These instructions assume you will be using a magnetic goniometer head that is suitable for Hampton Research 18 mm crystal caps. Our instrument has both Hampton Research and Huber goniometer heads. The Huber is normally installed on the goniometer head.

Centering an empty pin

• Open a command window from the toolbar by clicking on the appropriate icon
• Issue the command gt a 40 -30 80 -90 120 to configure the goniometer and detector for an optimal crystal mounting geometry.
  • See Quick Screening below for an alternative mounting geometry for rapid crystal screening.
• Attach an empty pin to the goniometer head
• On the computer workstation, press F12 to pass control of the goniometer to the remote box in the X-ray cabinet.
• Go to HOME and select 270° on the remote control box to gain access to locking screws and all goniometer adjustments.
• Loosen locking pins on the goniometer head and roughly adjust pin to center it in the microscope.
• Select LOWER on the remote control box to gain a better view of the sample loop
• Adjust goniometer to center sample loop and check at 180°, 90°, and 0° using the control box.
• Select UPPER on the remote control box to verify that crystal is still centered. Re-adjust height if necessary and re-check in the LOWER position.
• Go to the HOME position.
• Remove the empty cap and loop when adjustments have been completed.
• Close the F12 menu when finished

Mounting a crystal sample

• Open a command window from the toolbar by clicking on the appropriate icon
• Issue the command gt a 40 -30 80 -90 120 to configure the goniometer and detector for an optimal crystal mounting geometry.
  • See Quick Screening below for an alternative mounting geometry

Note: The arguments for the "goto angle" command are in the order omega, theta, phi, kappa, and distance.

• On the computer workstation, press F12 to pass control of the goniometer to the remote box in the X-ray cabinet.
• Using the vial clamp, remove the desired vial from the cryo-cane and keep it under the surface of the liquid nitrogen.
• Immerse the crystal wand into the liquid nitrogen until it stops bubbling vigorously. Use the crystal wand to remove the crystal cap with the mounted crystal. Keep the crystal cap under the surface of the liquid nitrogen at all times.
• Remove the empty vial and vial clamp from the liquid nitrogen and set aside. Keep the crystal cap totally submerged in liquid nitrogen during this and the next two steps.
• Insert the cryo-tongs into the liquid nitrogen and hold them there until all bubbling stops. This may take 30-60 seconds.The tongs will cool faster if they are held open during cooling. It is important that the cryo-tongs are fully cooled before proceeding.
• While keeping both tongs and crystal cap under the surface of the liquid nitrogen, open the tongs, grasp the crystal cap, and remove it from the crystal wand. The wand can now be removed from the liquid nitrogen and set aside.
• Quickly remove the cryo-tongs and the attached crystal cap from the liquid nitrogen and place the cap on the magnetic head of the goniometer. The tongs should be oriented so that when they are opened to release the cap, the cryostream can blow into the opening between the two halves of the tongs. This will prevent ice ring formation or crystal melting.
• Go to HOME and select 270° on the remote control box to gain access to locking screws and all goniometer adjustments.
• Roughly adjust pin to center crystal in the microscope.
• Select LOWER on the remote control box to gain a better view of the sample loop
• Adjust goniometer to center crystal and check at 180°, 90°, and 0° using the control box.
• Select UPPER on the remote control box to verify that crystal is still centered. Re-adjust height if necessary and re-check in the LOWER position.
• Go to the HOME position.
• Close the F12 menu when finished.

Screening crystals

Standard screening

This method will reset the goniometer to vertical position before collecting screening scans. The goniometer will return to the previously set position (usually the mounting geometry with OTKPD = 40 -30 80 -90 120) when the crystal centering dialog is closed. This can be time consuming if many crystals are screened, but the mounting geometry is very easy.

• Click on the Make Screening Scans... tool, and select make a phi scan, and click on start.
• Enter scan settings in the dialog box:
  • omega-theta-kappa angles (0 0 0 is suggested)
  • detector distance (60-80 mm is suggested)
  • Start angle (0 is suggested)
  • Width (0.5 degrees is suggested)
  • Exposure time (60-120 s is suggested)
  • Select the folder to store the data in using the Browse... button. Typically, you will create a crystal screening directory, e.g., hica-g41t-screen within your research group user directory.
  • Enter the base file name using the Edit button, e.g., hica-g41t-screen
• Click OK to start the phi scan
• If the phi scan is acceptable, proceed to collecting data.

Quick screening

This method will keep the goniometer in the LOWER crystal centering position during data collection, so that the goniometer geometry does not have to switch between the mounting geometry, the screening geometry, and the crystal centering geometry for each run. Mounting crystals on the goniometer in the LOWER crystal centering position is a little more difficult, but quite possible.

• Click on the Make Screening Scans... tool, and select make a phi scan, and click on start.
• Enter scan settings in the dialog box:
  • omega-theta-kappa angles (-57 0 134). This will keep the goniometer in the LOWER crystal centering position when the F12 crystal centering dialog box is closed, saving time.
  • detector distance (80-90mm is suggested)
  • Start angle phi (280 is suggested to minimize goniometer movement)
  • Width (0.5 degrees is suggested)
  • Exposure time (120 s is suggested)
  • Select the folder to store the data in using the Browse... button. Typically, you will create a crystal screening directory, e.g., hica-g41t-screen within your research group user directory.
  • Enter the base file name using the Edit button, e.g., hica-g41t-screen
• Click OK to start the phi scan
• If the phi scan is acceptable, proceed to collecting data.

Approximate resolution vs. camera distance for Oxford Diffraction Ruby detector at 1.54 Å

Distance (mm), R(edge), R(corner)

60, 2.26, 1.92

65, 2.39, 2.01

70, 2.52, 2.11

75, 2.66, 2.20

80. 2.79, 2.30

85, 2.93, 2.40

90, 3.07, 2.50

95, 3.21, 2.61

100, 3.35, 2.71

105, 3.49, 2.81

110, 3.64, 2.92

115, 3.78, 3.03

120, 3.92, 3.13

Note: the "edge" of the 135 mm ruby detector is about 100 mm when processed in CrysalisPro. The "corner" is actually the edge of the circular detector, about 135 mm; it does not extend to the corner of the square display area in CrysalisPro.

Collecting data

Collecting data with CrysalisPro is highly automated, and this mode of operation is encouraged. Collecting data typically involves collecting a pre-experiment to establish appropriate values for signal to noise ratio and resolution, selecting a strategy to accomplish the collection of adequate data in the time available, and then initiating the full experiment.

Running a Pre-Experiment

The pre-experiment will collect several frames of data at each of three omega angles to learn about the scattering properties and space group of your crystal, and provide guidance for selecting a suitable collection strategy.

• Begin the process by clicking on the Start/Stop bar on the upper right portion of the CrysalisPro screen and select Start New.
• In the dialog box, select the Browse root folder button and enter the root directory for the experiment. Typically, you will use a new directory that references the crystal number, e.g., hica-g41t-002.
• Under the "prior knowledge" area of the dialog box, check protein.
• Suggested tasks for manual configuration of the pre-experiment are:
  • Set width to 0.5 degrees
  • Set exposure time to 60-240 seconds/deg for all runs
• Select a detector distance
• Select Use Laue symmetry under "Experiment"
• Click on the Target I/sig button and enter the desired value. A good starting point is a value of 6-10.
• Click on and enter a Target resolution based on the initial crystal screen. A good starting value is usually 0.1-0.2 Å beyond the highest resolution spots clearly visualized on the screen image(s).

Note: The I/σ value in CryslalisPro refers to unmerged data without accounting for redundancy. The final value of I/σ for the scaled and merged data set can be expected to be approximately equal to the entered I/σ value times the square root of the redundancy. For example, an experiment with a target I/σ of 6 and a target redundancy of 4 will result in a scaled and merged I/σ value of approximately 12.

• Click on Start to begin the pre-experiment.
• During the pre-experiment, CrysalisPro will attempt to find your unit cell parameters, and will make an estimate of the necessary experiment time to meet the desired resolution and I/σ targets. The resolution and I/σ targets can be changed "on the fly" during the pre-experiment to develop new experimental time estimates.
• When the pre-experiment is completed, the Strategy module will open with a suggested strategy.

Selecting a Strategy

It is typically necessary to alter the initial suggested strategy before starting the experiment. Many options are available to configure the final experiment.

• Click on the Advanced button and check the following settings in the dialog box:
  • If the lattice type is known, select it in the Lattice Type drop-down menu. Setting the lattice type is critical if you want real-time processing to work correctly. The default is a P-lattice.
  • Change the scan width in the "Overlap Evaluation" area to the appropriate value for the final experiment (0.5 degree is typical)
  • To minimize loss of data from the dual beam stop, check the Use theta constraints and tick the positive theta only box.
  • To minimize ice formation on the crystal loop, consider setting Single kappa value restraints to -30 to +30 degrees. High kappa values (near 90 degrees) encourage the formation of "ice beards" on the crystal pins that may interfere with data collection.
  • Enter an exposure time in the default box that is appropriate for the desired target I/σ identified in the pre-experiment. Keep in mind that the expected I/σ will increase as the square root of the exposure time.
  • Click on OK to return to the strategy dialog box
• Enter the desired resolution for the final experiment.
• Uncheck Friedel mates are equivalent if anomalous scattering data is desired.
• Change the detector distance if desired.
• Select a strategy mode:
  • "Complete data" is the default mode. This mode will set up a strategy to collect 100% of the data to the desired resolution without regard to other constraints.
  • "Complete redundant data" will set up a strategy to collect 100% of data to the desired resolution and level of global redundancy.
  • "Total time constrained data" will set up a strategy to collect data to the desired resolution with a specific time constraint that may not be complete.
  • Complete total time constrained data" will set up a strategy to collect data to the desired resolution within the specific time constraint.
• Click on the Find Experiment button to generate the strategy. Examine the statistical table to see if the experiment meets your constraints acceptably.
• Click on Edit Runs to examine the strategy and verify that it meets the criteria you set. You can also change aspects of the run in this dialog box:
  • Click on Global width to change the scan width
  • Click on Global time to change the exposure time
  • Enter your crystal name under Name of experiment
  • It is recommended that Activate reference frames be deactivated

Note: If planning to process data in MOSFLM, ensure that all strategy runs are of the "omega scan" type.

• Click on OK to accept changes to the strategy.
• Click on Completeness to update the experimental time computation after Edit Runs
• If any of the strategy criteria are changed, click on Find Experiment again.
• When satisfied with the experimental settings, click on Start experiment to begin.

Running the Experiment

Data collection will start immediately after clicking on the Start experiment button in the strategy module. Progress of data collection can be followed by examining the information cards on the right of the CrysalisPro screen.

• The Crystal card provides information about the experiment name, crystal lattice, and beam center position
• The Data Collection card provides information about the status of the experiment, including:
  • anticipated time of experiment completion
  • number of frames collected and remeasured due to overflows
  • variation of X-ray power, CCD and cryojet temperature
• The Data Reduction card provides information about the quality of collected data, including:
  • 3D profile analysis (mosaicity)
  • Scale factor and empirical absorption correction variation
  • Unmerged I/σ, redundancy, and R(int)
  • space group

CrysalisPro will re-analyze the data after every 25 frames, and at the completion of each set of strategy runs

Shutdown

Perform the following operations when data collection is complete. If another user will follow you and require the cryojet, you may omit the cyrojet shutdown procedure.

• Open cabinet doors and remove crystal from the goniometer head.
• If using the Cu X-ray source, switch to the Mo X-ray source by clicking on the XRAY Cu button. Allow this operation to complete before proceeding.
• When source switching is complete, click on the XRAY status button and change the Mo X-ray power settings to 20 kV and 5 mA (standby power).
• Shut off helium flow by closing the main valve on the helium tank. Do not turn off helium flow by adjusting the flow control valve in the electronics cabinet.The status LEDs will turn from green to red after a few minutes.
• Change the cryojet flow rates to 2.0 L/min sample flow and 2.0 L/min shield flow
• Change the set point on the cryojet to 295K.
• You may change cryojet settings in one of two ways:
  • Click on the Cryo status button and change settings, OR,
  • On the cryojet control unit, set control to local and then change settings maunally by holding down the SET button and using appropriate RAISE and LOWER buttons.
• When the cryojet temperature reaches 295K the cryojet control unit may be switched off.
• Leave the main cooling water running.
• Shut down CrysalisPro and log off the computer.