1. In serial femtosecond crystallography (SFX) with hard X-ray free-electron laser as light source, a set of three-dimensional single-crystal diffraction data is produced by a huge number of crystal grains in micron size --- a typical powder sample. This is beneficial to protein crystallography by avoiding growth of large single crystal samples. This may also capable of enhancing the power of powder method, in solving crystal structures, from ~20 independent atoms to more than a thousand. Another important feature of SFX experiments is that diffraction patterns of a single experiment for multi-phase samples can be identified and grouped to independent subsets corresponding to each single phase. Simulating calculations were performed to exploring possibilities of SFX experiments. Encouraging results have been obtained. Phase identification and crystal-structure determination from a single SFX experiment for a mixture sample of two zeolites Sample: 4(TNU-9) + NU-87 TNU-9: Unit cell contents: Na, 24(SiO 2 ); space group: C2/m; Cell dimensions: a=27.845 Å, b=20.015 Å, c=19.597 Å, β=93.000° NU-87: Unit cell contents: 17(SiO 2 ); space group: P2 1 /c; Cell dimensions: a=14.324 Å, b=22.376 Å, c=25.092 Å, β=151.51° Simulation: The program pattern_sim in crystFEL package was used to simulate SFX diffraction data. Wavelength of X-ray used in the simulation is 0.7 Å; X-ray intensity is 10 11 photons per pulse; size of crystals is from 600 nm to 2000 nm. Two samples were calculated independently firstly. Then the simulation patterns were mixed randomly with a ratio of 4 to 1. One million of diffraction patterns were calculated in total, and 799139 of which belong to TNU-9. Identification: Single diffraction patterns were indexed separately by the program indexamajig in CrystFEL without information about cell dimensions. Each pattern that can be identified successfully defined a set of cell dimensions belong to itself. Volume of unit cell was chosen as an indicator to represent each set of cell dimensions. The distribution of number of patterns vs. unit cell volume was drawn as shown below. The two major peaks at 3800Å 3 and 5500Å 3 can be identified easily. Small peaks represent larger volume that are multiple of either of the two major peaks. Blue peaks belong to component 1; red ones belong to component 2. So the two kinds of components in the sample can be clearly identified. These are very closed respectively to the known values of TNU-9 and NU-87. Crystal structures of the two components were determined using the program SHELXD. Projections of the two structures are shown in the following figure. As can be seen, results from SHELXD are nearly complete, except the type of a few atoms should be corrected. SAD: phasing with impure sample In preparation of protein samples, especially heavy-atom derivatives for SFX, it is unavoidable that native and derivative(s) are mixed together. It is essential to extract diffraction patterns that belong to a single phase from a vast number of diffraction patterns yielded by a mixture sample. This can be done by a similar identification method proposed above. Simulating calculations were performed with a set of SFX SAD data from the mixture of Gadolinium derivative (90%) and native (10%) of lysozyme. When the whole set of mixed data were used in a SAD phasing, no reasonable result was obtained. However, if the subset of derivative data extracted by the identification process were used, the result is nearly a complete structure model. After analyzing the results of indexing, cell dimensions of the two components are refined as (1) a=17.30 Å, b=17.34 Å, c=19.46 Å, α=87.89 o, β=87.89 o, γ=70.84 o (2) a=13.66 Å, b=14.25 Å, c=22.35 Å, α=89.90 o, β=89.92 o, γ=61.87° All the diffraction patterns were re-indexed by the program indexamajig with information of cell dimensions obtained. The number of patterns that belong to component (1) is 675185, while that of component (2) is 159562. The ration is 4.40 : 1. Structure determination: The program XPREP was used to rearrange the cell dimensions and determine the space group. Results are as bellow (1) C2/m; a=28.23 Å, b=20.08 Å, c=19.46 Å,β=92.59 o (2) P2 1 /c; a=14.32 Å, b=22.38 Å, c=25.09 Å,β=151.50 o SIR: native & derivative data from a single SFX experiment In the single isomorphous replacement (SIR) case, we can used a mixture sample containing both native and derivative crystals. After the SFX experiment, diffraction patterns can be identified and grouped into two subsets, one corresponding to the native, while the other to the derivative. We thus obtained a set of SIR data from a single SFX experiment. Simulating calculation with the known protein LegC3N led to a satisfactory result. Similar process may also be applied in the multiple isomorphous replacement (MIR) case. E-mail: fanhf@cryst.iphy.ac.cn