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.
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.