1. 1 Catalog 1 Check the radiation damagep. 2 2 Check aggregation by AutoRg program. Calculate theoretical I 0, MW, and V tot with protein SAXS I 0 estimtaion.xls. p. 7 3 Check inter-particle interaction with concentration dependence I 0 /c p. 14 4 Profile merging merge the low and high concentration curves. p. 19 5 Crysol Program. Compare (fit) solution structure with PDB crystal structure. p. 29 6 Transform data to P(r) with GNOM program and data fitting. p. 38 7-1 Dammin Program An ensemble of dummy atom model simulation with P(r) output by GNOM. p. 47 7-2 Gasbor Program An ensemble of dummy residues model simulation with P(r) output by GNOM. p. 64

2. 1. Check the radiation damage 2

3. 1. Import multiple ACSII of output files of each frame and combined data 3

4. 2. Plot all curves in a graph 4

5. 3. Check the low-q region in the all profiles 5

6. If all profiles are overlapped well at low-q region, no radiation damage occurs. 6

7. 2. Check aggregation by AutoRg program. Calculate theoretical I 0, MW, and V tot with protein SAXS I 0 estimtaion.xls. 7

8. 1. Start program: Primus -> AutoRg 8

9. 2. Open the input file (file type: txt, dat) 9

10. 3. In Plot tab, read the raw data. 10

11. 4. In Guinier tab, check the aggregation and define the q min before unreliable range. 11

12. 5. In Info tab, read the sRg limits (Guinier region), I 0, and R g values. 12

13. 1 2 3 6 4 5 One can calculate theoretical MW, I 0, and V tot with protein SAXS I 0 estimtaion.xls. (spreadsheet edited by Dr. Jeng) 13

14. 3. Check inter-particle interaction with concentration dependence I 0 /c 14

15. 1. Import multiple ACSII of all different concentration curves. 15

16. 2. Plot all different concentration curves. 16

17. 3. Set each y and yEr column as I(q)/conc. 17

18. 4. Confirm if concentration dependence is distinguishable at low-q region. 18

19. 4. Profile merging merge the low and high concentration curves. 19

20. 1. Start program: Primus 20

21. 2. Click “Tools” to select the low and high concentration data for merging. 21

22. 3. Click “plot” to plot the two curves. 22

23. 4. Input the parameters into nBeg (begin point #) and nEnd (end point #) of Data Processing. 23

24. 4-2. Remove the points of inter-particle interference region and divergence region 24

25. Initially remove the unreliable points 25

26. 5. Scale the two curves. 26

27. 6. Fine tune the eEnd point to make sure that the endpoints are superimposed with the merged curve. 27

28. 7. Refine the point range for well-merge. 28

29. 5. Crysol Program Compare (fit) solution structure with PDB crystal structure. 29

30. 1. Enter your option :  30

31. 2. Select the PDB file (protein data bank) 31

32. 3. Maximum order of harmonics :  Order of Fibonacci grid :  Maximum s value :  Number of points :  Account for explicit hydrogens :  Fit the experimental curve :  32

33. 4. Enter data file (experimental dat file) 33

34. 5. Subtract constant :  Angular units in the input file :  Electron density of the solvent :  Plot the fit :  Another set of parameters :  Press “  ” to terminate the program 34

35. 35

36. 6. Plot the 1 st (experimental scattering vector) and 2 nd (theoretical intensity in solution) columns of fit file. fitted solution envelope of native cyt. c 36

37. 7. Open the log file to read the experimental and theoretical R g values. 37

38. 6. Transform data to P(r) with GNOM program and data fitting. 38

39. 1. Input data, first file : (select the file) 39

40. 2. Input Output file [ gnom.out ] : filename.out  40

41. 3. No of start points to skip [ 0 ] :  Input data, second file [ none ] :  No of end points to omit [ 0 ] :  Angular scale (1/2/3/4) [ 1 ] :  Plot input data (Y/N) [ Yes ] :  41

42. 4. File containing expert parameters [ none ] :  Kernel already calculated (Y/N) [ No ] :  Type of system (0/1/2/3/4/5/6) [ 0 ] :  Zero condition at r=r min (Y/N) [ Yes ] :  Zero condition at r=r max (Y/N) [ Yes ] :  42

43. 5. R max for evaluating p(r) : (input a proper value)  43

44. 6. Number of points in real space [ 101 ] :  Kernel-storage file name [ kern.bin ] :  Experimental setup (0/1/2) [ 0 ] :  Initial ALPHA [ 0.0 ] :  44

45. 7. Plot results (Y/N) [ Yes ] :  45

46. 8. GNOM fit shows probability distribution function P(r) 46

47. 7-1. Dammin Program An ensemble of dummy atom model simulation with P(r) output by GNOM. 47

48. 1. Select the Mode: ast, [S]low, [J]ag, [K]eep, [E]xpert :  48

49. 2. Input the Log file name : filename.log  49

50. 3. Input data, GNOM output file name : filename  50

51. File to be opened: m.out Project identificator : m 4. Enter project description :  Blue-colored text come from out file read by Dammin 51

52. Random sequence initialized from : 172235 ** Information read from the GNOM file ** Data set title: Merge of: cytc-5.txt cytc-10.txt Raw data file name: Merge01.dat Maximum diameter of the particle : 40.00 Solution at Alpha = 0.203E+01 Rg : 0.132E+02 I(0) : 0.388E-01 Radius of gyration read : 13.20 Number of GNOM data points : 428 5. Angular units in the input file: 4*pi*sin(theta)/lambda [1/angstrom] (1) 4*pi*sin(theta)/lambda [1/nm ] (2) :  Blue-colored text come from out file read by Dammin 52

53. Maximum s value [1/angstrom] : 0.3924 Number of Shannon channels. : 4.996 6. Portion of the curve to be fitted :  Blue-colored text come from out file read by Dammin 53

54. Number of knots in the curve to fit : 20 *** Warning: constant reduced to avoid oversubtraction A constant was subtracted : 1.788e-4 Maximum order of harmonics : 10 7. Initial DAM: type S for sphere [default], E for ellipsoid, C for cylinder, P for parallelepiped or start file name : (select one type)  Blue-colored text come from out file read by Dammin 54

55. 8. Symmetry: P1...19 or Pn2 (n=1,..,12) or P23 or P432 or PICO :  55

56. Sphere diameter [Angstrom]............................ : 35.00 Packing radius of dummy atoms.......................... : 1.300 Radius of the sphere generated......................... : 17.50 Number of dummy atoms.................................. : 1830 Number of equivalent positions......................... : 1 9. Expected particle shape: rolate, blate, or nknown :  Blue-colored text come from out file read by Dammin 56

57. ==== Simulated annealing procedure started ==== 57

58. 58

59. 59

60. 60

61. 61

62. 62

63. 63

64. 7-2. Gasbor Program An ensemble of dummy residues model simulation with P(r) output by GNOM. 64

65. 1. Computation mode (User or expert)…… :  65

66. 2. Input the Log file name :filename.log  66

67. 3. Select The Input Filename 67

68. File to be opened: m.out 4. Enter Project decsription:  68

69. 6. Angular units in the input file: 4*pi*sin(theta)/lambda [1/angstrom] (1) 4*pi*sin(theta)/lambda [1/nm ] (2) :  69

70. 7. Portion of the curve to be fitted…… :  70

71. 8. Initial DRM (CR for random)…… :  71

72. 9. Symmetry: P1…19 or Pn2 (n=1,..,12) or P23 or P432 or PIC0…. :  72

73. 10. number of residues in asymmetric part.. :  73

74. 11. Fibonacci grid order…… :  74

75. 12. Expected particle shape: rolate, blate, or nknown…… :  75

76. ==== Simulated annealing procedure started ==== 76

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