USING SINGLE WAVELENGTH ANOMALOUS SCATTERING DATA FOR IN-HOUSE PROTEIN STRUCTURE DETERMINATION

Introduction Over the past ten years, the use of anomalous dispersion information has become a routine means of determining a protein crystal structure using techniques such as Multi-wavelength Anomalous Dispersion (MAD) (Hendrickson, et al., 1990). The MAD method exploits the change in strength of an element's anomalous signal at wavelengths close to its X-ray absorption edge. Unfortunately, the MAD technique can not be used in the home laboratory since it requires a tunable X-ray source, such as a synchrotron. However, several elements (Table 1) have a significant anomalous signal (∆f") at 8 keV that can be used to generate phase information from carefully measured single wavelength anomalous scattering (SAS) data collected using Cu Kα radiation. The first protein structure determination using S-SAS data was reported in 1981 (Teeter and Hendrickson) for the small, sulfur rich protein crambin (5 kD, 6 sulfurs). The crambin structure was determined at 1.5 Å resolution using the Resolved Anomalous Phasing technique. This technique assumes that the phases for the protein structure are close to the phases of the anomalous scattering component (in this case sulfur, ∆f" = 0.56). Thus, it requires a relatively high percentage of anomalous scatterers in the protein and high resolution (>2 Å) data. Later, Wang (1985) introduced a different approach, which he termed the Iterative Single -wavelength Anomalous Scattering (ISAS) method that uses an iterative algorism to resolve the phase ambiguity associated with SAS data (see Fig. 1). In this approach, the anomalous scattering information is used to generate an image of the protein-solvent boundary (a low-resolution image of the protein) which in turn is used to select the correct protein phase from the bimodal SAS phase distribution. Because the ISAS method uses the protein image instead of the anomalous atomic substructure to resolve the SAS phase ambiguity, it should, in theory, be less dependent on the number of anomalous scatterers present in the protein (or crystal) or on the resolution of the data. This approach was successfully applied to the redetermination of Bence Jones protein Rhe (13 kDa, one disulfide) using simulated S-SAS data. Chen et al., (1991) applied this approach in the 2.8 Å structure determination of a neurophysin para-iodo-PheTyr-amide complex (46 kD, five iodine atoms) using rotating anode (Cu Kα) data. In this study, an iodinated peptide hormone mimic was used to introduce iodine (∆f" =6.8) into the hormone binding site at near 100% occupancy. The data set was broken up into a series of 10° data scans and the inverse beam technique was used to collect complementary (ω, ω+ 180°) data scans on an aligned crystal such that the Friedel related reflections were recorded close in time at the same position on the detector surface. More recently, Wu (2001) used rotating anode data and the ISAS method to determine the 2.35 Å structure of the human ferrochelatase (86 kD, 2 [2Fe2S] clusters) dimer. This study is interesting since the iron anomalous signal (∆f" =3.2) at 8 keV is similar in magnitude to the selenium anomalous