208

CRYOCRYSTALLOGRAPHY NOTES

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for the accuracy o f the claims made.

J. Appl. Cryst. (1996). 29, 208-210

A removable arc for mount ing and recovering f lash-cooled crystals

CHRISTIAN ENGEL, RIK WIERENGA AND PAUL A. TUCKER at Biological Structures Programme, EMBL, Meyerhofstrasse 1, D69012 Heidelberg, Germany. E-mail: tucker@embl-heidelberg.de

(Received 29 June 1995; accepted 22 September 1995)

Abstract

Macromolecular crystallographic data collection is increasingly and more routinely performed at temperatures around 1 O0 K. At these temperatures, radiation damage is greatly reduced and so the possibility of storing and reusing crystals exists. A simple device for the transfer of cryocooled crystals into and out of the data-collection position is described.

the goniometer head to the storage container and vice versa. Devices have been proposed that use a large-arc goniostat (Gamblin & Rodgers, 1993) or a rotatable ann (Mancia et al., 1995). The large-arc goniostat (marketed by Charles Supper Company) has the disadvantage of bulk and is not always suitable, for example on a MAR image-plate scanner, where rotation of the spindle would cause collision of the large arc

Most crystals of biological macromolecules are sensitive to X-ray irradiation, especially when high doses are received, as is the case in experiments conducted at synchrotron sources. The reduction in systematic errors that results when a complete data set is collected from one crystal is important both for the structure determination process itself and for the subsequent model refinement. For multiwavelength anomalous diffraction (MAD) experiments, the ability to collect several data sets from a single crystal is especially important. A way to greatly reduce (Young & Dewan, 1990), though not entirely eliminate (Gonzalez & Nave, 1994; Rodgers et al., 1995), radiation damage is to carry out data collection at cryogenic tempera- tures. Data collection of crystals of biological macromolecules close to liquid-nitrogen temperatures has become increasingly routine, not least because the reduction of radiation damage allows crystals to be stored and reused. Equally well, freshly grown crystals that are unstable in the original mother liquor can be stored at liquid-nitrogen temperatures for subsequent use. Ideally, cryocooled crystals are first characterized using in- house X-ray facilities, and subsequently stored and transported in liquid nitrogen, before the data collection is carried out at a synchrotron source.

In the cryocooling procedure, crystals are cooled extremely rapidly to prevent formation of ice crystals. In order to achieve this, the crystal is first transferred into a suitable cryoprotectant solution (Rodgers, 1994). Subsequently, the crystal is mounted on or in a loop (Teng, 1990) made of an amorphous material such as glass wool, rayon or nylon. This loop can be formed at the thin end of a pulled glass capillary such as a micro injection capillary (Sauer, 1995). The capillary is glued into a copper pin that is itself held in a ferrous metal base, referred to as the pin holder (Fig. 1). This pin holder can be quickly and smoothly mounted on a magnetic strip attached to the goniometer head (Gamblin & Rodgers, 1993). The crystals are flash cooled in a cold nitrogen gas stream or directly in a liquid cryogen such as propane, ethane, freon or nitrogen (Rodgers, 1994). A remaining problem is how to transfer the frozen crystal from

© 1996 International Union of Crystallography Printed in Great Britain - all rights reserved

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Fig. 1. The device mounted on a goniometer head. Labels indicate: 1 removable arc, 2 copper pin with loop (too thin to be visible) at its top, 3 ferrous metal pin holder, 4 magnetic strip, 5 pin-holder plate with heating block (visible on the right), 6 base plate, 7 goniometer head.

Journal of Applied Crystallography ISSN 0021-8898 © 1996

C R Y O C R Y S T A L L O G R A P H Y NOTES 209

(a)

(b)

(c) Fig. 2. The device mounted on a MAR scanner goniostat, viewed

approximately along the X-ray beam, towards the X-ray source. (a) The device is shown in data-collection mode; the arc is removed. The cold nitrogen stream comes from the nozzle visible at the right side of the picture. (b) The recovery mode is shown. The arc has been attached to the base plate and the pin holder has been moved upwards, with the pin pointing vertically downwards. The crystal is still in the cold gas stream. (c) The recovery procedure. A cryovial, filled with liquid nilxogen, is slipped over the pin and pin holder.

with the collimator or shutter housing. The rotatable ann was designed for a 60 ° rotation but for many goniostats a larger degree of rotation is preferable.

Here, we describe a device that uses a removable arc (Fig. 1). During data collection, the arc is removed and the device is small enough not to interfere with a rotation experiment on an instrument where the space available for the sample is small. In order to transfer the crystal to and from storage, the arc is attached to the device and the pin-holder plate is moved around the arc such that the pin points vertically downwards to allow convenient and quick transfer of the crystal. The device consists of three pieces: a base plate, a removable arc and a pin-holder plate including a heating block (Fig. 1). The brass base plate is attached to a standard goniometer head via a ferrous metal pin. The removable arc, also made from brass, can be attached to this base plate and is pinned and locked by a screw (Figs. 2a and b). It is used as a slide to move the brass pin-holder plate from the data-collection position (Fig. 2a) to the recovery position at the top of the arc (Fig. 2b). The pin-holder plate is an open-ended hollow right-rectangnlar prism with a slot on one side to accomodate the goniometer mounting pin (bottom side, as shown in Fig. 1), the magnetic strip and locating pin on the opposite side (top side, as shown in Fig. I), a locking screw on a third side and an additional brass extension to hold the power resistor on the remaining side. The goniometer mounting pin also defines the exact position of the pin-holder plate during data collection.

After removal of the arc for data collection, the whole device is only slightly longer than the goniometer head itself and therefore does not constrain the experimental set-up. The angular range of the arc can be varied in order to accommodate different goniostat geometries. For example, MAR image-plate scanners have a horizontal rotation axis, therefore a 90 ° rotation is convenient to set the pin pointing vertically downwards, which is optimal for quick transfer. In contrast, the three-axis goniostat of the Siemens X100A instrument has a rotation axis inclined at 45 ° to the vertical and therefore an arc of 135 ° is appropriate. The pin-holder plate can slide over the arc and can be fixed in any position with a screw. It contains a magnetic strip with a locating pin in the center, so the pin holder can be quickly and accurately moved on and off the pin-holder plate. The heating block is part of the pin-holder plate for good thermal transfer and during data collection it heats the whole device via an electric resistance (220 fl, 3 W power resistor, running at approximately 30V), which in turn prevents ice formation on the pin holder or goniometer head. The heater allows the device to be used without a dry enclosure around the instrument provided always that the cold gas stream is surrounded by an ambient temperature cylinder of dry gas.

For the recovery/storage procedure, the pin-holder plate can be unlocked and moved around the arc from the data-collection position (with the pin horizontal on a MAR scanner, Fig. 2a) to the recovery position (with the pin pointing vertically down- wards, Fig. 2b) where it can be locked. The loop is mounted on the device in such a way that the crystal always lies exactly in the midpoint of the circle the arc is describing (Fig. 2b). The cold gas stream is centered on this midpoint and therefore the crystal does not move out of the gas stream during the procedure. The essential step of the procedure is the transfer of the complete pin/pin-holder arrangement with the crystal mounted inside the loop out of or into the cryovial filled with liquid nitrogen (Fig. 2c). To mount the crystal on the

210 CRYOCRYSTALLOGRAPHY NOTES

goniometer head, the pin-holder plate with the magnetic strip is moved into the recovery position and is fixed with the screw. The pin holder in the cryovial is brought up under the pin- holder plate and after self-attachment of the pin holder to the magnetic strip the eryovial is quickly stripped off. The plate is moved back into the data-collection position and the arc is removed. The recovery of the crystal for storage is exactly the reverse of this procedure.

For storage, 1.8 ml Nunc cryovials with the thread on the outside of the tube can be used. The pin holder fits exactly into the opening of the cryovial and fixes the pin inside the tube, preventing the loop from touching the inner walls. The screw cap locks the pin holder rigidly into the cryovial. With this set- up, the crystals can be stored di~ctly in liquid nitrogen without any support of solidified propane or ethane around the crystal. In order to allow free exchange of evaporated nitrogen inside the cryovial with liquid nitrogen from the storage vessel, there are small holes drilled into the upper end of the Nunc tube. When using liquid nitrogen for storage, one has about 5 s to move the pin on or off the magnetic strip before the nitrogen boil off becomes too violent. The Nunc cryovials are fixed in standard cell-culture canes and stored in a dewar filled with liquid nitrogen.

This removable-arc set-up has been used routinely during several data-collection visits to synchrotrons. The device can also be used to collect data of the missing cusp region on an instrument with a single fixed rotation axis. For this purpose, the arc is attached to the base plate and the pin-holder plate is

slid upwards as for the recovery procedure. The region of reciprocal space originally inaccessible by rotation about the spindle axis is therefore now rotated 90 ° about the beam direction and can be collected in a second rotation experiment. In this position, the device can be rotated approximately 30 ° before the arc starts to shadow the detector.

A drawing of this device is available free of charge from the authors.

We thank Peter Everitt and the mechanical workshop of EMBL for their help, as well as our colleagues for useful discussions.

References

Gamblin, S. J. & Rodgers, D. W. (1993). Proceedings of the CCP4 Study Weekend 1993, pp. 28-32. SERC Daresbury Laboratory, Warrington, England.

Gonzalez, A. & Nave, C. (1994). Acta Cryst. DS0, 874-877. Mancia, E, Oubridge, C., Hellon, C., Woollard, T., Groves, J. & Nagai,

K. (1995). J Appl. Cryst. 28, 224-225. Rodgers, D. W. (1994). Structure, 15, 1135-1140. Rodgers, D. W, Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S.,

HeUmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995). Proc. Natl. Acad. Sci. USA, 92, 1222-1226.

Saner, U. (1995). Personal communication. Teng, T. (1990). J Appl. Cryst. 23, 387-391. Young, A. C. M. & Dewan, J. C. (1990). J. Appl. Cryst. 23, 215-218.