1. An Automated System to Mount Cryo-Cooled Protein Crystals on an SSRL Beam Line, Using Compact Sample Cassettes and a Small-Scale Robot Paul Phizackerley, Aina Cohen, Paul Ellis, Ashley Deacon, and Mitchell Miller Stanford Synchrotron Radiation Laboratory, SLAC, MS 69, 2575 Sand Hill Road, Menlo Park, CA 94025-7015 Introduction Many crystallographic projects would benefit enormously from more efficient ways of screening large numbers of crystals. As screening is typically one of the most tedious steps of a structure determination, the quality of the crystal selected for data collection can depend more on the patience of experimenters than on the number of samples available. This burden can be particularly onerous for researchers studying membrane proteins or macromolecular assemblies, such as viruses, as this often requires characterizing hundreds of crystals. An automated system for mounting and dismounting pre-frozen crystals has been implemented at the Stanford Synchrotron Radiation Laboratory (SSRL). It is based on a small industrial robot and compact cylindrical cassettes, each holding up to 96 crystals mounted on Hampton Research sample pins. For easy shipping and storage, the cassette fits inside several popular dry-shippers and long-term storage Dewars. A dispensing Dewar holds up to three cassettes in liquid nitrogen adjacent to the beam line goniometer. The robot uses a permanent magnet tool to extract samples from, and insert samples into a cassette, and a cryo-tong tool to transfer them to and from the beam line goniometer. The system is simple, with few moving parts, reliable in operation and convenient to use. Mount for 3 Cassettes in the Dispensing Dewar Most of the SSRL PX Group at Their Post The picture to the right shows a close up view of the dispensing Dewar that can hold up to three sample cassettes. Support platforms for the cassettes are mounted on a shelf well above the bottom of the Dewar to permit any ice particles that are generated in use to fall to the bottom and away from the mechanism. Each cassette is supported on a central cone and oriented about a vertical axis by means of a datum pin. Vertical rods help to guide the cassette down to the base of the mount when loading the cassette into the LN 2 which covers the cassette entirely in use. Each cassette rests on 6 balls to reduce the effect of ice, should a thin layer form on the bottom of the cassette during transfer from one cryo- container to another. Crystal Screening Interface The Robot In Action The sample pin being transported inside the cryo-tong to the phi-axis of the diffractometer. A sample pin being withdrawn by the strong picker magnet on the magnet tool. The cryo-tongs at the transfer post. The cryo-tong / magnet tool selector is about to pick up the magnet tool and take it to the requested sample port. Ideal Hampton Pin Lengths for Cassette The cassette is designed to accommodate samples mounted on off-the-shelf Hampton pins. Both CrystalCap Magnet and CrystalCap Copper Magnetic pins may be used. (The copper pins are recommended.) The drawings below show the ideal combinations of pin and Hampton Microtube length for use with the cassettes. However, small variations in these lengths can also be accommodated. Cassette Code Pin Rather than place a bar code on each cassette for identification, which is difficult to read through LN 2, we intend to use a code pin similar to the one in the drawing below, and place a unique one in port A1 of each cassette to identify the cassette. This code pin has multiple divisions, each being one of several standard diameters, rather like a door key in principle. After a cassette has been loaded, the robot will first load the code key on the beam line diffractometer and read the unique cassette code using the sample crystal viewing camera. We plan to use a number of additional divisions to provide a check sum for code verification. Robot and the Dispensing Dewar on BL11-1 The picture on the top shows the robot mounted on SSRL beam line 11-1. In the left foreground one can see a large Dewar, which is used to dispense samples from up to three cassettes (a total of up to 288 samples) located inside – shown more clearly in the bottom picture. We refer to this as the dispensing Dewar. This Dewar and the robot is supported on a rigid support stand that is attached to, and moves with, the experimental table on air bearings. The Kappa- geometry diffractometer can be seen to the right and is coloured green. The x-ray beam traverses from upper left to lower right and the x-ray area detector is out of the picture to the right - far enough away from the robot so that a collision is not possible. The robot is a commercial unit from Epson (Model ES550/320). It employs three vertical axes, giving a reach of 550mm in the horizontal plane, and the third axis incorporates a vertical translation of 320mm. Each of the four motions incorporates an absolute encoder providing an overall positional accuracy of better than 20 microns (3  ). A versatile and straightforward software language SPEL+ (which looks very similar to BASIC) is provided by Seiko for robot control. Optically isolated electronic I/O interfaces are used between the robot controller and pneumatic actuators and position sensors on the experimental apparatus. The vertical robot arm has been equipped with an SMC pneumatic gripper and a specially designed pair of tongs, that is used to select and mount the required samples. The dispensing Dewar is filled with LN 2 and will shortly be equipped with an automatic top-up system. The lid on top of the Dewar is pneumatically actuated and can be driven by either the computer or by a foot switch on the floor. The Dewar is conveniently situated near the experimental table and is easily accessible to the user. View of the entire robot mounting system. The crystal mounting robot Cassette being removed from CP100 dry shipping Dewar. Cassette being loaded into the K3 dispensing Dewar. The HC35 long term storage Dewar. The cassette and transfer handle. The Standard Cassette To provide maximum convenience and flexibility to the user and simplify transporting, storing and mounting a very large number of sample crystals, a compact “Standard” sample cassette has been developed and is shown below. Each cassette has ports for 96 samples mounted on standard Hampton Research CrystalCap Magnetic or CrystalCap Copper Magnetic Pins. There are 8 ports in the vertical direction and 12 ports around the circumference. Sectional plan and side views of the cassette are shown on the right to demonstrate the internal packing of the Hampton pins. Each pin is held in place by means of a NdFeB ring magnet (shown in blue in the detailed drawing on the bottom right) and the pin is slightly recessed for protection. The bottom of the cassette has a central conical hole used to position the cassette accurately on a location platform in the dispensing Dewar. A radial slot in the bottom is used to orient the cassette on an orientation pin. Each sample crystal is stored in a narrow tunnel within the cassette so that when LN 2 drains from the cassette during transfer from one cryo-container to another, the crystals will be maintained at ~LN 2 temperature. The cylindrical cassette was designed to fit snugly inside the inner cage of a Talor-Wharton CX100 dry shipping Dewar. Close up view of a single port. Installation at SSRL Transferring a Cassette from Shipping to Dispensing Dewar The MVE SC 4/2v or the Taylor Wharton CX100 transport Dewar have been adopted as our standard shipping Dewars for the cassettes. They have the ideal size for transporting a single cassette. It is convenient for the user to load and extract the cassette from the internal sleeve with a specially designed transfer handle shown in the pictures below. This handle locks (using a 90 o rotation) into a slot in the top of the cassette and is firmly attached until it is again unlocked. After the cassettes arrive at SSRL and they are required on the beam line, the user transfers the cassette into the dispensing Dewar after opening the lid using a handle on the lid. If, after arrival at SSRL, the cassettes that are not going to be used for a few days can be stored in a Taylor Wharton HC35 storage Dewar (pictured below) that is capable of storing up to 10 cassettes for several months without refilling. Description of the Robot Mounting Mechanism 1-5 11-1 9-2 9-1 Hard anodized aluminum cassette NdFeB ring magnet Hampton style pins Dispensing Dewar 4-Axis Robot Huber Goniometer Collimator Cryo-nozzle Sample Camera Gripper Arms Hutch Table Vertically opening gripper arms Fingers to Hold Dumbell Magnet Tool Cryo-tong Cavity SSRL is funded by: Department of Energy, Office of Basic Energy Sciences The Structural Molecular Biology Program is supported by: National Institutes of Health, National Center for Research Resources, Biomedical Technology Program NIH, National Institute of General Medical Sciences and by the Department of Energy, Office of Biological and Environmental Research. Aina Cohen Paul Ellis Mitchell Miller Ashley Deacon Paul Phizackerlery Keith Hodgson Michael Hollenbeck Peter Kuhn John Kovarik Michael Soltis Ross Floyd Ron Reyes Most of the SSRL PX Group at Their Post Vladimir Vinetskiy Henry Meier Renato Avelar Henry van den Bedem Güenter Wolf Timothy McPhillips Scott McPhillips A new screening tab has been added to BLU-ICE. This tab allows one to automatically collect images from crystals stored in the cassettes. An auto-centering routine is used to align the sample loops to the x-ray beam. Users may upload crystal information to SSRL using a web based form prior to their beamtime. The procedure for mounting a crystal: 1)The Dewar lid is opened and the tongs are moved from the de-icer. 2)The robot cools the cryo-tongs in the liquid nitrogen, within the dispensing Dewar, for approximately 20 seconds. 3)The robot picks up the dumb-bell magnet from the dumb-bell cradle with the fingers projecting from the cryo-tongs. 4)The robot positions the strong end of the dumb-bell magnet adjacent to the required sample pin. 5)The dumb-bell is moved radially away from the cassette. The strong end of the dumb-bell magnet tool holds the pin more tightly than the ring magnet in the cassette and the sample is removed from the port. 6)The dumb-bell magnet tool, with the sample pin still attached, is returned to the cradle and is released by the tongs. 7)The cryo-tongs, which are now fully cooled, move to surround the sample pin and are then closed around it. 8)The tongs are rotated about a vertical axis to release the pin from the dumb-bell magnet; they are then moved swiftly out of the liquid nitrogen and towards the beam line goniometer. 9)The base of the pin is positioned against the goniometer head permanent magnet. 10)The tongs are opened and moved away from the goniometer, leaving the sample centred in the nitrogen cryo-stream. 11)The tongs are de-iced and dried in preparation for the next operation. The entire mounting operation takes 40 seconds, not including the de-icing of the tongs, which takes an additional 40 seconds. The crystal is transported from under liquid nitrogen onto the goniometer in under 4 seconds The procedure for dismounting a crystal: 1)The Dewar lid is opened and the tongs are moved from the de-icer. 2)The robot cools the cryo-tongs in the liquid nitrogen for 30 seconds. 3)The tongs are closed and moved swiftly out of liquid nitrogen and towards the sample position at the goniometer. 4)When the tongs are near the sample, they open, move to surround the pin, and then close. 5)The tongs are rotated about a vertical axis to release the pin from the goniometer magnet, and are rapidly transferred back to the dispensing Dewar. 6)The sample pin is placed onto the weak end of the dumb-bell magnet tool. 7)The tongs are opened to release the pin onto the dumb-bell magnet. 8)The robot picks up the dumb-bell magnet tool from the cradle. 9)The robot places the pin within the appropriate sample port. 10)The dumb-bell is moved radially away from the cassette. The cassette ring magnet holds the pin more strongly than the weak end of the dumb-bell magnet tool and the sample remains behind. 11)The dumb-bell magnet tool is returned to the cradle and is released by the tongs. 12)The tongs are removed from the dispensing Dewar and the lid is closed. The tongs are then warmed in the de-icer and dried in preparation for the next cycle. The process takes approximately 45 seconds, not including de-icing and the tongs are out of liquid nitrogen for less than 7 seconds.