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CRYOCRYSTALLOGRAPHY PAPERS

Contributions intended for this section should be submitted to Dr P. C. Weber, Schering-Plough Research Institute, K-15-3/3855, 2015 Galloping Hill Road, Kenilworth, NJ 07033-0539, USA. They may be full research papers, short communications or brief notes. The International Union of Crystallography can assume no responsibility for the accuracy of the claims made.

J. Appl. Cryst. (1997). 30, 71-72

A simple method for making reproducible fibre loops for protein cryocrystallography

U. H. SAUER* AND T. A. CESKA at European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. E-mail: uwe@xray l.ucmp.umu.se

(Received 15 September 1995; accepted 25 July 1996)

Abstract

A practical, step-by-step description of the construction of loops for protein cryocrystallography using pulled-out micro- injection capillaries is described. The advantages of this method

are the high reproducibility of the loops, the ease of adjustment of the loop diameter and the formation of thin films owing to

* Present address: Ume~t University, Ume/t Center for Molecular Pathogenesis (UCMP), S-90187, Ume~, Sweden.

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© 1997 International Union of Crystallography Printed in Great Britain - all rights reserved

Fig. 1. How the loop is made. First, the tip of the pulled-out capillary is cut such that its diameter is about twice the diameter of the fibre. A second cut is made 10 mm below the tip with a diamond glass cutter. It is then temporarily attached to a glass slide with tape. (a) A 30 ram-long nylon fiber is threaded through the capillary from the wide end. A 15 mm length is allowed to emerge from the narrow opening at the tip. (b) A loop is formed by passing of the fibre back into the narrow opening until it emerges again at the wide end. (c) The loop diameter can be adjusted by pulling of both extruding fibre ends. Typical loop sizes serving most purposes are between 0.3 and 1.0 man. (d) A drop of low-viscosity instant glue is applied at the bottom end of the glass capillary. It will be drawn up to the tip by capillary action. When dry, the excess length of fibre is trimmed. (e) The capillary is inserted into the copper pin bore and the loop positioned about 2- 3 mm above the rim. (f) When positioned correctly, the gap between the copper pin and the capillary is sealed with instant glue. A mound of glue around the glass capillary mini- mizes turbulence in the cryostream. To pull out the capillaries, we use a microinjeetion micropipette puller from Sutter Instruments Company (Novato, California, USA), model P-87. Instrument settings on the micropipette puller are: heat 640; pull 50; velocity 30; time 100. Capillaries were obtained from Clark Electromedical Instruments (Reading, England), part number GC 120F-10.

Journal of Applied Crystallography ISSN 0021-8898 O 1997

72 CRYOCRYSTALLOGRAPHY PAPERS

the narrow base of the loop. During assembly, the loop does not make contact with either glue or cement.

1. Introduction

Cryogenic methods in protein crystallography have become more accepted and widely distributed as their advantages are revealed (Rodgers, 1994). The importance of cryocooling protein crystals will increase as higher-intensity synchrotron- radiation sources become available. Crystals are X-ray sensitive owing to the creation and subsequent diffusion of free radicals that cleave bonds in the protein. Freezing greatly reduces the diffusion of free radicals thus extending the crystal lifetime. For example, a good heavy-atom derivative can lead to enhanced sensitivity to radiation damage and freezing may be the only way to collect a complete data set from a single crystal. This was the case for mercury derivatives of PCD/DCoH (Ficner, Sauer, Stier & Suck, 1995). We also found that, under favourable circumstances, it is possible to freeze and thaw crystals more than once with little change in mosaic spread, intensity distribution and resolution. It was thus possible to collect native and derivative data sets from a single crystal.

In early reports of the construction of loops, a fine copper wire was used to suspend the crystals (Teng, 1990). Several techniques have recently been described for loop construction that use thin synthetic fibres (e.g. Blond, Pares & Kahn, 1995).

2. Description

The novelty of the method presented here is the use of a finely pulled-out microinjection capillary to support the fibre loop. The reduced cross section of the loop base minimizes the amount of liquid retracted with the loaded loop from the drop. The glass capillary is glued into a tapered copper pin. Fig. 1 shows the steps of the loop-making technique. Fig. 2 shows the loop and pin in a pin holder.

The pin holders commonly used in many laboratories are made of ferrous metal held in place via a magnetic base attached to the goniometer head. In our case, the pin holder has a central bore, allowing for z translation in order to compensate for variations of the loop position. The shape of the pin holder is designed to easily fit into and out of a Nunc tube for convenient storage of frozen crystals in liquid nitrogen. A removable arc device facilitating the transfer of the frozen crystal to a Nunc tube was described by Engel, Wierenga & Tucker (1996). This device is heated to prevent icing of the goniometer head. Besides supporting the loop, the glass capillary acts as a thermal insulator and allows a laminar flow of cryogen gas to pass the frozen crystal.

iii ii ̧ii

Fig. 2. The loop and pin in a pin holder. The pin can move in and out of the pin holder, and is secured by a screw in the side of the pin holder. The pin holder fits into a Nunc tube for storage in liquid nitrogen.

(iv) The noncircular teardrop shape of the loop allows some control of crystal alignment. (A general advantage of fibre loops is that they can be distorted by systematic pinching of the fibre. This may aid in the alignment of crystals if a particular orientation is desired.)

(v) The very small cross section of the base of the loop attracts little liquid. Hence, very even thin films are formed which vitrify more easily and show reduced background scattering.

(vi) The pin and mounts can be used with a goniostat heater, since the glass capillary acts as an insulator between the copper pin and the loop. We have found the loop-mounted pins a useful tool for gentle crystal handling.

A detailed technical drawing of the pins and pin holder is available from the authors.

We thank Paul Tucker (EMBL, Heidelberg), Thomas Schneider (EMBL, Hamburg) and Steve Ginnell (National Synchrotron Light Source, Brookhaven) for discussions that led us to the current design of our loops and pins.

3. Concluding remarks

We find there are several advantages of this method for making loops:

(i) The loops are highly reproducible. (ii) The loop is never in direct contact with glue during

assembly, which reduces the possibility of ice nucleation from imperfections on the surface of the loop.

(iii) The diameter of the loop is easily adjusted during loop making, allowing a variety of sizes to be made to accomodate the dimensions of the crystals available.

References

Blond, L., Pares, S. and Kahn, R. (1995). J. Appl. Cryst. 28, 653-654. Engel, C., Wierenga, R. & Tucker, P. A. (1996). J. Appl. Cryst. 29,

208-210. Ficner, R., Sauer, U. H., Stier, G. & Suck, D. (1995). EMBO J. 14,

2034-2042. Rodgers, D. W. (1994). Structure, 2, 1135-1140. Teng, T.-Y. (1990). J. Appl. Cryst. 23, 387-391.