Protein Crystal Mounting

Atanas Georgiev and Prof. Peter K. Allen
in collaboration with Prof. John Hunt and his group from the Dept. of Biological Sciences


Shovel mounting image

Fig.1: A crystal mounted on a microshovel

The Protein Crystal Mounting project addresses the need in the Protein Crystallography community for high-throughput (HTP) equipment which will help improve the execution of a specialized task, called crystal mounting. The goal of this project is to produce a microrobotic system capable of performing the task autonomously, quickly and robustly. We have built a robotic system for crystal mounting which relies on visual feedback from a camera looking through a microscope to control a micromanipulator with the tool used for mounting attached as its end-effector.

The motivation for this project, along with some background information, is described separately.

Task Description

Tools for crystal mounting

Fig.2: Crystal mounting tools

Telegrasping video

Video: A crystal mounting example (8MB)

Crystal mounting diagram

Fig.3: Crystal mounting in the HTP pipeline

Crystal mounting is simply described as the transfer of a selected protein crystal from its growth solution into a suitable mounting tool for data collection on a synchrotron. The task begins by placing a coverslip (usually a 21mm x 21mm square plastic slide, such as the ones used for 24-well Linbro plates) containing a droplet with protein crystals is placed under the microscope. The technician, looking through the microscope, uses a tool to catch and pick up the selected target crystal. The crystal is then quickly cryoprotected, frozen and stored for future data collection on an X-ray beamline.

The most-commonly used tool for mounting is the cryogenic loop (e.g. ones made by Hampton Research) though glass capillaries are also used. Recently, newly developed tools have been introduced, such as the micromounts manufactured by MiTeGen and our own microshovels. Fig.2 gives a visual comparison of the loop, the micromount and the microshovel.

The task is currently performed by skilled technicians. Mounting a crystal in a loop manually requires time, patience and excellent motor skills. Accuracy and speed are critical as the crystals are fragile and very sensitive to environmental changes. Dehydration quickly leads to crystal quality degradation. The video in the side panel is an example of what the task entails --- the loop in this video was installed on a micropositioner which was teleoperated. In reality, the task may be further complicated by the limited time for operation, "skins" forming on the surface of the droplet and crystals adhering to the coverslip.

Crystal mounting is a term that has been used to refer to both the task of picking up a protein crystal (a.k.a crystal harvesting) and the task of placing the tool with the crystal already on it on the beamline. In our work, we use the term to mean the former, and the latter we refer to as beamline mounting to avoid confusion (Fig.3).

System Design And Operation

We have designed and assembled a microrobotic system for protein crystal manipulation, which we use for our research and experiments. An earlier version of the system successfully demonstrated the use of our microshovels for autonomous mounting. The current implementation relies on a two-stage approach, where one tool (a glass pipette) is used to pick up the crystal from the incubation drop and transfer it to another tool (a micromount or a microshovel), which holds the crystal during X-ray data collection.

Crystal mounting setup image

Fig.4: Crystal mounting setup

Control system block diagram

Fig.5: Control system block diagram

Automatic crystal mounting video

Video: Automatic crystal mounting (1MB)

This method is beneficial for several reasons. First, it makes catching the crystal more robust, since the microinjector can adjust the amount of suction it applies via the pipette, compared to the methods that use a loop, a micromount or a microshovel directly, which rely on surface tension to hold the crystal. Second, the pipette aspirates some of the mother liquid along with the crystal and provides an enclosed natural environment for the crystal which protects it from dehydration when it is extracted from the drop; the crystal never gets exposed to the damaging effects of room conditions. Thirdly, this approach naturally combines two steps of the pipeline: crystal mounting and cryoprotection. And finally, problems typically associated with the use of pipettes in cryoprotection (e.g. difficulty in flash freezing because of lack of sufficient exposure) and data collection (e.g. excessive amount of liquid around the crystal) are avoided by transferring the crystal to a more appropriate tool for the purpose.

The crystal mounting procedure starts with the placement of the necessary tools and objects in the work space (Fig.4). First, a microbridge with cryoprotector is placed at its designated location on the tray. Next, a micromount is installed on the fixture to the right and is positioned adequately so it is immersed in the cryprotector at an angle of approximately 45 degrees and is ready to receive the crystal. Finally, the user places a coverslip with the droplet containing the protein crystals on the microscope tray such that they are in the field of view.

The user starts the program and specifies which crystal (among the possibly many in the drop) is to be mounted. After that, the system proceeds autonomously: It immerses the pipette into the drop, approaches the crystal, aspirates the crystal, transitions from the drop on the coverslip to the cryoprotector in the microbridge and deposits the crystal in the mounting tool. Some of these steps are performed in open-loop fashion because the system is calibrated for the locations and dimensions of the relevant objects and the system actuators meet the requirement for positioning accuracy. The aspiration of the crystal, however, is an example of a step that crucially depends on reliable sensory feedback. To determine the location of the crystal and detect when the crystal is inside the pipette, we use region trackers applied to the visual feed from the camera. A control loop tracks the motion of the crystal as it is drawn into the pipette and adjusts the suction applied by the microinjector accordingly until the crystal is safely inside. A block diagram of the control algorithm is shown in Fig.5. A video of the system in operation is shown in the side panel.