1. Direct-method
SAD phasing

2. Early attempts

3. 1985 1965： Fan, H. F. Chinese Physics, 1429.
1966： Karle, J. Acta Cryst. 21, 273.
1970： Hazell, A. C. Nature 227, 269.
1973: Sikka, S. K. Acta Cryst. A29, 211.
1978: Heinerman, J. J. L., Krabbendam, H., Kroon, J.
& Spek, A. L. Acta Cryst. A34, 447.
1982： Hauptman, H. A. Acta Cryst. A38, 632.
1983: Giacovazzo, C. Acta Cryst. A39, 585.
1985: Fan, H. F. and Gu, Y. X. Acta Cryst. A41, 280.
1993: Kyriakidis, C. E., Peschar, R. and Schenk, H.
Acta Cryst. A49, 557.
1998: Fan, H. F. et al. in Direct Methods for Solving
Macromolecular Structures, Kluwer
Academic Publishes, pp
1985

4. Direct-method phasing of the 2Å experimental SAD data of the protein aPP
Avian Pancreatic Polypeptide
Space group: C2
Unit cell:
a=34.18, b=32.92, c=28.44Å
b=105.3o
Protein atoms in ASU: 301
Resolution limit: 2.0Å
Anomalous scatterer: Hg, Zn
Wavelength: 1.542Å (Cu-Ka)
Df” = 7.686, 0.678
Phasing: direct methods
Acta Cryst. (1990). A46, 935.

5. Direct phasing of the 3Å SAD data of the
Space group: I222
Unit cell:
a=95.27; b=105.41, c= 47.56Å
Protein atoms in ASU: 1836
Resolution limit: 3.0Å
Anomalous scatterer: Se
l = Å; Df” = 3.663
Phasing:
direct methods +
solvent flattening +
non-crystallographic
symmetry averaging
Acta Cryst. (1995). D51, 342.
protein core streptavidin

6. A computer program for breaking
OASIS
(The first edition, 2000)
A computer program for breaking
the phase ambiguity in
One-wavelength Anomalous Scattering or Single Isomorphous Replacement (Substitution) protein data.

7. Mlphare + dm
Oasis + dm
Acta Cryst. (1999). D55,
The first example of solving an unknown protein by direct-method phasing of 2.1Å SAD data
Rusticyanin, MW: 16.8 kDa; SG: P21; a=32.43, b=60.68, c=38.01Å ; b=107.82o ;
Anomalous scatterer: Cu

8. tetragonal hen egg-white lysozyme
Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale J.-P. Ebel CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble CEDEX 01, France
Acta Cryst. D58, 1-9 (2002).
Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with
tetragonal hen egg-white lysozyme
OASIS
+ DM
Éric Girard, Laurent Chantalat, Jean Vicat and Richard Kahn

9. J. Mol. Biol. 348, 951–959 (2005)
Crystal Structures of Fms1 and its Complex with Spermine Reveal Substrate Specificity
Qingqiu Huang1, Qun Liu1 and Quan Hao1,2
1MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University Ithaca, NY , USA
2Institute of Physics, Chinese Academy of Sciences, Beijing , China

10. Basis for structural diversity in homologous RNAs
Science, Vol. 306, Issue 5693, (2004)
Basis for structural diversity
in homologous RNAs
Andrey S. Krasilnikov*, Yinghua Xiao*, Tao Pan†, and Alfonso Mondragón*
* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA
† Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA
For SAD phasing, the positions of the first 3 Ba+2 sites were identified using SOLVE, 6 more Ba+2 sites were identified and added during heavy atom refinement with SHARP. The phase ambiguity in SAD phasing was partially resolved using OASIS and solvent flattening with SOLOMON as implemented in SHARP. Further improvement of the phases was achieved by doing iterative cycles of phase refinement incorporating phase information from
partially built models followed by solvent flattening.

11. CrKα線を用いたSAD 法による位相決定
構造生物 Vol.10 No 年2 月発行
CrKα線を用いたSAD 法による位相決定
理学電機(株) 山野昭人、佐藤貴久、長谷川智一
初期位相の決定はSHARP やOASIS が成績が良い。MLPHARE でも可能との事だが、筆者の使用法の問題だと思われるが、これまで成功した経
験がない。
図2 はサウマチンの解析例である。1 フレームを0.5度振動、1 分露光で180 度分測定したイメージをHKL2000 で処理。15-3Åのデータを用いてSHELX97 を実行。8 個のS-S の位置と1 個の硫黄原子の位置全てが決定できた。これらの座標を基にOASIS を実行。初期位相を決定した。DM およびSOLOMON に
より位相を改良した。

12. OASIS-2004 A direct-method program for
Institute of Physics, CAS, Beijing, P.R. China
A direct-method program for
ab initio phasing and reciprocal-space
fragment extension with SAD/SIR data

13. Difficult SAD phasing SAD phasing at Bijvoet ratio ~ 0.56%
An originally unknown protein with 1206 residues in the ASU solved automatically using Cr-Ka sulfur-SAD data

14. OASIS-2004 application Xylanase Contoured at 1s Space group: P21
Unit cell: a = 41.07, b = 67.14, c = 50.81Å
b = 113.5o
Resolution limit: 1.75Å; Multiplicity: 15.9
Anomalous scatterer: S (5 )
X-rays: synchrotron radiation
l = 1.488Å; D f ” = 0.52
Bijvoet ratio: <|DF |>/<F > = 0.56%
Phasing: OASIS DM (Cowtan)
Model building: RESOLVE BUILD &
ARP/wARP found 299 of the total 303
residues at the 6th cycle of iteration
Data courtesy of Dr. Z. Dauter,
National Cancer Institute, USA

15. TT0570 OASIS-2004 application Space group: P21212 Unit cell:
Data courtesy of Professor Isao Tanaka & Dr. Nobuhisa Watanabe
Graduate School of Science, Hokkaido University, Japan
Space group: P21212
Unit cell:
a =
b =
c = Å
Number of residues
in the ASU: 1206
Resolution range: Å
Multiplicity: 20.9
Anomalous scatterer: S (22)
Wavelength:
l = 2.291Å; Df ” = 1.14
Bijvoet ratio: <|DF|>/<F> = 1.16%
Phasing:
OASIS DM (Cowtan)
Model building:
RESOLVE BUILD & ARP/wARP
ARP/wARP found 1153 of the total 1206 residues after 2 cycles of iteration
OASIS-2004
application

16. OASIS-2004
Features of

17. Better initial SAD phases

18. Phase information available in SAD
Bimodal distribution
of SAD
The phase of F”
Phase information available in SAD
Cochran
distribution
Peaked at
any where
from 0 to 2p
Peaked at
Sim
distribution

19. Two different kinds of initial SAD phases + PSim PBimodal
Sim-modified phases
PSim PCochran P+ P+ + P
P+-modified phases

20. Cover figure of Acta Cryst. D60, Part 11 (2004)
Se-SAD
Histone Methyltransferase Set 7/9
Space group: P212121
Unit cell: a = 66.09, b = 82.83, c = Å
Number of residues in ASU: 560
Number of independent reflections: 16352
Resolution limit: 2.8Å
Multiplicity: 3.8
Anomalous scatterer: Se(12)
l = Å; Df’ = -7.5, Df” = 6.5
Bijvoet ratio: <|DF|>/<F> = 7.03%
SAD phasing by OASIS DM
Data provided by Dr. S. J. Gamblin
and Dr. B. Xiao
Cover figure of Acta Cryst. D60, Part 11 (2004)

21. Comparison of the two kinds of initial phases
using 4 typical reflections from the protein
histone methyltransferase SET 7/9

22. Comparison on cumulative phase errors
sorted in descending order of Fobs
Number of reflections
Errors of Sim-modified phases ( o )
Errors of P+-modified phases ( o )
1500
57.1
45.8
3000
57.1
49.1
4500
56.5
50.0
6000
57.0
51.2
7500
57. 8
52.9
9000
58.7
54.1
10500
59.4
55.6
12000
60.8
56.9
13500
61.9
58.4
15000
63.4
60.2
16352
65.2
62.3

23. 2. Inclusion and auto balance of the
lack-of-closure error in the direct-method
phasing formula

24. Automatic solution of protein structures
OASIS DM
by a single run of
RESOLVE
(Build only)
and/or
ARP/wARP +

25. Br-SAD OASIS-2004 application Pepstatin-insenstive carboxylproteinase
Contoured at 1s
Pepstatin-insenstive carboxylproteinase
Space group: P62
Unit cell: a = b = 97.31, c = 82.94Å, g = 120o
Resolution limit: 1.8Å; Multiplicity: 5.45
Anomalous scatterer: Br (13)
X-rays: synchrotron radiation
l = Å; D f ” = 5.0
Bijvoet ratio: <|D F |>/<F > = 7.06%
Phasing: OASIS DM (Cowtan)
Model building:
ARP/wARP found 358 of the total 372 residues
Data courtesy of Dr. Z. Dauter,
National Cancer Institute, USA

26. Xe-SAD OASIS-2004 application Porcine Pancreatic Elastase
Contoured at 1s
Porcine Pancreatic Elastase
Space group: P212121
Unit cell: a = 50.2, b = 58.1, c = 74.3Å
Resolution limit: 1.94Å;
Total rotation range: 360o
Anomalous scatterer: Xe (1)
X-rays: synchrotron radiation
l = 2.1Å; D f ” = 11.8
Bijvoet ratio: <|D F |>/<F > = 5.76%
Phasing: OASIS DM (Cowtan)
Model building:
ARP/wARP found 236 of the total 240
residues
Data courtesy of Dr. M. S. Weiss, EMBL
Hamburg Outstation, c/o DESY, Germany

27. S-SAD OASIS-2004 application Lysozyme Contoured at 1s
Space group: P43212
Unit cell: a = 78.81, c = 36.80Å
Atoms in the asymmetric unit: 1001
Resolution limit: 1.53Å; Multiplicity: 23
Anomalous scatterer: S (10), Cl (7)
X-rays: synchrotron radiation
l = 1.54Å; D f ” = 0.56, 0.70
Bijvoet ratio: <|D F |>/<F > = 1.55%
Phasing: OASIS DM (Cowtan)
Model building:
ARP/wARP found 128 of the total 129
residues
Data courtesy of Dr. Z. Dauter,
National Cancer Institute, USA

28. 302 residues found automatically
OASIS-2004 application
YfbpA
Space group: P212121
Unit cell: a = ,
b = ,
c = Å
Resolution range: – 1.42Å
Anomalous scatterer: Fe (1)
Wavelength: 1.542Å
Df ” = 3.20
<|DF|>/<F> ~ 1.4%
Phased by:
OASIS + DM (Cowtan)
Automatic model building by:
ARP/wARP
Data provided by Dr. Cheng Yang
Rigaku/MSC, USA
Cu-Ka Fe-SAD
302 residues found automatically

29. 3. Dual-space fragment extension
Partial
model No
Yes
Reciprocal-space
fragment extension by
OASIS DM
Real-space
RESOLVE BUILD
and/or ARP/wARP
OK?
End

30. Examples
Case study
and

31. Azurin
Cu-SAD
Synchrotron l = 0.97Å
Cycle 3
95%
Azurin
Cu-SAD
Synchrotron l = 0.97Å
Cycle 0
42%
Xylanase S-SAD
Synchrotron
l = 1.49Å
99%
Cycle 6
25%
Cycle 0
Xylanase S-SAD
Synchrotron
l = 1.49Å
Lysozyme
S-SAD
Cr-Ka
98%
Cycle 6
52%
Cycle 0
Lysozyme
S-SAD
Cr-Ka
Glucose
isomerase
S-SAD
Cu-Ka
17%
Cycle 0
97%
Cycle 6
Glucose
isomerase
S-SAD
Cu-Ka
Cr-Ka
Se, S-SAD
Alanine racemase
Cycle 4
97%
Cr-Ka
Se, S-SAD
Alanine racemase
Cycle 0
52%

33. OASIS-2004 application Xylanase Contoured at 1s Space group: P21
Unit cell: a = 41.07, b = 67.14, c = 50.81Å
b = 113.5o
Resolution limit: 1.75Å; Multiplicity: 15.9
Anomalous scatterer: S (5 )
X-rays: synchrotron radiation
l = 1.488Å; D f ” = 0.52
Bijvoet ratio: <|DF |>/<F > = 0.56%
Phasing: OASIS DM (Cowtan)
Model building: RESOLVE BUILD &
ARP/wARP found 299 of the total 303
residues at the 6th cycle of iteration
Data courtesy of Dr. Z. Dauter,
National Cancer Institute, USA

34. Xylanase: average phase error decreased during dual-space iteration
Phase error in degrees 70 60 50 40 30 20 80 10
Cycle 5 1 6 2 4 3
Xylanase: average phase error decreased
during dual-space iteration
Is OASIS
necessary
here?
Is OASIS
necessary
here?
Yes
What would it be
without using
RESOLVE (build only)?
- OASIS-2004
- DM
- Partial model from
RESOLVE BUILD
or ARP/wARP No

35. OASIS-DM-ARP/wARP Iteration
Xylanase sulfur-SAD phasing
Synchrotron radiation l = 1.49Å, <DF>/<F> = 0.56% 70 60 50 40 30 20 80 10 2 12 4 8 6 14 16
Phase error in degrees
Cycle
- OASIS-2004
- DM
- Partial model from
ARP/wARP

36. Improvement on electron-density map and automatic model building
Cycle 0
Cycle 3
Cycle 6
Improvement on electron-density map and
automatic model building

37. Inside direct-method
SAD phasing

38. SAD formulation

39. SAD formulation F”
F +
F +
- F”
F* - F’
F o

40. SAD formulation
F + ?

42. P+ formula

44. Maximizing P(Djh) Þ
Djh=b

45. Replacing Ehexp(ia) with
mhEhexp(iabest) Þ

46. Fan, H.F. & Gu, Y.X.,
Acta Cryst. A41, (1985)

47. Fan, H.F. & Gu, Y.X.,
Acta Cryst. A41, (1985)

48. Thank you!