Capillaries, mounting crystals

Two weeks are enough for droplets of about 200 nl to 3 jl1 to equilibrate imder any conditions (Mikol et al., 1990). During this period, droplets should be inspected daily to foUow up the appearance of crystals. Crystals may still form after 2 weeks, but this is less likely in the case of oligonucleotides. Crystals can then be cryoprotected and frozen or capillary-mounted to be tested. Fluidigm markets crystallization chips dedicated to crystal growth optimization which can sustain and are transparent to X-rays in order to discriminate between salt and macromolecule crystals. Extracting the crystal from the chip is performed only for crystals deserving data collection. 

The classical method of mounting crystals is to transfer them into a fine glass capillary along with a droplet of the mother liquor. The capillary is then sealed at both ends and mounted onto a goniometer head (see Fig. 4.20, and Chapter 4, Section III.D), a device that allows control of the crystal s orientation in the X-ray beam. The droplet of mother liquor keeps the crystal hydrated. 

Figure H.4. The crystals are manipulated by scooping them up with a small loop of nylon that is glued to the end of a pin. Surface tension firom the liquid will hold the crystal in the loop, but the crystal can also be held by using a loop that is smaller in size than the crystal of interest. This technique will work particularly well with fragile crystals, thin plates for example, that would normally fall apart in a capillary mount. Once the crystal is frozen, it is placed on an axis in line of both an X-ray source and a stream of nitrogen set to about 100,000to keep the crystal frozen. The crystal is rotated in increments during the data collection procedure to collect a full data set (typically one or two degrees per frame, depending on the resolution limits, mosaicity of the crystal, unit cell lengths, etc.).

FIGURE 7.7. Methods of mounting crystals, (a) A crystal mounted in a glass fiber, as used for a small-molecule crystal that does not decompose on exposure to air. (b) Diagram of the mounting of a crystal in a capillary tube, (c) and (d). A crystal of a chemically modified horse hemoglobin enclosed with mother liquor in a thin walled glass capillary. (Courtesy J. J. Stezowski). 

In low temperature studies, it is preferable to mount the crystal in a flow cell [215]. This permits the gradual change of cryoprotectant concentration as the temperature is lowered and substrate is introduced into the crystal. A quartz capillary is attached to a brass support by epoxy cement and a tight-fitting polyethylene tube sealed to the bottom of the capillary. The capillary is filled with protein mother liquor solution. The crystal is introduced to the top and allowed to settle either on to a bed of pipe cleaner fibres placed on top of the polyethylene tube as support or on to a support made by previously introducing a constriction into the quartz capillary. The crystal may be secured further by additional fibres. The inlet polyethylene tube is sealed to the top of the capillary. The inlet and outlet tubes are then connected to the reservoir and sink, respectively (Fig. 13). 

Recrystallisation of [(CH3)4N]2 [p-t-butylcalix[6]arene - 2H] from acetonitrile provided a mixture of relatively large rhombs and some very fine needles. The structure determination was performed on a capillary-mounted specimen of the larger crystals. 

Figure 5.8 A Debye-Scherrer powder camera for X-ray diffraction. The camera (a) consists of a long strip of photographic film fitted inside a disk. The sample (usually contained within a quartz capillary tube) is mounted vertically at the center of the camera and rotated slowly around its vertical axis. X-rays enter from the left, are scattered by the sample, and the undeflected part of the beam exits at the right. After about 24 hours the film is removed (b), and, following development, shows the diffraction pattern as a series of pairs of dark lines, symmetric about the exit slit. The diffraction angle (20) is measured from the film, and used to calculate the d spacings of the crystal from Bragg s law.

Cryostats have been much used in small-molecule crystallography because they allow rapid cooling to very low temperatures with minimal cold gas consumption and offer great advantages in the area of frost prevention. Unfortunately, most designs employ beryllium shrouds or other nontransparent material, and are of a size which does not lend itself to crystals mounted in capillary tubes and flow cells. A recent advance is the description of a Mylar cryostat specifically designed for... 

In contrast, it is often possible to achieve a modest amount of cooling of crystals mounted in capillaries using an air cooling system, such as the FTS air jet cooler (FTS Systems Inc., P.O. Box 158, Rt 209 Stone Ridge, NY 12484, USA), which allows investigation of the effect of temperatures close to 273 K on a crystal s lifetime and diffraction (Diprose etal., 1999). 

Fig. 6. Lane diffraction pattern re-cordered from a 30 x 35 x 10 jun crystal of gramicidin A mounted in the usual way, sealed in a capillary with mother liquor. Exposure time 0.5 s...

Bearing in mind the technical difBculties arising from volatile organic solvents as precipitants, we also looked for ribosomes that are stable under high salt concentrations they could perhaps be crystallized using conventional precipitants such as ammonium sulfate or other non-volatile agents. Thus the crystals could be handlai with less difficulties and mounted in the conventional way in X-ray capillaries. 

When the Gandolfi camera is used, a single crystal or crystal fragment is mounted on a glass capillary (Refs 11 19). This camera is thus of great value when a very small amount of the phase to be investigated is available... 

The next step is for a protein crystallographer to mount a small perfect crystal in a closed silica capillary tube and to use an X-ray camera to record diffraction patterns such as that in Fig. 3-20. These patterns indicate how perfectly the crystal is formed and how well it diffracts X-rays. The patterns are also used to calculate the dimensions of the unit cell and to assign the crystal to one of the seven crystal systems and one of the 65 enantiomorphic space groups. This provides important information about the relationship of one molecule to another within the unit cell of the crystal. The unit cell (Fig. 3-21) is a parallelopiped... 

Until recently, only two reports existed for 1 1 adducts of silver halides with amines, namely AgT piperidine57 and Agl-morpholine.381 The first had a tetrameric cubane structure whilst the second was described as a stair polymer adduct. The range has now been extended to include 2-and 3-methylpyridine, quinoline and triethylamine.382 In each case the adduct was obtained by recrystallization of silver(I) iodide from neat base. The colourless crystals were found to lose base readily on exposure to the atmosphere and structural data were collected from crystals mounted in argon-filled capillaries, containing mother liquor. 

In all forms of data collection, the crystal is mounted on a goniometer head, a device that allows the crystallographer to set the crystal orientation precisely. The goniometer head (Fig. 4.20) consists of a holder for a capillary tube containing the crystal two arcs (marked by angle scales), which permit rotation of the crystal by 40° in each of two perpendicular planes and two dovetailed sledges, which permit small translations of the arcs for centering the crystal on the rotation axis of the head. 

Protein crystals, either sealed in capillary tubes with mother liquor or flash-frozen in a fiber loop, are mounted on the goniometer head, which is adjusted to center one face of the crystal perpendicular to the X-ray beam and to allow rotation of the crystal while maintaining centering. Flash-frozen crystals are held in a stream of cold nitrogen gas emerging from a reservoir of liquid nitrogen. 

Measuring Raman spectra of polymorphs requires little or no sample preparation. Spectra can be measured from single crystals and from powders. Moreover, samples can be contained in glass capillaries or mounted on a goniometer. As mentioned earlier, fiber optics can be used to interface the instrument with the samples. Thus the measurements are straightforward and easy to perform, whereas the analyses produce information on structures and spectral fingerprints for straightforward identifications. 

Of course, the solubilities of AY, BX, and BY have to be considerably larger than that of AX. As is shown in Fig. 1, the double-infusion crystallizer consists of a thermostated (filled with oil) Pyrex vessel containing the saturated solution and eventually some seed crystals mounted on a stirrer. The solutions AY and BX are introduced through glass capillary tubes by means of a micropump t... 

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