International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 9.2, pp. 231-233

Section 9.2.2. Robotic sample loaders

T. Earnesta* and C. Corka

aStructural Proteomics Development Group, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Correspondence e-mail:  TNEarnest@lbl.gov

9.2.2. Robotic sample loaders

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Significant amounts of synchrotron beam time can be lost due to crystal manipulation and misalignment by the beamline user, which becomes more critical with the increase in brightness and stability of the newer synchrotron sources. Crystal-screening and data-collection runs have become significantly shorter. Therefore, more efficient mounting and alignment tools have been developed that reduce the fraction of time spent changing samples, including the implementation of robotic sample loaders (automounters), which can greatly facilitate the throughput and production of crystallography beamlines and other X-ray sources.

There are several benefits to using an automounter system, particularly on bright synchrotron beamlines:

  • (i) It facilitates optimum use of synchrotron beam time. Crystallography experiments are installed in radiation-shielded hutches, which are inaccessible during data collection. Changing the crystal manually requires opening and closing the hutch, initiating the interlocks and performing a hutch search, which typically takes several minutes. The automounter significantly reduces both the sample mounting time and the number of required hutch accesses.

  • (ii) It facilitates advanced data-collection techniques. An experimental station can be fully automated, including integrated data collection and processing whereby the structure-solving software can influence the data-collection process (e.g. crystal ranking and data-collection strategy determination). Remote data collection, where the researchers send cryoprotected crystals (maintained near liquid-nitrogen temperatures) to the beamline for robotic mounting and automated (or semi-automated) data collection, can proceed in a flexibly scheduled manner.

  • (iii) It facilitates the collection of higher-quality data. The rapid crystal-interchange mechanism enables the researcher to evaluate a large pool of samples and to select the best crystals from the set.

  • (iv) It reduces risk to crystals. Automated mounting and dismounting of crystals can be done much more reliably than manual handling.

  • (v) It facilitates systematic studies of experimental protocols. Alternative protocols can be performed in a manner and number that would be impractical for humans to perform manually. Furthermore, this can allow for an intelligent system to `learn' improved methods of data collection and processing.

The major challenge in the design of automation hardware for the mounting and alignment of crystals of biological samples is the necessity to maintain the sample at approximately 100 K, since cryogenic data collection is required at synchrotron beamlines and usually at home sources as well. The process involves the optical screening of several candidate crystals under a microscope, followed by `freezing' the selected crystal in an amorphous glass formed by the mixing of reservoir and cryoprotectant solutions. In this manner, it is possible to manipulate small or fragile crystals more easily. More importantly, the frozen crystals are far more resistant to X-ray induced radiation damage, a common problem at synchrotron sources. The frozen crystals can then be stored indefinitely in liquid nitrogen and handled with minimum difficulty at cryogenic temperatures. There are established procedures for the manual manipulation and mounting of the crystal while at cryogenic temperatures. At the beamline, the crystals are transferred from the transport container to a small holding Dewar. Next, they are mounted in a secondary sample-transfer tool, and then they are transferred to the X-ray diffractometer. These can be very time-consuming steps when performed manually. Consequently, instrumentation has been developed to minimize the extent of manual manipulations of frozen crystals at the beamline (Snell et al., 2004[link]; Cork et al., 2006[link]) (Fig. 9.2.2.1[link]). Groups at Abbott (Muchmore et al., 2000[link]), at the SSRL (Cohen et al., 2002[link]), at European facilities (Cipriani et al., 2006[link]) and in Japan (Ueno et al., 2006[link]) have similarly produced robotic crystal-mounting systems with a diversity of approaches to achieve the same goal. Commercial systems have also been developed (e.g. the Rigaku ACTOR and the IRELEC CATS).

[Figure 9.2.2.1]

Figure 9.2.2.1 | top | pdf |

(a) Hardware for the storage and transport of crystals. Each puck holds up to 16 crystals, and seven can be shipped in a standard cryo-shipper. (b) The robotic crystal mounter implemented on Beamline 5.0.2 at the Advanced Light Source.

There are two basic approaches that have been pursued: (i) the use of commercial multi-axis robots (Muchmore et al., 2000[link]; Cohen et al., 2002[link]; Rigaku ACTOR) and (ii) modular purpose-built systems, such as the one developed at the Lawrence Berkeley Laboratory Advanced Light Source and shown in Fig. 9.2.2.1[link](b) (Snell et al., 2004[link]). All systems must take into consideration that the procedures used to transfer samples from a crystal tray and mount them on the goniometer need to ensure that the crystals be maintained in a frozen frost-free environment and transferred rapidly. For example, the system developed at the Advanced Light Source can be adapted for use with a variety of experimental station configurations. The only significant constraint in the use of this design is that the nitrogen cold stream subsystem must be oriented off-axis from the primary goniometer axis. The first automounter was installed on experimental station BL5.0.3 at the Advanced Light Source, a fixed wavelength station with significant access constraints. This system has been in operation since March 2001. Shortly thereafter, systems were installed on a second monochromatic experimental station, BL5.0.1, and also on a multiwavelength experimental station, BL5.0.2 (Snell et al., 2004[link]). The automounter configuration is essentially identical for all three experimental stations with a high degree of standardization. The sample-mounting robot consists of three main components: the gripper, which holds the sample during transport from the Dewar to the goniometer while keeping it at low temperature and frost-free; the XY–θ stage, which performs the actual transport; and the Dewar stage, which supports the samples in a regular array submerged in liquid nitrogen and positions the selected sample for access by the transporter (Fig. 9.2.2.1[link]b).

The gripper contains a conically shaped brass collet that can be opened and closed using a small pneumatic actuator (Fig. 9.2.2.2[link]). The collet is precooled to ∼100 K before engaging the sample holder. A sensor inside the collet is used to monitor its temperature. The inner tube is surrounded by a low thermal capacity outer shroud (a very thin stainless steel tube), which provides a sheath flow of warm dry gas to reduce icing and frost formation during exposure to ambient moisture-laden air. During continuous use, ice is periodically removed from the gripper by means of an air-jet heater (cf. Fig 9.2.2.1[link]b). Additionally, the gripper has a small low-force linear stage (labelled `small move' in Fig. 9.2.2.2[link]). This stage is used for the final positioning of the gripper on the sample during mounting and dismounting. The sample gripper was originally designed around the standard Hampton metal cap, which is held on the goniometer by a permanent magnet. A modified cap design, which has tighter tolerances and a conical shape rather than a ledge, has been designed to increase automounter reliability (also available from Hampton Research as part No. HR4–779).

[Figure 9.2.2.2]

Figure 9.2.2.2 | top | pdf |

Crystal gripper assembly with low-force actuator (labelled `small move').

The gripper is mounted on a pneumatic XY–θ stage, which is used to transport samples between the goniometer and the storage Dewar. A vertical Y stage moves the gripper in and out of the Dewar, a 90° rotational θ stage orients the gripper either horizontally or vertically, and a long horizontal X stage moves the gripper between the Dewar and the goniometer mount points.

When they are not mounted on the goniometer for data collection, the samples are maintained in a small cylindrical Dewar (see Fig. 9.2.2.1[link]b). A custom cassette or puck (Fig. 9.2.2.1[link]a) facilitates automated handling and bulk transport. Up to seven cassettes (112 samples) can be loaded into the sample Dewar. The Dewar is mounted on an R–θ motorized stage, which is used to position the selected sample for access by the gripper. The Dewar can also be positioned such that the gripper will be inserted into an unoccupied space for a precooling action when boiling of liquid nitrogen might endanger the samples. The Dewar is automatically filled directly from the facility's liquid-nitrogen supply system. During normal operations, an insulated and heated cover reduces icing and evaporation of liquid nitrogen. The gripper reaches into the Dewar through a small heated hole in the cover. The sample cassettes are moved in or out of the Dewar through a small access port in the cover.

The Robosync web site (http://smb.slac.stanford.edu/robosync/ ), maintained by the SSRL Structural Molecular Biology Group, features a list of biological crystallography beamlines with automated crystal mounting systems.

Important for the efficient and productive use of robotic crystal mounters is their integration into the beamline control system (McPhillips et al., 2002[link]; Snell et al., 2004[link]). The different motions of the automounter can typically be actuated from a software interface, either individually or through scripts which run a sequence of actions. The development of more fully automated capabilities requires the integration of the automounter and beamline control system with the data collection and analysis software. To this end, software has been developed that provides a framework for indexing integration and symmetry determination, along with suggested strategies for data collection (González et al., 2008[link]). When coupled with bright synchrotron beamlines and automounters, the underlying requirements for building fully automated and optimized data collection are mostly in place. The development of intelligent automated control (through e.g. artificial-intelligence approaches) should permit fully automated data-collection facilities to collect optimized data from a wide range of structural projects in the most efficient and cost-effective way and with minimal human intervention prior to the scientific analysis of the structures obtained.

References

Cipriani, F., Felisaz, F., Launer, L., Aksoy, J.-S., Caserotto, H., Cusack, S., Dallery, M., di-Chiaro, F., Guijarro, M., Huet, J., Larsen, S., Lentini, M., McCarthy, J., McSweeney, S., Ravelli, R., Renier, M., Taffut, C., Thompson, A., Leonard, G. A. & Walsh, M. A. (2006). Automation of sample mounting for macromolecular crystallography. Acta Cryst. D62, 1251–1259.
Cohen, A. E., Ellis, P. J., Miller, M. D., Deacon, A. M. & Phizackerley, R. P. (2002). An automated system to mount cryo-cooled protein crystals on a synchrotron beamline, using compact sample cassettes and a small-scale robot. J. Appl. Cryst. 35, 720–726.
Cork, C., O'Neill, J., Taylor, J. & Earnest, T. (2006). Advanced beamline automation for biological crystallography experiments. Acta Cryst. D62, 852–858.
González, A., Moorhead, P., McPhillips, S. E., Song, J., Sharp, K., Taylor, J. R., Adams, P. D., Sauter, N. K. & Soltis, S. M. (2008). Web-Ice: integrated data collection and analysis for macromolecular crystallography. J. Appl. Cryst. 41, 176–184.
McPhillips, T. M., McPhillips, S. E., Chiu, H.-J., Cohen, A. E., Deacon, A. M., Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P., Soltis, S. M. & Kuhn, P. (2002). Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Rad. 9, 401–406.
Muchmore, S. W., Olson, J., Jones, R., Pan, J., Blum, M., Greer, J., Merrick, S. M., Magdalinos, P. & Nienaber, V. L. (2000). Automated crystal mounting and data collection for protein crystallography. Structure, 8, R243–R246.
Snell, G., Nordmeyer, R., Cornell, E., Meigs, G., Cork, C., Yegian, D., Jaklevic, J., Jin, J., Stevens, R. C. & Earnest, T. (2004). Automatic sample mounting and alignment system for biological crystallography at a synchrotron source. Structure, 12, 536–545.
Ueno, G., Kanda, H., Hirose, R., Ida, K., Kumasaka, T. & Yamamoto, M. (2006). RIKEN structural genomics beamlines at the SPring-8; high throughput protein crystallography with automated beamline operation. J. Struct. Funct. Genom. 7, 15–22.








































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