C. D. Zheng

SAD phasing by combination of direct methods with the Solve/Resolve procedure

In the initial stage of SAD phasing, the essential point is to break the intrinsic phase ambiguity. The presence of two kinds of phase information enables the discrimination of phase doublets from SAD data prior to density modification. One is from the heavy atoms (anomalous scatterers), while the other is from the direct-methods phase relationships. The former can be expressed by the Sim distribution, while the latter can be expressed by the Cochran distribution. Typically, only the Sim distribution has been used to yield initial phases for subsequent density modification. However, it has been demonstrated that using direct-methods phases based on the product of the Sim and Cochran distributions can lead to improved initial phases. In this paper, the direct-methods phasing procedure OASIS has been improved and combined with the SOLVE/RESOLVE procedure. Experimental SAD data from three known proteins with expected Bijvoet ratios / in the range 1.4-7.0% were used as test cases. In all cases, the phases obtained using the program RESOLVE beginning with initial phases based on experimental phases plus Sim and direct-methods information were more accurate than those based on experimental plus Sim phase information alone.

Structural features of the incommensurate modulation in the Pb-doped Bi-2223 high-Tc phase revealed by direct-method electron diffraction analysis

Structural modulation waves in the Pb-doped Bi-2223 (Bi2Sr2Ca2Cu3Ox) phase are observed for the first time on the potential distribution function projected along the a axis. The newly developed multidimensional direct methods were used. Unlike the previous techniques, there is no need to assume any modulation model before the modulation waves can be seen directly from the resulting Fourier map. The symmetry of the title compound belongs to the four-dimensional space group P111Bbmb with three-dimensional unit cell parameters a=5.49 AA, b=5.41 AA, c=37.1 AA, alpha = beta = gamma =90 degrees and the modulation wavevector q=0.117b*. Occupational and positional modulations are obvious for most atoms. Disordered oxygen atoms arranged perpendicular to the b axis bridging the Cu(2)-Ca-Cu(1)-Ca-Cu(2) layers were observed.

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.

Image processing in high-resolution electron microscopy using the direct method. III. Structure-factor extrapolation

A resolution-enhancement method has been proposed which makes use of the Sayre equation [Sayre (1952). Acta Cryst. 5, 60-65] to extrapolate both phases and magnitudes of structure factors. The starting point of the procedure is just a single deconvoluted electron microscopic image. No preliminary knowledge other than the chemical composition of the sample is necessary. A simulation on a theoretical image of copper perchlorophthalocyanine shows that the image resolution can be enhanced from 2 to 1 A, resolving clearly individual atoms.

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.

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.

Optimizing the error term in direct-method SAD phasing.

The probability formula of the direct-method SAD (single-wavelength anomalous diffraction) phasing proposed by Fan & Gu (1985, Acta Cryst. A41, 280-284) contains an error term which is related to the lack-of-closure error. This error term is used as a weighting function in the phase derivation and in the subsequent calculation of electron-density maps. Previously, there has been a constant in the error term that has had to be determined empirically for each particular case. It has been found that improper choice of the constant often leads to failure of the direct-method SAD phasing. The problem is resolved by introducing a modified error term and a method of automatically tuning the associated scaling factor.

Incommensurate modulation of the Bi-2223 superconducting phase observed by direct-method electron diffraction analysis

Structure details of the incommensurate modulation in the Pb-doped Bi-2223 high Tc superconducting phase have been revealed by a multidimensional direct-method analysis of the electron diffraction pattern of the (100) reciprocal plane. Occupational and positional modulations have been found for most atoms. Disordered oxygen atoms arranged perpendicular to the b axis bridging the Cu(2)-Ca-Cu(1)-Ca-Cu(2) layers were observed. This implies the existence of large amount of O-vacancies on the Cu(1)-O plane.

A new iterative procedure for combining direct methods with solvent flattening - Dealing with the phase ambiguity in protein crystallography

A new procedure for combining direct methods with the solvent-flattening technique is proposed for phasing single isomorphous replacement (SIR) or one-wavelength anomalous scattering (OAS) data of proteins. The new procedure differs from the previous one [Zheng, Zheng, Gu, Mo, Fan & Hao (1997). Acta Cryst. D53, 49-55] in that the direct method not only provides input phases to but also accepts feedback phases from solvent flattening, thus forming an iterative process for breaking the ambiguities and refining the values of phases. The new procedure was tested with the experimental SIR data of the known structure ribonuclease Sa. For the strongest 1000 of the total 7264 reflections, the mean F(obs)-weighted phase error is 7.5 and 9.4 degrees lower than that of the previous procedure and that of solvent flattening alone, respectively.