Operating Procedure: RaxisIV++/Yale Mirror System

Safety

	The X-ray generator is usually left making X-rays at lower power (20 kV/10 mA = 200 W). If either of the red X-RAY ON lights are lit on either the front panel or on the tower of warning lights, the generator is making X-rays. Be extremely careful. X-rays will not escape the anode tower unless the shutter opens, however, and the shutter will not open if the hutch door is open and the interlocks are engaged. So if you are working in the hutch, leave the door open and the shutter cannot open. Conversely you'll need to close the door once you leave the hutch, in order to take images.
	If the yellow SHUTTER OPEN light is lit on the warning light stand, then the shutter is open X-rays are hitting the crystal - do not enter the hutch. There's also a small red light on top of the x-ray shutter assembly but this is unobtrusive - the shutter is also open when this is lit. The safety mechanisms are arranged such that if the bulbs are blown, the features will not work (X-rays will not be generated or shutter will not open). These features are tested every 6 months. he system is surveyed for background scatter at least annually - the bottom line is that outside of the hutch the scatter is minimal.
	Each machine has an experimental hutch with a safety interlock associated with it. The X-ray shutter will not open if any of the hutch doors are open. Before you start working with the machine, make sure the safety interlocks are not overridden. Check that the appropriate override lock is set to ENABLE the interlocks and that the keys are not in the lock. Never operate the machine if the interlocks are bypassed or if there are keys in the override lock. Always leave the hutch door open when you are inside the hutch - this will prevent any possible opening of the X-ray shutter by mistake. The interlocks are tested every 6 months.
	On the warning light tower, the top red light indicates X-RAYS ON - the generator is making x-rays. The middle two yellow lights indicate SHUTTER OPEN (we only use the right port, so the left shutter is never open) - i.e. x-rays are hitting your sample when the lights are lit. The bottom (green) lamp is READY, which is lit only when the anode is spinning and the x-rays are off - you'll rarely see this lit. In the photo the X-rays are on and the shutter is open - this is a warning that you should stay way from the X-ray optics and out of the hutch.

Also, the software is often rather unobtrusive while it is working, so you need to double check that the software is truly "idle" before attempting to mount your crystal. Leaving the hutch door open whenever you are working in there, and closing it only after you've left the hutch, is a good failsafe method in case the computer still thinks it is controlling the machine.

Which Machine am I using ?

With your back to the door,the machine to the right is the Raxis IIc, an older smaller area detector. The machine in front of you is the Raxis IV++ with a newer larger area detector. Optically they are equivalent, but the detector material and the readout is more sensitive on the IV++ so it's the "machine of choice". Technically the IIc is not covered in this SOP but it's operation is very similar - this system was mothballed in June 2006.

Before You Start

Helium for mirrors and collimator
X-ray generator settings
Oxford cryo settings

Turn on the helium tank. This sits in the Raxis IIc enclosure so you'll have to negotiate with anyone using the IIc (who would need to interrupt data collection). Do not just walk in there without checking otherwise you'll trip the shutter closed Check the Helium level/pressure on the Helium regulator on the gas tank. The pressure on the right-hand gauge on the regulator (i.e. nearest the tank) is an indication of the helium level in the tank. If the level is less than 750 psi tell Phil or email/call Receiving at mol-receiving@exchange.princeton.edu or 8-2807 to request another tank and switch to the spare one (sometimes inside, sometimes outside the hutch). Make sure they know they are replacing Helium - sometimes they think it is Argon. Prominently label the empty tank EMPTY and move it out of the hutch.

Now flush the mirror box with helium. The mirror box is the large white box containing the X-ray optics to the left of the image. Disconnect the small plastic hose at the right-hand end of the mirror box that connects the mirror box to the collimator. Disconnect it (unscrew it) at the mirror box end, not the collimator end.

After you've disconnected the hose, turn the helium flow valve to FLOOD (points to the left) and let it flush for 10 minutes.

Then turn the flow valve to PURGE (points to the right) and reconnect the hose to the mirror box. Make sure the hose is disconnected when you FLUSH the mirror box. Helium reduces X-ray scatter in the mirror box and protects the mirror surface from oxidation. DO NOT OPEN THE X-RAY SHUTTER UNLESS THERE IS HELIUM IN THE MIRROR BOX - IT WILL TRASH THE OPTICS Make sure that you leave the helium running on PURGE throughout your experiment. Check the pressure at the regulator at the helium tank periodically.

Check that the generator is running. On the warning light stand, the red X-RAY ON light should be lit. On the front panel it should read Tube Voltage 20 KV and Tube Current 10 mA (unless someone has left the machine at full power - in the photo it's almost at full power). Note also the X-RAY ON light on this front panel photo:

If the generator power is at 20 kV and 10 mA, bring the generator slowly up to full power at 50 kV and 100 mA. Increase by 2 kV and 4 mA at a time, once every 5-10 seconds. Check the vacuum gauge on the extreme far right of the front of the generator (next to the Helium flow manifold) to make sure that it doesn't spike while you do that, although it should drift up as you increase power. Generator Front Panel

If any ALARM condition LEDs are lit, this is helful in diagnosing the error (TC and OL lights indicate instabilities in the generation of x-rays - normally the generator can just be restarted; VC means there's an issue with the vacuum system; FC means the filament has blown; CW means there's an issue with the cooling water). If you are going to call Phil to report an error, please make sure you know what this error state is.

Now check that there is enough liquid nitrogen in the cryo dewar to run the cryo system. On the table outside the hutch, the control box/level meter reads how much lN2 there is in the small dewar. Never operate the cryo system if the lN2 level is below 5 and do not leave it operating below 6. If it is low, fill the small dewar (inside the hutch) from the large one (outside the hutch) by flipping the toggle switch to OPEN on the control box. Do not leave a filling dewar unattended. Do not fill the small dewar beyond 15 (inches). Recently I've changed the fill mode to AUTO, which automatically fills the small dewar from the large one when the level gets too low. Nevertheless you should always check the reading on the level controller, and make sure there is enough lN2 in the large dewar.
Now start the cryo unit. This takes about 90 minutes to come down to operating temperature from room temperature. Turn on the cryo dry air generator (see photo) that sits at the far end of the enclosure. Then turn on the cryo controller box (power switch at the rear right). Wait for it to self-check. Then press the PROGRAM button. Using the arrow keys and the ENTER button, program the cryo to RAMP at 120 K/hr down to 100 K. Then on the next line put a HOLD entry at 100 K (do not press the HOLD button !!). Then on the 3rd line put an END entry at 129 K/hr to 293 K. This sets up the entire cycle of the experiment including the shut-down procedure:
RAMP 120 100 HOLD 100 END 120 293
Once you've programmed it press the START button and the cryo should slowly ramp down to operating temperature. Because of the algorithm, it rarely stays locked at precisely 100K but tends to wander a few K around this point. That's not a problem. Once you've finished with the cryo unit, pressing the START button again will send it to the END statement and it will do a controlled shutdown.

If the cryo unit cannot reach your target temperature (100K) during the RAMP phase it will not progress to the HOLD phase. This usually happens if the vacuum is starting to fail on the cryo head. You can still use the unit as long as it is capable of maintaining <110K, but in order to move to the next phase (for shutdown) you'll need to press the HOLD button to force the end of the ramp (it will try and maintain the current temperature), and then press the START button to initiate the END sequence including ramp up to 293K and shutdown.

If the temperature is varying more than 0.5 K around the target temperature after 2 hours of you starting the machine, you probably have to do the EvapT fix. This involves changing the default setting of EvapT. Instructions are given in this link.

If there's enough Helium and both X-ray generator and Oxford cryostream are working OK, then you can proceed.

Mounting a crystal

Treat the hardware nicely !!
Mount an empty loop and check for alignment
Mount a crystal and align it

Do not bash, tap, bend or otherwise abuse the optics or the collimator. It's a very expensive precision device and if you push on it, it will misalign. No X-rays for you ! Similarly, abuse of the beam stop is likely to irrevocably damage the detector via direct beam burns.

Unlock the phi axis via the Locked/Free dial. It's locked when the word "LOCK" is on the right of the dial, and unlocked when "FREE" is there instead. What this does is engage/disengage the phi motor from the phi axis spindle from the. You need to lock the phi axis before collecting data otherwise the computer has no way to control the crystal. If the control software shows an error during the initialization phase this is the first thing to check - it's a very commonly made mistake. However make sure you stop the software before locking down the phi axis - trying to lock it down while the motor is spinning may strip the threads.

Find a goniometer head (of Supper or Huber design, according to your preference) and mount it on the phi axis. Make sure it's tight enough so that the head does not waggle on the axis. Often a goniometer head will already be in place. Put an empty loop on the head and align it in the microscope. Because the cold stream is set at an angle to the phi axis, the goniometer head does not freeze. Therefore you can align the loop/crystal at your leisure. Unlock the phi axis if it is locked down (the small black grub screw on the phi axis body). Translate the goniometer head vertically via the thinner gnurled ring around the phi axis. Translate the loop/crystal via the sleds on the goniometer head. The center is always the point which the loop/pin/crystal precesses around so it's actually easy to center - spin the loop back and forth through 180 degrees, and move the translations (the ones that move the xtal left/right in the microscope - the ones perpendicular to your viewpoint) until it spins around it's own center. Then do the same thing after rotating the loop 90 degrees.
Note that you should only change the sled that moves the loop left/right across the microscope view direction - changing the other sled (along the viewing direction) just moves the loop along the viewpoint and you won't be able to tell the center reliably - the xtal will just go in and out of focus.

Visually check that the cold stream nozzle is centered on the loop when the loop is centered in the microscope. If it's not you'll have to get Phil to recenter it for you. But you can ajust the up/down position of the cryo head (i.e. it's distance from your crystal) via the large black knob at the rear of the cryo head slide. Generally speaking the closer the nozzle is to the crystal the better, although you'll want it at least 7mm away from the crystal to avoid it clipping the X-ray beam.

With the loop approximately centered, you're ready to mount the crystal. Simply remove the empty loop and mount your crystal. Center the crystal as indicated above. Lock down the phi axis using the LOCK/FREE dial and optionally the phi axis translation screw after everything is centered using the Locked/Free dial and then you're ready to take some images. Check that the right hand shutter switch (SHUTTER panel on front of generator) is down at the EXT setting (EXTernal control). Exit the hutch and close the door.

Controlling the RaxisIV++ with the Software

Crystal Clear is the software that controls the computer. It's one of Rigaku/MSC's less-than-perfect implementations.

On the Pentium 3 computer (now with flat screen!) there is a CrystalClear icon on the Desktop (top left corner unless someone moves it. Double click on it and start the software. It will ask you for a username (pli) and a password (check the log book on p.30 for the current one).

The software has the idea of a Project and a Sample. For the Project you can use your name or the name of your protein. If you've not used the machine before, select New Project from the File menu. If you have used the machine before and have an existing project name you can use New Sample to create a new sample directory for your experiment. You don't actually have to use a new sample for each crystal, since you can just change the filenames at the data collection stage.

Once you have selected your Project and Sample, you can select what you want to do. I never mess around with the other procedures - I just click on the Collect Data button. A few protocols exist: 0 degrees (one frame), 0/90 degrees (two frames), 0/90/180/270 (four frames), and "custom". You'd use the latter for general data collection. I use 0/90 for crystal screening.

Turn on the helium tank. This sits in the Raxis IIc enclosure so you'll have to negotiate with anyone using the IIc (who would need to interrupt data collection). Do not just walk in there without checking otherwise you'll trip the shutter closed Check the Helium level/pressure on the Helium regulator on the gas tank. The pressure on the right-hand gauge on the regulator (i.e. nearest the tank) is an indication of the helium level in the tank. If the level is less than 750 psi tell Phil or email/call Receiving at mol-receiving@exchange.princeton.edu or 8-2807 to request another tank and switch to the spare one (sometimes inside, sometimes outside the hutch). Make sure they know they are replacing Helium - sometimes they think it is Argon. Prominently label the empty tank EMPTY and move it out of the hutch.
Now flush the mirror box with helium. The mirror box is the large white box containing the X-ray optics to the left of the image. Disconnect the small plastic hose at the right-hand end of the mirror box that connects the mirror box to the collimator. Disconnect it (unscrew it) at the mirror box end, not the collimator end.
After you've disconnected the hose, turn the helium flow valve to FLOOD (points to the left) and let it flush for 10 minutes.
Then turn the flow valve to PURGE (points to the right) and reconnect the hose to the mirror box. Make sure the hose is disconnected when you FLUSH the mirror box. Helium reduces X-ray scatter in the mirror box and protects the mirror surface from oxidation. DO NOT OPEN THE X-RAY SHUTTER UNLESS THERE IS HELIUM IN THE MIRROR BOX - IT WILL TRASH THE OPTICS Make sure that you leave the helium running on PURGE throughout your experiment. Check the pressure at the regulator at the helium tank periodically.

If any ALARM condition LEDs are lit, this is helful in diagnosing the error (TC and OL lights indicate instabilities in the generation of x-rays - normally the generator can just be restarted; VC means there's an issue with the vacuum system; FC means the filament has blown; CW means there's an issue with the cooling water). If you are going to call Phil to report an error, please make sure you know what this error state is.
Now check that there is enough liquid nitrogen in the cryo dewar to run the cryo system. On the table outside the hutch, the control box/level meter reads how much lN2 there is in the small dewar. Never operate the cryo system if the lN2 level is below 5 and do not leave it operating below 6. If it is low, fill the small dewar (inside the hutch) from the large one (outside the hutch) by flipping the toggle switch to OPEN on the control box. Do not leave a filling dewar unattended. Do not fill the small dewar beyond 15 (inches). Recently I've changed the fill mode to AUTO, which automatically fills the small dewar from the large one when the level gets too low. Nevertheless you should always check the reading on the level controller, and make sure there is enough lN2 in the large dewar.
Now start the cryo unit. This takes about 90 minutes to come down to operating temperature from room temperature. Turn on the cryo dry air generator (see photo) that sits at the far end of the enclosure. Then turn on the cryo controller box (power switch at the rear right). Wait for it to self-check. Then press the PROGRAM button. Using the arrow keys and the ENTER button, program the cryo to RAMP at 120 K/hr down to 100 K. Then on the next line put a HOLD entry at 100 K (do not press the HOLD button !!). Then on the 3rd line put an END entry at 129 K/hr to 293 K. This sets up the entire cycle of the experiment including the shut-down procedure: RAMP 120 100 HOLD 100 END 120 293 Once you've programmed it press the START button and the cryo should slowly ramp down to operating temperature. Because of the algorithm, it rarely stays locked at precisely 100K but tends to wander a few K around this point. That's not a problem. Once you've finished with the cryo unit, pressing the START button again will send it to the END statement and it will do a controlled shutdown.