Use of iterative OASIS with low resolution SAD laboratory X-ray source (Cr Kα) data sets

Abstract

Anomalous scattering methods are widely used for phasing macromolecular structures. OASIS program works on a direct methods procedure and is used to break the phase ambiguity intrinsic in a single wavelength anomalous diffraction (SAD) experiment. An iterative phasing/model-building procedure for processing SAD data includes the initial direct method phasing of SAD data, density modification, automatic model building and direct methods phasing of SAD data with feed back from partial structure information. This dual-space procedure has been tested on two experimental Cr Kα SAD data sets, 2.3 A for S-SAD and 2.4 A for I-SAD, of an enzyme glucose isomerase with an approximate molecular weight of 44 kDa (388 residues). HYSS in PHENIX was used to locate the anomalous scatterers for both the data sets (11 sulfurs for 2.3 A data and 9 iodines for 2.4 A data). Phasing and density modification were carried out using OASIS, DM and model building was then carried out using ARP/wARP. Two iterative cycles were necessary for the successful structure solution and refinement of the 2.3 A data set which built 384 residues out of 388. Regular ARP/wARP failed for the 2.4 A resolution data and hence the modified version of REFMAC5 was used in ARP/wARP. A successful model could be built after three iterative cycles of OASIS using this modified version which built 382 residues out of 388 residues. Minimal manual model building was required at this stage and the structure determination was completed using regular REFMAC5. All the computations mentioned here were carried out using Pentium IV PC.