Research Article: Rapid cadmium SAD phasing at the standard wavelength (1 Å)

Date Published: July 01, 2017

Publisher: International Union of Crystallography

Author(s): Saravanan Panneerselvam, Esa-Pekka Kumpula, Inari Kursula, Anja Burkhardt, Alke Meents.


Single-wavelength anomalous dispersion (SAD) phasing experiments were successfully carried out at the standard wavelength of 1 Å by using cadmium ions as anomalous scatterers.

Partial Text

High-throughput crystallography projects demand rapid data collection and robust experimental phasing procedures that are suitable for various target proteins. Recent developments in sample preparation, beamline instrumentation and data-processing programs have improved the success rate in macromolecular crystallography (reviewed in Su et al., 2015 ▸). The availability of microfocused X-ray beams (1–20 µm) at synchrotrons and the emergence of X-ray free-electron laser sources enable us to push the limits of crystal size required for diffraction experiments (Helliwell & Mitchell, 2015 ▸; Stellato et al., 2014 ▸). However, the ‘phase problem’ still remains a hurdle in macromolecular crystallography. Experimental phasing, especially single-wavelength anomalous dispersion (SAD) phasing using either heavy atoms or naturally occurring atoms (for example S, Cl, P etc.), has become a fast and dominant method for macromolecular crystal structure determination owing to the advantage that one single crystal is often sufficient for successful phasing (Nagem et al., 2001 ▸; Rose et al., 2015 ▸). Currently, most SAD phasing experiments are routinely carried out using X-rays tuned to the absorption edge of the heavy atom being used or longer wavelengths (1.7–2.5 Å) to utilize natively occurring light elements. Heavy-atom derivatization of protein crystals normally requires incorporation via protein expression (for example selenomethionine labelling) or time-consuming soaking procedures. Furthermore, phasing at longer wavelengths often leads to radiation damage owing to increased absorption, beam instability and loss of high-resolution data (Wang et al., 2006 ▸), and in some cases requires additional instrumentation such as a helium cone (Liebschner et al., 2016 ▸) or an in-vacuum setup (Wagner et al., 2016 ▸) to overcome absorption-related issues. Hence, the possibility of performing SAD phasing experiments at the standard wavelength of 1 Å would be a great advantage for high-throughput crystallography.

The crystals of our three samples diffracted to high resolution. In practice, most protein crystals do not diffract as strongly, and typical data sets used for phasing are at resolutions in the range 2–3 Å. An optimal X-ray wavelength of 1.7–2.1 Å has been proposed for sulfur SAD experiments (Rose et al., 2015 ▸; Mueller-Dieckmann et al., 2007 ▸). Owing to the long wavelength of the incident beam, the high-resolution data cannot be recorded in most sulfur SAD experiments. In order to better understand the resolution dependence, we further tested the effect of the resolution cutoff at different steps of the phasing procedure. The resolution-cutoff analysis was carried out in two different modes. In the first mode of analysis, data sets were scaled together without any resolution cutoff and used directly for phasing. In the substructure-determination step, the desired low-resolution range was selected with the parameter SHEL. The resulting substructures were then used for SHELXE runs with the corresponding full resolution data sets. This mode of analysis estimates the correctness of initial substructure determination by SHELXD at low resolution. In the second mode of analysis, the data sets were scaled and truncated with the desired resolution limit at the scaling step. These truncated data sets were then used for the entire phasing procedure with the same resolution limit as in the scaling step. The results are illustrated in Fig. 5 ▸. The SHELXE CC values were used as an indicator of successful phasing (Thorn & Sheldrick, 2013 ▸).

High-throughput crystallography projects demand a rapid data-collection setup and robust experimental phasing of various target proteins. In this work, we showed that a single data set collected at the standard wavelength of 1 Å (12 keV) is sufficient for experimental phasing as well as final structure refinement. Cadmium ions, as used here, provide two benefits: (i) they promote crystal growth and/or improve crystal quality by mediating intermolecular bridges and (ii) their anomalous signal at the standard wavelength is very well suited for experimental phasing.