1

Catalog1 Check the radiation damage p. 2

2 Check aggregation by AutoRg program. Calculate theoretical I0, MW, and Vtot with protein SAXS I0 estimtaion.xls.

p. 7

3 Check inter-particle interaction with concentration dependence I0/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 ProgramAn ensemble of dummy atom model simulation with P(r) output by GNOM.

p. 47

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

p. 64

2

1. Check the radiation damage

0.01 0.1 11E-6

1E-5

1E-4

1E-3

0.01

0.1

1

Cyt c-10 mg15keV(10 sec / 10 frames)tm = 0.61124calibrated thickness: 3.254 mm

G86_0 G86_1 G86_2 G86_3 G86_4 G86_5 G86_6 G86_7 G86_8 G86_9 combined

I(q) (

cm-1)

q (Å-1)

3

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

4

2. Plot all curves in a graph

5

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

6

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

7

2. Check aggregation by AutoRg program.

Calculate theoretical I0, MW, and Vtot with protein SAXS I0 estimtaion.xls.

8

1. Start program: Primus -> AutoRg

9

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

10

3. In Plot tab, read the raw data.

11

4. In Guinier tab, check the aggregation and define the qmin before unreliable range.

12

5. In Info tab, read the sRg limits (Guinier region), I0, and Rg values.

13

1

2

3

6

4

5

One can calculate theoretical MW, I0, and Vtot with protein SAXS I0 estimtaion.xls. (spreadsheet edited by Dr. Jeng)

14

3. Check inter-particle interaction with concentration dependence I0/c

15

1. Import multiple ACSII of all different concentration curves.

16

2. Plot all different concentration curves.

17

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

18

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

19

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

20

1. Start program: Primus

21

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

22

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

23

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

24

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

25

• Initially remove the unreliable points

26

5. Scale the two curves.

27

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

28

7. Refine the point range for well-merge .

29

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

30

1. Enter your option <0>:

31

2. Select the PDB file (protein data bank)

32

3. Maximum order of harmonics <15>: Order of Fibonacci grid <17>: Maximum s value <0.5>: Number of points <51>: Account for explicit hydrogens <no>: Fit the experimental curve <yes>:

33

4. Enter data file (experimental dat file)

34

5.Subtract constant <no>: Angular units in the input file <1>: Electron density of the solvent <0.334>: Plot the fit <yes>: Another set of parameters <no>: Press “ ” to terminate the program

35

36

6. Plot the 1st (experimental scattering vector) and 2nd (theoretical intensity in solution) columns of fit file.

fitted solution envelope of native cyt. c

37

7. Open the log file to read the experimental and theoretical Rg values.

38

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

39

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

40

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

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 ] :

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=rmin (Y/N) [ Yes ] : Zero condition at r=rmax (Y/N) [ Yes ] :

43

5. Rmax for evaluating p(r) : (input a proper value)

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 ] :

45

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

46

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

47

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

48

1. Select the Mode: <[F]>ast, [S]low, [J]ag, [K]eep, [E]xpert < Fast >:

49

2. Input the Log file name < .log >: filename .log

50

3. Input data, GNOM output file name < .out >: filename

51

File to be opened: m.outProject identificator : m4. Enter project description :

Blue-colored text come from out file read by Dammin

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 : 40.00Solution at Alpha = 0.203E+01Rg : 0.132E+02 I(0) : 0.388E-01 Radius of gyration read : 13.20Number of GNOM data points : 4285. Angular units in the input file: 4*pi*sin(theta)/lambda [1/angstrom] (1) 4*pi*sin(theta)/lambda [1/nm ] (2) < 1 >:

Blue-colored text come from out file read by Dammin

53

Maximum s value [1/angstrom] : 0.3924Number of Shannon channels . : 4.9966. Portion of the curve to be fitted < 1.000 >:

Blue-colored text come from out file read by Dammin

54

Number of knots in the curve to fit : 20*** Warning: constant reduced to avoid oversubtractionA constant was subtracted : 1.788e-4Maximum order of harmonics : 107. Initial DAM: type S for sphere [default], E for ellipsoid, C for cylinder, P for parallelepiped or start file name <dammin.pdb >: (select one type)

Blue-colored text come from out file read by Dammin

55

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

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 ......................... : 19. Expected particle shape: <P>rolate, <O>blate, or <U>nknown < unknown >:

Blue-colored text come from out file read by Dammin

57

==== Simulated annealing procedure started ====

58

59

60

61

62

63

64

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

65

1. Computation mode (User or expert)……<User>:

66

2. Input the Log file name <.log>:filename .log

67

3. Select The Input Filename

68

File to be opened: m.out4. Enter Project decsription:

69

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

70

7. Portion of the curve to be fitted……<1.000>:

71

8. Initial DRM (CR for random)……< gasbor .pdb>:

72

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

73

10. number of residues in asymmetric part..< 62>:

74

11. Fibonacci grid order……<9>:

75

12. Expected particle shape: <P>rolate, <O>blate, or <U>nknown……<Unknown>:

76

==== Simulated annealing procedure started ====

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