Section 19.3.3.3. Experimental considerations

Sample volumes required for one measurement are between 10 to 50 µl, occasionally more, depending on the specific design of the sample cell used. The concentration required to record a scattering curve with satisfactory statistics depends primarily on the molecular weight of the protein and the beam flux. Approximately 1 mg ml⁻¹ of a small protein (10–20 kDa) is the lower limit for recording a scattering pattern with satisfactory statistics when experiments are performed with a typical synchrotron instrument equipped with a gas-chamber detector. Somewhat lower concentrations of larger molecular weight proteins may be used. Higher concentrations will improve statistics significantly and reduce exposure times, but interparticle interference may result from high concentrations. Time-resolved experiments benefit dramatically from higher sample concentrations. In addition to the scattering power of the sample, the signal-to-background ratio and overall stability of an instrument (from X-ray source and optics to detector) limit the lowest concentration for a given experiment. Although higher concentrations add dramatically to the scattering and improve statistics, sample solutions must be monodisperse. Small-angle solution scattering is not well suited to the study of polydisperse systems, which give scattering of the entire molecular population weighted by the square of the mass, although a few distinct populations of substantially different sizes may be resolved with good-quality data. Chemical components that may have been carried along in a sample preparation, such as ammonium sulfate, sucrose, chloroform or caesium chloride, should be removed. The presence of such compounds may change the electron-density contrast and X-ray absorbency of the sample. In general, this can be most effectively avoided by exhaustive dialysis with the desired buffer solutions. The outer solution used for the final dialysis should be used for the blank measurements. Scattering contributions from the buffer solution, the sample cell and parasitic scattering must be subtracted from the measured scattering curve; these can be measured accurately from a well prepared blank. Extra buffer solution should be available for sample dilution. The data quality is improved and problems with radiation-sensitive samples are readily detected when protein concentrations and biological activities of samples are measured before and after the scattering experiment. Accurate protein concentration measurements permit scattering intensities from different samples to be scaled together accurately. This is particularly important in determining molecular weight.

Sample holders used in solution scattering are either Lindeman glass or quartz capillaries, or machined cells equipped with flat windows (Fig. 19.3.3.6). Glass capillaries have been widely used to contain a sample solution. A beam size significantly smaller than the diameter of the capillary is required to minimize strong parasitic scattering from the round edge of the capillary. A large beam size in the horizontal direction may be used with capillaries to obtain stronger scattering intensity without adding parasitic background. Capillary cells are suitable for measuring scattering primarily in the direction perpendicular to the long axis of the cell. The advantage of the capillary cell is the small volume of sample solution required for measurements. About 4 µl of a sample solution in a 1 mm-diameter capillary provides sufficient material for the experiment. By employing a specialized holder, a capillary cell can be placed under a vacuum, minimizing air scattering (Dubuisson et al., 1997). This feature is useful for solution scattering studies of small proteins. Anaerobic samples may be sealed in a capillary.

Figure 19.3.3.6| top | pdf | (a) Flat-window cells for solution scattering and (b) a diagram of a stopped-flow rapid mixer for time-resolved solution scattering. The solution cell to the left in (a) is made of polycarbonate and is equipped with two synthetic mica windows. A sample solution is injected through one of two sample loading channels using a microsyringe. The black cell to the right is made of coloured polyoxymethylene for light-activated, time-resolved studies and has a smaller sample chamber. In (b), two solutions, e.g. an enzyme solution and a substrate solution, are put in sample reservoirs, loaded into individual syringes and wait for a trigger signal from the data-acquisition system. Then the two solutions are rapidly mixed, transferred to the observation cell, typically within 5 ms or less, and a trigger signal initiates a series of time-sliced scattering-data acquisitions. This stopped-flow mixer is also equipped with optical paths to monitor absorption or fluorescence from the protein solution in the observation cell.

Another common sample cell holds the solution between a pair of flat windows. This cell offers two improvements over capillary cells – a larger beam cross section may be used, which increases the number of photons incident on the sample, and a two-dimensional detector can be effectively used for recording the scattering. Flat-window cells are available that require only about 10 µl of solution. Both capillary and flat-window cells require a holder with temperature regulation. The choice of window materials for the sample container is important because of the weak sample scattering. An X-ray-transparent material is required that has little intrinsic scattering within the scattering-angle range of the sample. Common window materials include synthetic mica of high purity and certain types of polypropylene and polyamide. Etched high-quality beryllium has also been used. The windows must be thin enough to transmit X-ray photons, but rigid enough to keep a constant beam-path length through sample solutions. A valuable comparison of window materials has been published recently (Henderson, 1995). A protein solution scattering pattern and a corresponding blank scattering curve should be measured in the same sample cell, unless the cells and windows are identical.

Special sample-handling devices have been built for time-resolved studies. Stopped-flow mixers for solution X-ray scattering are routinely used by a number of research groups (Kihara, 1994), and a high-pressure solution cell has been constructed (Czeslik et al., 1996). The assembly process of tobacco mosaic virus has been studied using a temperature-jump apparatus (Hiragi et al., 1988).

The angular range for data collection should first be determined on the basis of the structural information required, and an instrument should be configured accordingly. Most camera systems have the flexibility to use more than one sample-to-detector distance, and they are occasionally equipped with an additional detector to cover higher angles.

The exposure time is usually determined empirically, taking into account the data statistics required, the number of conditions to be investigated and the total amount of beam time available. Typical exposure times run from a few minutes with a synchrotron source to several hours with a laboratory source. Exposure time depends strongly on the sample molecular weight, the concentration and the angular scattering range of interest. It is essential to measure the blank solvent, i.e., the buffer solution in which the protein of interest is dissolved. It should be recorded with the same statistical significance as the protein solution scattering. This blank is subtracted from the observed protein solution scattering intensity, and therefore its intensity contributes to the overall counting statistics. Accurate blank measurements can take significant amounts of time and should not be ignored when planning the experiment. Blank solution scattering patterns should be recorded as often as possible. They serve as an internal control to detect systematic errors during periods of instrumental instability. In addition, the intensity of the incident X-ray beam should be integrated during the exposure time and used to scale the scattering data. Although it is useful to record the beam intensity transmitted through the solution sample, absorption is generally not a significant problem for solution scattering.

Scattering intensity measurements should be performed with a series of experiments. Solutions at different concentrations will demonstrate that the anticipated trends are observed at a qualitative level, even before the data are processed. Time-dependent aggregation or degradation are detected by recording scattering curves in a series, with a short exposure for each curve (e.g., recording 10 successive scattering curves every 30 s for one protein solution). Proteins in solution may degrade in the full flux of a synchrotron-radiation beam and may exhibit radiation-induced aggregation. This is easily recognized by inspecting the very small angle region of scattering patterns that are recorded successively using short exposure times. Radiation damage can be reduced or eliminated either by attenuating the primary beam or by adding small amounts of antioxidants, which may help remove free radicals. There are several cases in which a small amount of dithiothreitol or β-mercaptoethanol prevented radiation-induced aggregation. The effects of the sulfur-reducing agents are consistent with the observation by a time-resolved single-crystal study that the sulfur atom disappears first from an electron-density map (Weik et al., 2000). Temperature control is essential for most experiments. Irradiation by a 9 keV X-ray beam at a rate of 10¹¹ photons s⁻¹ will heat up an aqueous solution of a few µl by a fraction of a Kelvin per second if a constant-temperature device is not employed.

Preliminary data processing should take place immediately with each sample measurement. Processing `on the fly' is particularly important when experiments are performed at a synchrotron where access may be limited. Visual inspections or statistical evaluation should be made for time-dependent aggregation at a low scattering angle. A determination of the sample molecular weight by estimating I(0) and the radius of gyration from the Guinier plot would also be useful. In addition to making measurements with a blank, scattering from a standard sample should be measured, allowing the relative molecular weight of the unknown to be determined. A standard, stable, well defined known sample should be recorded prior to any other measurements. Running the same standard sample in every data-collection period makes it easier to compare and scale data sets recorded at different times. Sample solutions should be recovered from sample cells and stored separately, so that post-irradiation concentrations can be measured, as well as their biological activity if this can be assayed. A standard sample that gives sharp diffraction peaks of known spacing should be measured to allow the conversion of the detector channel number to Q values. A dried collagen fibre or a non-biological or more stable specimen such as cholesterol myristate is commonly used. Determining the direct beam position at the detector plane is critical in converting a detector pixel number to a Q value. The symmetric centre of a diffraction pattern of a powder or fibre sample may be used, or a thick metal foil may be inserted to attenuate the primary beam intensity and the beam position measured directly with the detector. In order to place the scattering intensity on an absolute scale, i.e., to determine the scattering cross section, data from a calibrated standard sample need to be recorded.

Processing the solution scattering data first requires scaling the raw intensity measurements to the incident or transmitted beam flux, then averaging scattering curves recorded from identical samples and subtracting measurements from the blanks described above. Corrections for detector non-uniformity or image distortion, if any, must be made, and then the detector channel numbers are converted to Q values (or inverse Bragg-scattering spacing) through the use of the scattering standard for absolute scattering-angle determination. Many facilities provide data-processing support at least to this level. Except for the image-distortion correction, commercially available analysis and visualization software can be used for this purpose. Data formats currently vary among facilities and depend on the detector systems used, but efforts are underway to establish a standard format that would enable the wide use of common data-reduction software. Common formats will make it straightforward to perform data reduction `on the fly' and to monitor the quality of the data closely. Unfortunately, the size of the biological small-angle scattering community is not conducive to costly development of commercial software, so user friendliness has not generally been a priority in its development.

Plots of I versus Q that are corrected for all the known experimental factors are obtained from the data processing, and structural parameters may be derived from these data. The molecular weight and the radius of gyration can be derived immediately from the Guinier approximation. Calculation of the optimal electron pair distribution function by computing the Fourier transform of the intensity function provides the only `real-space' data directly obtainable from the experiment. The program GNOM developed by D. Svergun is widely used for this purpose and may be obtained from the program author. An algorithm developed by P. Moore has been widely implemented, and software developed by Glatter et al. (Glatter, 2004) is available. Table 19.3.3.1 lists software suites frequently used in interpreting solution X-ray scattering data.

Table 19.3.3.1| top | pdf | List of commonly used software for solution scattering Program Function Reference OTOKO † Data evaluation for noncrystalline diffraction Koch (1988) SAPOKO Data evaluation for solution scattering Koch & Svergun (1992) GNOM Indirect Fourier transform for P(r) Svergun (1992) IFT Indirect Fourier transform for P(r) Glatter (2004) CRYSOL Calculation of I(Q) from PDB files Svergun et al. (1995) SASHA Spherical harmonics structure determination Svergun, Volkov et al. (1997) ASSA and ALM22INT Three-dimensional modelling in real space Kozin et al. (1997) DAMMIN Simulated annealing structure determination Svergun (1999) DALAI_GA Genetic algorithm structure determination Chacón et al. (1998) †An X-terminal compatible version of OTOKO has been developed at Daresbury Laboratory.