Direct-method SAD phasing of proteins enhanced by the use of intrinsic bimodal phase distributions in the subsequent phase-improvement process.

A modified SAD (single-wavelength anomalous diffraction) phasing algorithm has been introduced in the latest version of the program OASIS. In addition to direct-method phases and figures of merit, Hendrickson-Lattman coefficients that correspond to the original unresolved bimodal phase distributions are also output and used in subsequent phase-improvement procedures in combination with the improved phases. This provides the possibility of rebreaking the SAD phase ambiguity using the ever-improving phases resulting from the phase-improvement process. Tests using experimental SAD data from six known proteins showed that in all cases the new treatment produced significant improved results.

OASIS and molecular-replacement model completion.

A method of dual-space molecular-replacement model completion has been proposed which involves the programs ARP/wARP, REFMAC, OASIS and DM. OASIS is used in reciprocal space for phase refinement based on models built by ARP/wARP. For this purpose, the direct-method probability formula of breaking SAD/SIR phase ambiguities has been redefined. During the phase refinement, phi(h)('') in the expression phi(h) = phi(h)('') +/- |Delta phi(h)| is redefined as a reference phase calculated from a randomly selected 5% of the atoms in the current structure model, while |Delta phi(h)| is defined as the absolute difference between the phase of the current model and phi(h)(''). The probability formula P(+)(Delta phi(h)) = (1/2) + (1/2) tanh {sin |Delta phi(h)| x [Sigma (h('))m(h('))m(h - h('))kappa(h,h(')) sin(Phi'(3) + Delta phi(h'best) + Delta phi(h - h'best)) + chi sin delta(h)]} is then used to derive the sign of Delta phi(h). In this way the '0-2pi' phase problem is reduced to a 'plus or minus' sign problem. The redefinition implies that during the refinement phases close to the true values will probably be kept unchanged, while those distant from the true values will probably undergo a large shift. This is the desired property of phase refinement. The procedure has been tested using protein diffraction data without SAD/SIR signals. The results show that dual-space MR-model completion making use of OASIS is much more efficient than that without.

SAD phasing by OASIS-2004: case studies of dual-space fragment extension.

The principle of dual-space phasing is used in dealing with protein SAD data. Four programs are involved in iterative dual-space fragment extension to improve automatic model building. OASIS-2004 is used to break the phase ambiguity intrinsic in the SAD experiment. In the initial cycle, discrimination of SAD phase doublets is performed by the direct method incorporating the known anomalous-scattering substructure. In subsequent cycles, discrimination is performed by the direct method incorporating both the known anomalous-scattering substructure and the partial protein structure obtained from model building in the preceding cycle. DM is used to improve direct-method phases via density modification. RESOLVE is used for initial model building and ARP/wARP is used to complete the structure. Case studies with three sets of difficult SAD data showed that the procedure is beneficial to high-throughput protein-structure determination and all of the four programs involved make their unique contribution to the process.

Comparison of phasing methods for sulfur-SAD using in-house chromium radiation: case studies for standard proteins and a 69 kDa protein.

Phasing of the crystal structures of four standard proteins (lysozyme, trypsin, glucose isomerase and thaumatin) and a novel 69 kDa protein from Thermus thermophilus, TT0570, was performed using the single-wavelength anomalous diffraction of S atoms intrinsically present within the native protein molecules. To utilize the sulfur anomalous diffraction, the data sets were collected using the loopless data-collection method with chromium Kalpha X-rays of wavelength 2.29 A. Three phasing methods, MLPHARE, SHARP and OASIS-2004, were tested in combination with the DM or SOLOMON density-modification method. The results showed that the solvent contents are still an important factor for phasing with the S-SAD method, even when longer wavelength Cr Kalpha radiation is used. Of the three procedures, the improved direct phasing of OASIS-2004 with its implemented fragment feedback to the direct-method probability calculation gave the best results in determining the initial phases. For all five proteins, almost the entire models could be built automatically.