The difference between the data collected with the regular and MDS strategies can turn out to be marginal and therefore a sensitive method is required to measure the subtle difference and assess the impact of this difference on the structure solution. We decided to use sulfur’s anomalous signal in zinc-free insulin crystals as a probe to assess the data quality of diffraction data collected using both strategies. Sulfur’s anomalous signal is comparatively weak if the diffraction data are collected using the usual X-ray wavelength (0.97–2.0 Å), but this shortcoming has not stopped researchers from using sulfur’s anomalous signal as a phasing probe. It has been explored experimentally by Hendrickson & Teeter (1981

) and theoretically by Wang (1985

). More successful cases were reported in the 1990s (Dauter

*et al.*, 1999

; Liu, 2000

). Therefore, sulfur atoms’ weak anomalous signal can serve as a sensitive probe to distinguish the subtle difference in the diffraction data collected with different strategies. The efficiencies of the two data-collection strategies can be evaluated by measuring and comparing the strengths of the anomalous signal recorded in the diffraction data. The rationale for choosing insulin crystals is as follows: (i) a Zn-free insulin crystal has high symmetry (

*I*2

_{1}3 space group) and is suitable for collecting data with both strategies without introducing too much radiation damage to the crystal; (ii) it is easy to obtain an insulin sample and grow crystals, and the diffraction resolution (around 2.0 Å) of an insulin crystal is suitable for the evaluation of data quality; (iii) there are three disulfide bonds per insulin molecule and the anomalous signal from those three disulfide bonds is a perfect probe for the evaluation of data quality. Three parameters were proposed to evaluate the quality of the data collected using the different strategies:

(1) Relative peak height (RPH): RPH is the ratio of the average peak height of three disulfide bonds (the top three highest peaks) and the average peak height of the last three (seventh, eighth and ninth) in the first nine highest peaks in the anomalous difference Fourier map calculated at 50.0–2.5 Å resolution using anomalous data and rigid-body-refined model phases calculated by the program

*FFT* in the

*CCP4* suite (Collaborative Computational Project, Number 4, 1994

). The idea here is to compare the anomalous peak densities of the three ‘specific’ disulfide bonds (top three) in relation to the ‘representative’ noise peaks in the map. It is expected that a higher RPH value means stronger anomalous signals from three disulfide bonds were recorded and thus a set of better-quality data was collected.

The fourth, fifth and sixth highest peaks were not selected in the calculation because of the consideration that they may be more affected by experimental conditions. For example, any metal ions from either the insulin sample, buffer or crystallization solutions may contribute to the higher level of background anomalous signals. Therefore, peaks 4, 5 and 6 are more likely to be affected than are peaks 7, 8 and 9; in other words, the seventh, eighth and ninth peaks are more eligible to ‘represent’ the noise level in the map.

(2) Map correlation coefficient (Map cc): Map cc is the map correlation coefficient between the model-phased 2

*f*
_{o} −

*f*
_{c} electron-density map and the S-SAD-phased experimental map calculated at the same resolution range (50.0–2.5 Å). It is used to measure the deviations between the experimentally S-SAD-phased map and the theoretically calculated ideal map. It is an indirect indication of the data quality collected using different strategies. The model-phased map was calculated using the Fourier synthesis method with equation (11)

:

where

*p* is the electron-density function,

*w* is the figure of merit (FOM) calculated from the rigid-body refinement process,

*F* is the difference of the two times’ measured amplitude in the diffraction data minus the calculated diffraction factor (2

*f*
_{o} −

*f*
_{c}), ϕ represents the phases calculated from the refined model. The S-SAD experimentally phased map was calculated using the same equation (11)

and the same amplitude

*F*, but the FOM and phases were calculated using sulfur atoms’ anomalous scattering signals in each data set (Wang, 1985

). The sulfur atoms’ coordinates were obtained from the rigid-body-refined models. The Map cc is the correlation coefficient between two maps, calculated using

*Overlapmap* in the

*CCP4* suite (Collaborative Computational Project, Number 4, 1994

). It is defined by equation (12)

where

*x* represents the density values from one map and

*y* represents the values from the other map,

represents the mean value of the quantities inside the parentheses.

(3) Ratio of map correlation coefficient (Rcc): Rcc is defined as the ratio of Map cc calculated for data collected using the MDS strategy to the Map cc of data collected using the regular strategy and is expressed as

where Map cc

_{MDS} and Map cc

_{reg} are the map correlation coefficients of data collected with the MDS and regular strategies, respectively, of the same crystal. It is designed to compare the effectiveness of MDS and regular data collection. A larger value of Rcc indicates a bigger difference between the two data-collection strategies.