Preparations for Phasing
Structural Molecular Biology Laboratory, ChemM230D
Michael R. Sawaya, Duilio Cascio
1) Print out the powerpoint presentation handout_phasing.ppt  and distribute as handouts.

Copy necessary files to the student's directory.  See /auto_nfs/data1/users/sawaya/HTML/m230d/Phasing/FILES

total 11830
-rwxr-xr-x    1 sawaya   eisenber     1922 Feb 22 23:54
-rwxr-xr-x    1 sawaya   eisenber     7846 Feb 22 23:54 helix.pdb
-rwxr-xr-x    1 sawaya   eisenber      234 Feb 22 23:54
-rw-r--r--    1 sawaya   eisenber       86 Feb 22 23:54 mapmove
-rw-r--r--    1 sawaya   eisenber      369 Feb 22 23:54 omacro
-rw-r--r--    1 sawaya   eisenber 12096896 Feb 22 23:54
-rw-r--r--    1 sawaya   eisenber       95 Feb 22 23:54 xcontur.defaults

Mike's comment for next year- give the students a handout with real & imaginary axis, lengths of FP and FPH vectors, and vector for heavy atom FH.  Have them determine phase graphically with compass.

2) We will check that the heavy atom site you solved can predict the position of all the Patterson peaks in the isomorphous difference Patterson map.  Apply all the difference vector equations we saw from last week.

3) Calculate Best phases and figure of merit for this site.  Use the isomorphous differences as well as anomalous differences. This much should be familiar to you.  Just using Harker Constructions as seen in the anomalous scattering lecture last Friday.  This would be an example of SIRAS phasing.  However, we have to resolve the ambiguity of handedness of the electron density map.  Remember, the coordinate xyz as well as -x,-y,-z are both consistent with the Patterson map.  This ambiguity arises because Patterson map has an additional center of symmetry that the electron density map does not have.  Only one of these choices is correct.  We must test both choices. We can do this by looking at the map to see which has the features of a protein. Another way to determine the correct handedness is to perform a cross difference Fourier with a second derivative. With these phases calculated from one derivative, calculate a cross difference Fourier with the other derivative.  Peaks in map will correspond to second derivative site.  Notice the peak height.

4) Check the inverted hand with inverted space group.  Peaks in map will correspond to noise.  Check the peak height.

5)Choose the correct hand. This is important for handedness and for getting the two heavy atoms refered to the same origin.

6) Produce a combined phasing map which includes phasing information from both derivatives.  This will have the best phases.

7) Perform density modification.

8) Observe map.

9) Record phasing statistics.

Back to phasing experiment

Back to CHEM M230D course syllabus 

[Overview] ·[Facilities] · [People] · [Services] ·[Lectures] · [BioLinks] · [Stats] ·[Search]