Phasing, Density Modification, & Electron Density Map Calculation
Structural Molecular Biology Laboratory, ChemM230D

MIR phasing circle.
Suggested Reading Materials

1) Heavy-atom location and phase determination with single-wavelength diffraction data by B.W. Matthews. International Tables of Crystallography, Volume F, page 293-298.

2) Phase improvement by iterative density modification by K.Y.J. Zhang, K.D. Cowtan, and P. Main.  International Tables of Crystallography, Volume F, page 311-316.

3) Chapter 6 (page 101-127) from Crystallography Made Crystal Clearby Gale Rhodes. 

4) MLPHARE manual by Zbyszek. Otwinowski.

5) DM manual by Kevin Cowtan.
6) Mike's introduction to Phasing lab in powerpoint format.


Assignment & Procedures
3rd Assignment: 
Phasing Statistics Table
due week of February 16, 2004
Objective: To report phasing statistics describing your first experimental electron density map. 

Method:  For each derivative, report the number of heavy atom sites, Rcullis (centric/acentric), Phasing power (centric/acentric), and the overall figure of merit (before and after density modification) from the log file of MLPHARE.  Submit your Table 2 to Mike or Duilio by Tuesday, February 19, 2002.  See notes below on the meaning of these phasing statistics and what  the values of these phasing statistics are in the case of good/bad  quality phases.

An example from assignment three. Adapted from Steegborn et al., 2001.  Crystal Structure of Transcription Factor MalT DomainIII: A Novel Helix Repeat Fold implicated in Regulated Oligomeriztion, Structure, Vol. 9, 1051-1060.

Part One:
Verify your heavy atom coordinates.
Objective: To verify that the heavy atom coordinates that you calculated are consistent with the experimental isomorphous difference Patterson map.

 Open a Konsole window. Change directories to your personal working directory. Here, you will find the Eu anomalous difference Patterson map (, calculated as you did in the last meeting, but this time using the XtalView xfft GUI.  Make a file containing the coordinates for your heavy atom (e.g. eu.sol).  Now use XtalView's xpatpred GUI to convert these XYZ coordinates to UVW coordinates of Patterson space (xpatpred eu.sol eu.pred).  Display the difference Patterson map with the coordinates of the predicted Patterson peaks superimposed (xcontur eu.pred).  If your heavy atom coordinates were correct,  there will be a cross mark over all the major peaks in the difference Patterson map. 



Part Two:
Calcluate Phases and Choose Handedness
Objective: To refine heavy atom positions, calculate phases, and calculate electron density maps.

Strategy:  The quality of an experimental electron density map will generally improve with the number of derivatives added to the phasing calculation.  To combine the heavy atom derivative data sets FPH1, FPH2, FPH3, etc. for phasing two tasks must be accomplished (1) determining the correct handedness of the heavy atom model and (2) placing the heavy atoms from each derivative on the same origin.  Both tasks can be accomplished using the cross-difference Fourier method. In this method, protein phases (aP) are first calculated using the native data set (FP) and structure factors and anomalous differences from only one of the heavy atom derivative data sets (FPH1   and  DPH1).   (These phases are refered to as SIRAS phases because they are calculated using single isomorphous replacement with anomalous scattering.) A cross-difference Fourier map is then calculated using coefficients FP-FPH2and phases aP.  The highest peak on this cross-difference Fourier map should correspond to the metal position of the second derivative (PH2). However, there is a 50/50 chance that we chose the incorrect hand of the first heavy atom (i.e. the heavy atom positions used for the phasing calculation).  We can judge whether the handedness we chose is correct by comparing the height of the highest cross-difference Fourier peak with the height of the highest peak from a second cross-difference Fourier map.  The second map has the same coefficients as the first cross difference Fourier map (FP-FPH2) but with phases aP obtained from using the opposite hand of the first heavy atom (i.e. -x-y-z instead of x,y,z) .  The cross difference Fourier map that produces the highest peak is the correct hand.  You may have expected that the peak heights in the two cross-difference Fourier maps would be identical because of Friedel's law. That is, the phases of the protein calculated in either of the opposite hands should differ by only a negative sign.  Hence, you would expect the cross difference Fourier peaks to be the same height in either hand.  But, as you will see from the experimental evidence this is clearly not true.  The reason that there is a small but significant difference in peak heights is due to the anomalous scattering contribution DPH1 used in calculating the protein phases.  So the protein phases output in the two phasing runs differ by more than just a negative sign (though not by much more).

Calculate SIRAS (single isomorphous / anomalous scattering) phases using the Europium position and calculate the cross difference Fourier map to find the pHMBA sites all in one script.    Choose the MLPHARE button in the CCP4 GUI.  Choose cross peak maps option.  Input coordinates for the Europium site (negate all signs), then select the option to "change the space group to opposite hand before running Mlphare".  Look at the log file.  Peaks from the cross difference Fourier map will be listed in decreasing order, with the highest peak on the top.  Note the position and height of the top peak.  Now, repeat the calculation but DEselect the option to "change the space group to opposite hand before running Mlphare".  Note that the highest peak in this run is higher than in the previous run.  Therefore, the space group should NOT be changed to the opposite hand.  Note the position of the highest peak and use these coordinates as the pHMBA site to input in the COMBINED phasing calculation.  Because the coordinates for the pHMBA site were taken from a difference Fourier map, they will be on the same origin as the Eu.  Deselect the option for cross-peaks maps.  Add a second derivative to the list by pressing the button to "Add Another Derivative".  Input the heavy atom site for the pHMBA.  Look at the log file and record the phasing statistics.


MlPhaRe phasing and
cross-difference Fourier calculation (inverted hand).

MlPhaRe phasing and
cross-difference Fourier calculation (original hand).

 MlPhare phasing combined Eu and pCMBS derivatives.

Part Three:
Density modification and Map calculation.
Objective: To improve the experimental phases by imposing some constraints on the electron density distribution.

 Use the DM window in the programs list of ccp4i.  Run  View map with O.  Use @omacro.  See bones.  Use stereo glasses.



A command file to extend the electron density map to cover the unit cell, produce bones, and convert to O format.  To run it, type ccp4mapfilename

#!/bin/csh -f
extend MAPIN $1 MAPOUT dm.ext << END-extend
XYZLIM 0 1  0 1.0  0 1

mapman -b mapsize 10000000<<eof
re m1 dm.ext ccp4
no m1
map m1 dm.omap
bo sk m1 1.3 0.7 100
bo co dm.odb skl 5

Figure of Merit statistics from MLPHARE log file.  Phasing with Hg only.

Lack of Closure Analysis from MLPHARE log file --Phasing on Hg only.

Taken from

Instructor's preparations

Back to CHEM M230D course syllabus 

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