SIRAS treatment based on low-resolution SAD phasing

        SIRAS phasing is important in solving membrane protein structures. Because anomalous diffraction signals from intrinsic sulphur atoms are usually not enough to ensure a successful SAD phasing while preperation of MIR specimens is often difficult.
        In theory, diffraction phases can be derived from a set of protein SIRAS data without ambiguity. However in practice, there may be imperfection of isomorphism between the native protein and the heavy-atom derivative and, the high-resolution outoff of the derivative may be much lower than that of the native. A case study on the treatment of protein SIRAS data showed that effects of the above problems can be eliminated by the SIRAS phasing starting from a low-resolution SAD phasing followed by a phase extension from the derivative to the native (more details).


New results on SAD phasing

        The known protein TTHA1012 with 213-residues was solved in a test of the pipeline IPCAS based on the anomalous scattering signals from 2 sulphur atoms under Cr-Kα radiation.
        Ten cycles iterative SAD phasing were performed uisng the programs OASIS, DM, RESOLVE (build only) and ARP/wARP. The resultant best model (shown on the left of the following figure) consists of 166 residues (166 of which sequenced) with R/Rfree=0.267/0.343. Based on this model, ten cycles of iterative fragment extension running with OASIS, DM and Buccaneer led to a model (on the right of the following figure) containing 200 residues (198 of which sequenced) with R/Rfree=0.246/0.303.

Data by courtesy of Professor Nobuhisa Watanabe, Department of Biotechnology and
Biomaterial Chemistry, Nagoya University, Japan


OASIS (v4.2) - IPCAS (v1.0) released

        OASIS 4.2 - a program of direct-method phasing based on a partial model with or without SAD/SIR information;
        IPCAS 1.0 - a pipeline for "Iterative Protein Crystal-structure Automatic Solution" developed in the
"Institute of Physics, Chinese Academy of Sciences".


I. New expression of bimodal SAD-phase distributions by the sum of two von Mises distributions

        An important step in direct-method SAD phasing is to express SAD-phase distributions by the sum of two unimodal distributions. Up to the moment, Guassian distribution has been used for this purpose in the program OASIS. However this could cause large errors when Δφ is small. Much better results can be obtained by replaceing the Guassian distribution with the von Mises distribution. In the following figure the solid line denotes the experimental bimodal phase distribution; Black dots indicate the approximation by the sum of two von Mises distributions; Blank diamonds indicate the approximation by the sum of two Gaussian distributions. As is seen the von Mises approaximation is much better than the Guassian one. The new expression will be used in the next version of OASIS. Click on the figure to read the original publication.

Click on the map for a copy of the PDF file of Chin. Phys. B. 19, 086102 (2010).

II. Combination of SAD- and MR-iteration

        Ribbon structure models of TTHA1012 plotted by PyMOL are shown in the following figure. Left: the best model from cycles 0-20 of SAD iteration; Right: the best model from 1-10 cycles MR iteration based on 0-10 cycles SAD iteration. As is seen the result from SAD+MR iteration is better than that from SAD iteration alone. Click on the figure to read the original publication.

Click on the map for a copy of the PDF file of Chin. Phys. B. 19, 096101 (2010).


New version of OASIS: OASIS4.0

Click on the map for a copy of the PDF file of Chin. Phys. B. 19, 086103 (2010).

I. Improved SAD phasing

Click on the map for a copy of the PDF file of Acta Cryst. D65, 1213-1216 (2009).


II. GUI for controlling and monitoring OASIS dual-space iteration

         Control panel


         Monitoring board


         Details for finished cycles


         Real-time model display

          Detailed description of the OASIS GUI


I. Combining SOLVE/RESOLVE with OASIS/DM at low resolution

Click on the map for a summary of the sample

II. SAD phasing and fragment extension at different resolutions down to 3Å and below

Click on the map for a summary of the sample


[1] Y. He, Y.X. Gu, Z.J. Lin, C.D. Zheng and H.F. Fan, Chinese Physics 16, 3022-3028. (2007) PDF file
[2] D.Q. Yao, H. Li, Q. Chen, Y.X. Gu, C.D. Zheng, Z.J. Lin, H.F. Fan, N. Watanabe and B.D. Sha, Chinese Physics B 17, 0-0 (2008) PDF file


Direct-method dual-space fragment extension for MR model completion

       The direct-method dual-space fragment extension has been extended to use in the case where SAD/SIR information are not available. Test with the protein 1UJZ showed that the technique is very efficient in model completion starting with a partial structure (~20% of the total) obtained from molecular replacement.

Click on the map for a summary of the test


Y. He, D.Q. Yao, Y.X. Gu, Z.J. Lin, C.D. Zheng & H.F. Fan, Acta Cryst. D63, 793-799. (2007) PDF file


Dual-space fragment extension using SAD data

       Extensive case studies have been carried out. Results have been summarized in References.

(Click on the thumbnail for an enlarged picture)
Flow chart Azurin Lysozyme Xylanase

Staged iteration results of the above samples


[1] D.Q. Yao, S. Huang, J.W. Wang, Y.X. Gu, C.D. Zheng & H.F. Fan, N. Watanabe & I. Tanaka, Acta Cryst. D62, 883-890. (2006) PDF file
[2] N. Watanabe, Y. Kitago, I. Tanaka, J.W. Wang, Y.X. Gu, C.D. Zheng & H.F. Fan, Acta Cryst. D61, 1533-1540. (2005) PDF file


I. Auto-tuning the error term in direct-method SAD phasing

       The probability formula of direct-method SAD phasing used in the program OASIS contains an error term, which is related to the lack-of-closure error. Previously in the error term, there is a constant to be determined empirically for each particular case. It has been found that improper choice of the constant led often to the failure of phase derivation. The problem is resolved by introducing a modified error term and a method of automatically tuning the associated scaling factor.

Electron density maps obtained by a default run of the modified OASIS followed by a default run of DM (Cowtan, K.).

(Click on the thumbnail for an enlarged picture)

II. Direct-method SAD phasing with reciprocal-space fragment extension

       Structure fragments obtained from automatic model building based on direct-method SAD phasing are fed back to the direct-method probability formula to strengthen the phasing power and to benefit automation.

(Click on the thumbnail for an enlarged picture)
Flowchart Xylanase synchrotron S-SAD data
Rusticyanin synchrotron Cu-SAD data Thaumatin Cr-Ka S-SAD data


[1] J.W. Wang, J.R. Chen, Y.X. Gu, C.D. Zheng, F. Jiang and H.F. Fan, Acta Cryst. D60, 1987-1990. (2004) PDF file
[2] J.W. Wang, J.R. Chen, Y.X. Gu, C.D. Zheng, and H.F. Fan, Acta Cryst. D60, 1991-1996. (2004) PDF file


SAD phasing, why direct methods?

        SAD phasing is becoming popular in solving protein structures. The essential point to success is to break the phase ambiguity intrinsic in SAD data. Mostly this is done using density-modification techniques. However, density modification does not directly make choice between the two possible SAD phases for each reflection. The use of density-modification techniques alone leads often not to the best results. Two kinds of phase information enable discrimination of phase doublets from SAD data. One is from the heavy atoms (anomalous scatterers), while the other from the phase relationship of direct methods. The former can be expressed by the Sim distribution, while the latter can be expressed by the Cochran distribution. Usually only the Sim distribution is used to yield initial phases for the subsequent density modification. We use instead the product of the Sim and the Cochran distributions to produce initial phases, which have been proved much better than that from only the Sim distribution. Density modification based on such phases has been proved capable of yielding much better results than that based on only the Sim distribution.

Comparison of SAD phasing with and without direct methods

(Click on the thumbnail for an enlarged picture)
Phase ambiguity intrinsic in SAD Phase information available in SAD Two different kinds of initial SAD phases Phase probability disributions
phase errors
HMT SET7/9 Rusticyanin Azurin


[1] J.W. Wang, J.R. Chen, Y.X. Gu, C.D. Zheng, F. Jiang, H.F. Fan, T.C. Terwilliger and Q. Hao, Acta Cryst. D60, 1244-1253. (2004) PDF file

Enlarged image 2002:
        Conventional MIR phases of the known protein Rpe have been significantly improved by the direct-method procedure DMIR

Y.X. Gu, W.R. Chang, T. Jiang, C.D. Zheng & H.F. Fan, Acta Cryst. A58, 547-551 (2002) PDF file
Enlarged image 2001:
        The Fourier map from conventional MAD phasing of 4-wavelength anomalous diffraction data was improved by direct-method phasing of only the 2-wavelength subset of data

Fan Hai-fu, lecture at the Euro Conference on Phasing Biological Macromolecules, Martina Franca, Italy, June 2001 PowerPoint slides

Y.X. Gu, F. Jiang, B.D. Sha & H.F. Fan, Z. Krist. 217, 710-714 (2002) PDF file
Enlarged image 2001:
        Direct-method aided MAD phasing --- the first example of improving MAD phases by direct methods

Y.X. Gu, Y.D. Liu, Q. Hao. S.E. Ealick & H.F. Fan, Acta Cryst. D57, 250-253 (2001) PDF file
OASIS 2000:
        The CCP4 supported program for direct-method phasing of One-wavelength Anomalous Scattering or Single Isomorphous Substitution (replacement) Data

Hao, Q., Gu, Y.X., Zheng, C.D. & Fan, H.F., J Appl. Cryst. 33, 980-981 (2000) PDF file
Enlarged image 1999:
        The first example of breaking the protein SIR phase ambiguity when replacing atoms are in centric arrangement

Liu Yu-dong, Gu Yuan-xin, Zheng Chao-de, Q. Hao and Fan Hai-fu, Acta Cryst. D55, 846-848 (1999) PDF file
Enlarged image 1999:
        Comparing the result of breaking SAD-phase ambiguity using direct methods and that without using direct methods

Liu Yu-dong, I. Harvey, Gu Yuan-xin, Zheng Chao-de, He Yi-zong, Fan Hai-fu, S. S. Hasnain, and Q. Hao, Acta Cryst. D55, 1620-1622 (1999) PDF file
Enlarged image 1997:
        The first example of solving an originally unknown protein by direct-method phasing of the 2Å SAD data

H. F. Fan, Q. Hao, I. Harvey, S. S. Hasnain, Y. D. Liu, Y. X. Gu, C. D. Zheng & H. Ke, in "Direct Methods for Solving Macromolecular Structures" Ed. by S. Fortier, Kluwer Academic Publishes, The Netherlands, 1998, pp. 479-485. PDF file
Enlarged image 1996:
        Direct-method phasing and solvent flattening treatment of the 1.9Å SAD data of the protein azurin led to an easily traceable Fourier map

Zheng Xiao-feng, Fan Hai-fu, Q. Hao, F.E. Dodd & S.S. Hasnian, Acta Cryst. D52, 937-941 (1996) PDF file
Enlarged image 1995:
        Test with the SAD data from the protein core streptavidin, which was originally solved using 3-wavelength MAD data

Sha Bing-dong, Liu Shen-ping, Gu Yuan-xin, Fan Hai-fu, Hengming Ke, Yao Jia-xing & M. M. Woolfson, Acta Cryst. D51, 342-346 (1995) PDF file
Enlarged image 1990:
        The first traceable Fourier map from direct-method phasing of the experimental 2Å SAD data of the protein aPP

Fan Hai-fu, Hao Quan, Gu Yuan-xin, Qian Jin-zi, Zheng Chao-de & Hengming Ke, Acta Cryst. A46, 935-939 (1990) PDF file
        The first successful test on direct-method phasing of the 3Å experimental SIR data from the native Bence Jones protein Rhe and its Au-derivative

Collaborative work by H.F. Fan and B.C. Wang, 1988 (see M.M. Woolfson and Fan, Hai-fu, Physical and Non-physical Methods of Solving Crystal Structures, Cambridge University Press, 1995. Pp. 164-166.) details
        The first successful test on direct-mothod phasing of the 2Å experimental SAD data from the protein aPP

Fan Hai-fu & Gu Yuan-xin, Acta Cryst. A41, 280-284 (1985) PDF file
        It was proposed that the phase ambiguity due to single-wavelength anomalous diffration (SAD) or single isomorphous replacement (SIR) could be resolved by direct methods.

Fan Hai-fu, Chinese Phys. 1429-1435 (1965) PDF file
see also Fan Hai-fu, Acta Phys. Sin. 21 1114-1118 (1965). (In Chinese) PDF file