Planning a SAD experiment with plan_sad_experiment



plan_sad_experiment is a tool for estimating the anomalous signal that you might get from your SAD experiment and for predicting whether this signal would be sufficient to solve the structure. plan_sad_experiment is normally used along with scale_and_merge and anomalous_signal to plan a SAD experiment, scale the data, and analyze the anomalous signal before solving the structure.


How plan_sad_experiment works:

Output from plan_sad_experiment

plan_sad_experiment provides a summary of the scattering expected from your crystal and a summary of the anomalous signal expected if you are able to measure your data with the suggested overall I/sigI. You can set the maximum I/sigI to look for. Here is an example setting max_i_over_sigma=30:

   ----------Dataset overall I/sigma required to solve a structure----------

Dataset characteristics:
  Target anomalous signal:    30.0
  Residues: 325
  Chain-type: PROTEIN
  Solvent\_fraction:    0.50
  Atoms: 2642
  Anomalously-scattering atom: se
  Wavelength:  0.9792 A
  Sites: 7
  f-double-prime:    3.84

Target anomalous scatterer:
  Atom: se  f": 3.84  n:    7   rmsF:   10.2

Other anomalous scatterers in the structure:
  Atom:  C  f": 0.00  n: 1674   rmsF:    0.1
  Atom:  N  f": 0.01  n:  445   rmsF:    0.1
  Atom:  O  f": 0.01  n:  514   rmsF:    0.3
  Atom:  S  f": 0.23  n:   10   rmsF:    0.7

Normalized anomalous scattering:
  From target anomalous atoms rms(x**2)/rms(F**2):     2.97
  From other anomalous atoms rms(e**2)/rms(F**2):      0.24
  Correlation of useful to total anomalous scattering: 1.00

  ----------Dataset <I>/<sigI> needed for anomalous signal of 15-30----------

-------Targets for entire dataset-------  ----------Likely outcome-----------

                              Anomalous    Useful    Useful
                            Half-dataset  Anom CC   Anomalous
 Dmin   N     I/sigI sigF/F     CC       (cc*\_anom)  Signal   P(Substr)   FOM
                      (%)                                        (%)

 6.00    852    29    3.0      0.58        0.64        7         51       0.22
 5.00   1473    29    3.0      0.62        0.66        9         79       0.15
 3.00   6821    29    3.0      0.64        0.66       19         89       0.22
 2.50  11787    29    3.0      0.70        0.68       25         96       0.19
 2.00  23021    28    3.2      0.62        0.66       29         97       0.17
 1.50  54569    13    6.7      0.18        0.42       29         97       0.15

Note: Target anomalous signal not achievable with tested I/sigma (up to 30 )
for resolutions of  2.50 A and lower. I/sigma shown is value
of max\_i\_over\_sigma.

This table says that if you collect your data to a resolution of   2.0 A with
an overall <I>/<sigma> of about  28 then the half-dataset anomalous
correlation should be about  0.62 (typically within a factor of 2).  This
should lead to a correlation of your anomalous data to true anomalous
differences (CC*\_ano) of about  0.66, and a useful anomalous signal around
 29 (again within a factor of about two). With this value of estimated
anomalous signal the probability of finding the anomalous substructure is
about  96% (based on estimated anomalous signal and actual outcomes for
real structures.), and the estimated figure of merit of phasing is 0.17.

The value of sigF/F (actually rms(sigF)/rms(F)) is approximately the inverse
of I/sigma. The calculations are based on rms(sigF)/rms(F).

Note that these values assume data measured with little radiation damage or at
least with anomalous pairs measured close in time. The values also assume that
the anomalously-scattering atoms are nearly as well-ordered as other atoms.
If your crystal does not fit these assumptions it may be necessary to collect
data with even higher I/sigma than indicated here.

Note also that anomalous signal is roughly proportional to the anomalous
structure factors at a given resolution. That means that if you have 50%
occupancy of your anomalous atoms, the signal will be 50% of what it otherwise
would be.  Also it means that if your anomalously scattering atoms only
contribute to 5 A, you should only consider data to 5 A in this analysis.

What to do next:

1. Collect your data, trying to obtain a value of I/sigma for the whole
   dataset at least as high as your target.
2. Scale and analyze your unmerged data with phenix.scale\_and\_merge to get
   accurate scaled and merged data as well as two half-dataset data files
   that can be used to estimate the quality of your data.
3. Analyze your anomalous data (the scaled merged data and the two
   half-dataset data files) with phenix.anomalous\_signal to estimate
   the anomalous signal in your data. This tool will again guess the
   fraction of the substructure that can be obtained with your data,
   this time with knowledge of the actual anomalous signal.  It will also
   estimate the figure of merit of phasing that you can obtain once you
   solve the substruture.
4. Compare the anomalous signal in your measured data with the
   estimated values in the table above. If they are lower than expected
   you may need to collect more data to obtain the target anomalous signal.


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