Catalog

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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

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

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

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2. Check aggregation by AutoRg program.

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

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1. Start program: Primus -> AutoRg

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

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3. In Plot tab, read the raw data.

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

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

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

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

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1. Import multiple ACSII of all different concentration curves.

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2. Plot all different concentration curves.

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

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

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

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1. Start program: Primus

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

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

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

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

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• Initially remove the unreliable points

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5. Scale the two curves.

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

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7. Refine the point range for well-merge .

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

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1. Enter your option <0>:

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2. Select the PDB file (protein data bank)

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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>:

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4. Enter data file (experimental dat file)

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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

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

fitted solution envelope of native cyt. c

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

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

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

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

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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 ] :

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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 ] :

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

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

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

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

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

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

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

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

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

Blue-colored text come from out file read by Dammin

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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

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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

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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

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

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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

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

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

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

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

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3. Select The Input Filename

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

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

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

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

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

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

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11. Fibonacci grid order……<9>:

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

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

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