Preparations for Phasing
Structural Molecular Biology Laboratory, ChemM230D
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Michael R. Sawaya, Duilio Cascio
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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
COREY:FILES[985] ls
total 11830
-rwxr-xr-x 1 sawaya eisenber
1922 Feb 22 23:54 cp.com
-rwxr-xr-x 1 sawaya eisenber
7846 Feb 22 23:54 helix.pdb
-rwxr-xr-x 1 sawaya eisenber
234 Feb 22 23:54 mapman.com
-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 sas.map
-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.
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