1. Phasing Today’s goal is to calculate phases (  p ) for proteinase K using PCMBS and EuCl 3 (MIRAS method). What experimental data do we need? 1) from native crystal we measured |F p | for native crystal 2) from PCMBS derivative we measured |F PHg(h k l) | 3) also from PCMBS derivative we measured |F PHg(-h-k-l) | 4) f Hg for Hg atom of PCMBS 5) from EuCl 3 derivative we measured |F PEu(h k l) | 6) also from EuCl 3 derivative we measured |F PEu(-h-k-l) | 7) F Eu for Eu atom of EuCl 3 How many phasing triangles will we have for each structure factor if we use all the data sets ? F P ( h k l) = F PH ( h k l) - F H ( h k l) is one type of phase triangle. F P ( h k l) = F PH(-h-k-l) * - F H (-h-k-l) * is another type of phase triangle.

2. Four Phase Relationships PCMBS F P ( h k l) = F PHg ( h k l) - f Hg ( h k l) isomorphous differences F P ( h k l) = F PHg(-h-k-l) * - f Hg (-h-k-l) * from Friedel mates (anomalous) EuCl 3 F P ( h k l) = F PEu( h k l) - f Eu ( h k l) isomorphous differences F P ( h k l) = F PEu (-h-k-l) * - f Eu (-h-k-l) * from Friedel mates (anomalous)

3. |F p ( h k l) | = 1.8 Real axis Imaginary axis F P ( h k l) = F PH1 ( h k l) - f H1 (h k l) 1 2 2 1 -2 -2 Harker construction 1) |F P ( h k l) | –native measurement

4. Real axis Imaginary axis Harker construction 1) |F P ( h k l) | –native measurement 2) F H (h k l) calculated from heavy atom position. FHFH |F p | F P ( h k l) = F PH1 ( h k l) - f H1 ( h k l) |F p ( h k l) | = 1.8 |f H ( h k l) | = 1.4  H (hkl) = 45°

5. Real axis Imaginary axis fHfH |F p | |F PH | F P ( h k l) = F PH1 ( h k l) - f H1 ( h k l) |F P ( hkl) | = 1.8 |f H1 ( hkl) | = 1.4  H1 (hkl) = 45° |F PH1 (hkl) | = 2.8 Harker construction 1) |F P ( h k l) | –native measurement 2) f H1 (h k l) calculated from heavy atom position. 3) |F PH1 (hkl)|–measured from derivative. |F PH | Let’s look at the quality of the phasing statistics up to this point.

6. Imaginary axis SIR Real axis fHfH |F p | |F PH | 0 90 180 270 360 Which of the following graphs best represents the phase probability distribution, P(  )? a) b) c)

7. Imaginary axis SIR Real axis fHfH |F p | |F PH | 0 90 180 270 360 The phase probability distribution, P(  ) is sometimes shown as being wrapped around the phasing circle. 90 0 180 270

8. Imaginary axis SIR Real axis fHfH |F p | |F PH | Which of the following is the best choice of F p ? 90 0 180 270 90 0 180 270 90 0 180 270 90 0 180 270 a) b) c) Radius of circle is approximately |F p |

9. Imaginary axis SIR Real axis fHfH |F p | |F PH | 90 0 180 270 best F = |F p |e i  P(  )d   Sum of probability weighted vectors F p Usually shorter than F p

10. 90 0 180 270 SIR Sum of probability weighted vectors F p Best phase best F = |F p |e i  P(  )d  

11. 90 0 180 270 SIR a)1.00 b)2.00 c)0.50 d)-0.10 Which of the following is the best approximation to the Figure Of Merit (FOM) for this reflection? FOM=|F best |/|F P | Radius of circle is approximately |F p |

12. Which phase probability distribution would yield the most desirable Figure of Merit? 0 90 180 270 + + 0 90 180 270 + + a) b) c) 90 0 180 270

13. F best |F PH | Imaginary axis SIR Real axis |F p | a)2.50 b)1.00 c)0.50 d)-0.50 Which of the following is the best approximation to the phasing power for this reflection? Lack of closure = |F PH |-|F P +F H | = 0.5 (at the  P of F best ) |F p | fHfH |f H ( h k l) | = 1.4 Phasing Power = |f H | Lack of closure

14. F best |F PH | Imaginary axis SIR Real axis |F p | a)2.50 b)1.00 c)0.50 d)-0.50 Which of the following is the most desirable phasing power? |F p | fHfH What Phasing Power is sufficient to solve the structure? >1 Phasing Power = |f H | Lack of closure

15. fHfH SIR a)-0.5 b)0.5 c)1.30 d)2.00 Which of the following is the R Cullis for this reflection? R Cullis = Lack of closure isomorphous difference From previous page, LoC=0.5 Isomorphous difference= |F PH | - |F P | 1.0 = 2.8-1.8 |F P ( hkl) | = 1.8 |F PHg (hkl) | = 2.8 F best Imaginary axis Real axis |F p | |F PH |

16. Real axis Imaginary axis f H ( hkl ) F p (hkl) We will calculate SIRAS phases using the PCMBS Hg site. F P –native measurement f H (hkl) and f H (-h-k-l) calculated from heavy atom position. F PH (hkl) and F PH (-h-k-l) –measured from derivative. Point to these on graph. Isomorphous differences Anomalous differences To Resolve the phase ambiguity f H (-h-k-l)* F PH (-h-k-l)* SIRAS F P ( h k l) = F PH(-h-k-l) * - f H (-h-k-l) *

17. Isomorphous differences Anomalous differences Real axis Imaginary axis f H ( hkl ) F p (hkl) f H (-h-k-l)* F PH (-h-k-l)* SIRAS 0 90 180 270 360 Which P(  ) corresponds to SIR? Which P(  ) corresponds to SIRAS?

18. Isomorphous differences Anomalous differences Real axis Imaginary axis f H ( hkl ) F p (hkl) f H (-h-k-l)* F PH (-h-k-l)* SIRAS Which graph represents FOM of SIRAS phase? Which represents FOM of SIR phase? Which is better? Why? 90 0 180 270 90 0 180 270

19. Calculate phases and refine parameters (MLPhaRe)

20. A Tale of Two Ambiguities We can solve both ambiguities in one experiment.

21.  Remember, because the position of Hg was determined using a Patterson map there is an ambiguity in handedness.  The Patterson map has an additional center of symmetry not present in the real crystal. Therefore, both the site x,y,z and -x,-y,-z are equally consistent with Patterson peaks.  Handedness can be resolved by calculating both electron density maps and choosing the map which contains structural features of real proteins (L- amino acids, right handed  -helices).  If anomalous data is included, then one map will appear significantly better than the other. Note: Inversion of the space group symmetry (P4 3 2 1 2 →P4 1 2 1 2) accompanies inversion of the coordinates (x,y,z→ -x,-y,-z) Center of inversion ambiguity Patterson map

22. Choice of origin ambiguity I want to include the Eu data (derivative 2) in phase calculation. I can determine the Eu site x,y,z coordinates using a difference Patterson map. But, how can I guarantee the set of coordinates I obtain are referred to the same origin as Hg (derivative 1)? Do I have to try all 48 possibilities?

23. Use a Cross difference Fourier to resolve the handedness ambiguity With newly calculated protein phases,  P, a protein electron density map could be calculated. The amplitudes would be |F P |, the phases would be  P.  x  =1/V*  |F P |e -2  i(hx+ky+lz-  P ) Answer: If we replace the coefficients with |F PH2 -F P |, the result is an electron density map corresponding to this structural feature.

24.  x  =1/V*  |F PH2 -F P |e -2  i(hx-  P ) What is the second heavy atom, Alex. When the difference F PH2 -F P is taken, the protein component is removed and we are left with only the contribution from the second heavy atom. This cross difference Fourier will help us in two ways: 1)It will resolve the handedness ambiguity by producing a very high peak when phases are calculated in the correct hand, but only noise when phases are calculated in the incorrect hand. 2)It will allow us to find the position of the second heavy atom and combine this data set into our phasing. Thus improving our phases.

25. Phasing Procedures 1)Calculate phases for site x,y,z of Hg and run cross difference Fourier to find the Eu site. Note the height of the peak and Eu coordinates. 2)Negate x,y,z of Hg and invert the space group from P4 3 2 1 2 to P4 1 2 1 2. Calculate a second set of phases and run a second cross difference Fourier to find the Eu site. Compare the height of the peak with step 1. 3)Chose the handedness which produces the highest peak for Eu. Use the corresponding hand of space group and Hg, and Eu coordinates to make a combined set of phases.

26. Real axis Imaginary axis Anomalous Deriv 1 F p (hkl) Isomorphous Deriv 1 Isomorphous Deriv 2 EuCl 3 F PH = F P +f H for isomorphous differences MIRAS f H1 (-h-k-l)* F PH1 (-h-k-l)* f H1 (hkl) F PH1 (hkl) f H2 (hkl) F PH2 (hkl)

27. Real axis Imaginary axis Anomalous Deriv 1 Isomorphous Deriv 1 Isomorphous Deriv 2 F p (hkl) Anomalous Deriv 2 EuCl 3 F P ( h k l) = F PH1 (-h-k-l) * - f H1 (-h-k-l) * Harker Construction for MIRAS phasing (Multiple Isomorphous Replacement with Anomalous Scattering) f H1 (-h-k-l)* F PH1 (-h-k-l)* f H1 (hkl) F PH1 (hkl) f H2 (-h-k-l)* F PH2 (-h-k-l)* f H2 (hkl) F PH2 (hkl)

28. Density modification A) Solvent flattening. Calculate an electron density map. If  solvent If  >threshold -> protein Build a mask Set density value in solvent region to a constant (low). Transform flattened map to structure factors Combine modified phases with original phases. Iterate Histogram matching

29. MIR phased map + Solvent Flattening + Histogram Matching MIRAS phased map

30. MIR phased map + Solvent Flattening + Histogram Matching MIRAS phased map

31. Density modification B) Histogram matching. Calculate an electron density map. Calculate the electron density distribution. It’s a histogram. How many grid points on map have an electron density falling between 0.2 and 0.3 etc? Compare this histogram with ideal protein electron density map. Modify electron density to resemble an ideal distribution. Number of times a particular electron density value is observed. Electron density value

32. HOMEWORK

33. Barriers to combining phase information from 2 derivatives 1)Initial Phasing with PCMBS 1)Calculate phases using coordinates you determined. 2)Refine heavy atom coordinates 2)Find Eu site using Cross Difference Fourier map. 1)Easier than Patterson methods. 2)Want to combine PCMBS and Eu to make MIRAS phases. 3)Determine handedness (P4 3 2 1 2 or P4 1 2 1 2 ?) 1)Repeat calculation above, but in P4 1 2 1 2. 2)Compare map features with P4 3 2 1 2 map to determine handedness. 4)Combine PCMBS and Eu sites (use correct hand of space group) for improved phases. 5)Density modification (solvent flattening & histogram matching) 1)Improves Phases 6)View electron density map