Handling of Crystals

To be able to measure crystal diffraction, it is necessary to remove the crystal from the drop in which it has crystallized and introduce it to the x-ray camera. As first observed by Bernal and Crowfoot, protein crystals tend to be fragile and are particularly sensitive to humidity changes. It is therefore necessary to maintain sufficient moisture within the crystal

Figure 12 (a) A typicalin-house x-ray crystallographic facility: (a) rotating anode; (b) confocal mirror optics; (c) x-ray collimator; (d) microscope for aligning crystal in x-ray beam; (e) cryo-cooling equipment; (i) image plate detector. (b) close up of crystal: (c) x-ray collimator exit; (d) microscope; (e) cryohead; (f) goniometer head for positioning crystal; (g) loop containing frozen crystal; (h) beamstop to prevent primary x-ray beam reaching detector. (c) Characteristic x-ray spectra for Cu and Mo anodes; note the sharp inner shell transition lines superimposed on the white Bremsstrahlung radiation.

during data collection. While it is sometimes possible to remove the crystal from the crystal plate and bring it directly to the camera, this can be risky, particularly if several crystals are present in the drop. In most cases, it is wise to prepare a harvesting buffer. This differs from the mother liquor in which the crystal grew in one crucial respect - the mother liquor contains saturating concentrations of dissolved protein in equilibrium with crystalline material. To compensate for this lack of protein, the harvesting buffer contains a higher concentration of the precipitating agent used for crystallization, so that transfer should not lead to dissolution of the crystal (which can be the case if the reservoir solution of the crystallization setup is used to mount the crystal). The harvesting buffer is also used for soaking

10000 100000

10000 100000

Xp Xi

Xp Xi

X-ray energy (eV)

Figure 13 (a) Comparison of the radiation spectrum from a synchrotron source and a conventional rotating anode; not only is there a tremendous increase in intensity, but there is also a wide spectral range. (b) A typical anomalous scattering curve for Se (atomic number 34). Theoretically, the absorption maximum occurs at 12.6578 keV (peak 1p = 0.9795 A) and the dispersive maximum at inflection \ = 0.97935 A; these values can vary due to the atomic environment or the synchrotron and should therefore always be measured experimentally through an x-ray fluorescence scan. For MAD experiments, additional data can be collected below (low remote, 1jr) or above (high remote, l^r) the absorption edge.

X-ray energy (eV)

Figure 13 (a) Comparison of the radiation spectrum from a synchrotron source and a conventional rotating anode; not only is there a tremendous increase in intensity, but there is also a wide spectral range. (b) A typical anomalous scattering curve for Se (atomic number 34). Theoretically, the absorption maximum occurs at 12.6578 keV (peak 1p = 0.9795 A) and the dispersive maximum at inflection \ = 0.97935 A; these values can vary due to the atomic environment or the synchrotron and should therefore always be measured experimentally through an x-ray fluorescence scan. For MAD experiments, additional data can be collected below (low remote, 1jr) or above (high remote, l^r) the absorption edge.

experiments, where a crystal is placed in a buffer in which heavy atoms, halides, substrates, ligands, or inhibitors are dissolved, allowing diffusion of the compound into the crystal prior to subsequent measurement.

To handle crystals, it is helpful to have a polarizing stereo microscope, a set of micro tools (e.g., fine mounted needles such as used by dentists, siliconized capillaries, wax, and/or fine nylon loops) and a steady hand. Once the crystallization vessel has been opened, it is necessary to work quickly, as the drops can dry out and extinguish all hopes of obtaining a reasonable diffraction pattern, so all materials should be at hand before mounting. Crystals often tend to stick to the crystallization plate base or cover slips, but can be prised off gently using a needle. It is also sometimes necessary to cut crystals that have grown together - this can usually be achieved by applying gentle pressure to the interface between the two crystals with a fine needle.

For measurements at room temperature, the crystal can be drawn into x-ray capillaries. Although the crystal must be maintained in a humid environment, there is a high risk of crystal slippage during data collection if too much liquid is present, which is detrimental to obtaining a useful data set. Moisture must therefore be removed carefully from the crystal (using drawn-out glass capillaries or thin strips of filter paper) until only a thin film of liquid surrounds it that adheres it to the capillary wall. A drop of harvesting buffer is then introduced to one end of the capillary, which is then sealed at both ends using wax, making sure to avoid heating of the crystal. The crystal can then be placed in the x-ray beam by positioning the capillary on the goniometer head, the interface between crystal and x-ray camera (Figure 12).

Today, most crystals are measured using Cryocrystallography43: due to reduced radiation damage at low temperatures, more data can be collected from a single crystal. As a result of the high solvent content of protein crystals, it is usually necessary to prepare harvesting buffers containing a cryoprotectant to prevent the formation of ice crystals, which would lead to crystal deterioration and cracking. Common cryoprotectants are glycerol (5-30%), ethylene glycol (20-40%), polyethylene glycol (PEG)400 (10-30%), and paraffin oil (100%).44 The choice of protectant and concentration is dependent on the harvesting buffer composition and on crystal compatibility; it is therefore advisable to prepare a range of these. Prior to crystal mounting, the minimum concentration of cryoprotectant should be tested at which the solution freezes as a glass. Cryo conditions are achieved by introducing the solution into a small nylon loop of dimension slightly larger than the crystal to be examined, and mounting it in a carefully controlled stream of nitrogen gas at about 100 K (Figure 12). Once the appropriate cryobuffer has been chosen, the crystal can be fished out of its mother liquor using the loop, introduced to the cryobuffer for a few seconds, then mounted on the goniometer head into the cryostream and x-ray beam, ready for measurement.

Freezing of crystals can lead to a deterioration of the diffraction pattern, although this can only be ascertained if a separate measurement is made at room temperature. The resolution of the diffraction pattern can however be improved by 'crystal annealing' - the frozen crystal is removed from the cryostream, allowed to thaw, and then returned to the stream. While this does not work for every crystal, it is always worth attempting when desperation sets in. Unfortunately, this process is difficult to control; a more promising method seems to be the free mounting system, where the crystal is placed in a stream of air of controlled humidity.45 The humidity can then be manipulated while monitoring the x-ray diffraction pattern, allowing possible transitions in crystal quality to be followed.

Was this article helpful?

0 0
Lower Your Cholesterol In Just 33 Days

Lower Your Cholesterol In Just 33 Days

Discover secrets, myths, truths, lies and strategies for dealing effectively with cholesterol, now and forever! Uncover techniques, remedies and alternative for lowering your cholesterol quickly and significantly in just ONE MONTH! Find insights into the screenings, meanings and numbers involved in lowering cholesterol and the implications, consideration it has for your lifestyle and future!

Get My Free Ebook


Post a comment