Detail check procedures (ppt)

Report
Catalog
1
Check the radiation damage
p. 2
2
Check aggregation by AutoRg program.
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.
p. 29
6
Transform data to P(r) with GNOM program and
data fitting.
p. 38
7-1
Dammin Program
p. 47
7-2
Gasbor Program
p. 64
Calculate theoretical I0, MW, and Vtot with protein SAXS I0 estimtaion.xls.
Compare (fit) solution structure with PDB crystal structure.
An ensemble of dummy atom model simulation with P(r) output by GNOM.
An ensemble of dummy residues model simulation with P(r) output by GNOM.
1
1. Check the radiation damage
G86_0
G86_1
G86_2
G86_3
G86_4
G86_5
G86_6
G86_7
G86_8
G86_9
combined
1
0.1
-1
I(q) (cm )
0.01
1E-3
1E-4
1E-5
1E-6
0.01
Cyt c-10 mg
15keV
(10 sec / 10 frames)
tm = 0.61124
calibrated thickness: 3.254 mm
0.1
1
-1
q (Å )
2
1. Import multiple ACSII of output files of each frame and combined data
3
2. Plot all curves in a graph
4
3. Check the low-q region in the all profiles
5
•
If all profiles are overlapped well at low-q region, no radiation damage occurs.
6
2. Check aggregation by AutoRg program.
Calculate theoretical I0, MW, and Vtot with protein SAXS I0 estimtaion.xls.
7
1. Start program: Primus -> AutoRg
8
2. Open the input file (file type: txt, dat)
9
3. In Plot tab, read the raw data.
10
4. In Guinier tab, check the aggregation and define the qmin before unreliable
range.
11
5. In Info tab, read the sRg limits (Guinier region), I0, and Rg values.
12
One can calculate theoretical MW, I0, and Vtot with protein SAXS I0 estimtaion.xls.
(spreadsheet edited by Dr. Jeng)
3
2
4
1
5
6
13
3. Check inter-particle interaction
with concentration dependence I0/c
14
1. Import multiple ACSII of all different concentration curves.
15
2. Plot all different concentration curves.
16
3. Set each y and yEr column as I(q)/conc.
17
4. Confirm if concentration dependence is distinguishable at low-q region.
18
4. Profile merging
merge the low and high concentration curves.
19
1. Start program: Primus
20
2. Click “Tools” to select the low and high concentration data for merging.
21
3. Click “plot” to plot the two curves.
22
4. Input the parameters into nBeg (begin point #) and nEnd (end point #)
of Data Processing.
23
4-2. Remove the points of inter-particle interference region and divergence region
24
•
Initially remove the unreliable points
25
5. Scale the two curves.
26
6. Fine tune the eEnd point to make sure that the endpoints are superimposed with
the merged curve.
27
7. Refine the point range for well-merge .
28
5. Crysol Program
Compare (fit) solution structure with PDB crystal structure.
29
1. Enter your option <0>: 
30
2. Select the PDB file (protein data bank)
31
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>: 
32
4. Enter data file (experimental dat file)
33
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
34
35
6. Plot the 1st (experimental scattering vector) and 2nd (theoretical intensity in
solution) columns of fit file.
fitted solution envelope of native cyt. c
36
7. Open the log file to read the
experimental and theoretical Rg values.
37
6. Transform data to P(r) with
GNOM program and data fitting.
38
1. Input data, first file : (select the file)
39
2. Input Output file [ gnom.out ] : filename.out 
40
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
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 ] : 
42
5. Rmax for evaluating p(r) : (input a proper value) 
43
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
7. Plot results (Y/N) [ Yes ] : 
45
8. GNOM fit shows probability distribution function P(r)
46
7-1. Dammin Program
An ensemble of dummy atom model simulation with P(r) output by GNOM.
47
1. Select the Mode: <[F]>ast, [S]low, [J]ag, [K]eep, [E]xpert < Fast >: 
48
2. Input the Log file name <
.log >: filename .log
49
3. Input data, GNOM output file name < .out >: filename 
50
File to be opened: m.out
Project identificator : m
4. Enter project description : 
Blue-colored text
come from out file
read by Dammin
51
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) < 1 >: 
Blue-colored text
come from out file
read by Dammin
52
Maximum s value [1/angstrom] : 0.3924
Number of Shannon channels . : 4.996
6. Portion of the curve to be fitted < 1.000 >: 
Blue-colored text
come from out file
read by Dammin
53
Blue-colored text
Number of knots in the curve to fit : 20
come from out file
*** Warning: constant reduced to avoid oversubtraction
read by Dammin
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 <dammin.pdb >: (select one type) 
54
8. Symmetry: P1...19 or Pn2 (n=1,..,12) or P23 or P432 or PICO < P1 >: 
55
Sphere diameter [Angstrom] ............................ : 35.00
Blue-colored text
come from out file
Packing radius of dummy atoms .......................... : 1.300
read by Dammin
Radius of the sphere generated ......................... : 17.50
Number of dummy atoms .................................. : 1830
Number of equivalent positions ......................... : 1
9. Expected particle shape: <P>rolate, <O>blate, or <U>nknown < unknown >: 
56
==== Simulated annealing procedure started ====
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60
61
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7-2. Gasbor Program
An ensemble of dummy residues model simulation with P(r) output by GNOM.
64
1. Computation mode (User or expert)……<User>: 
65
2. Input the Log file name <.log>:filename .log
66
3. Select The Input Filename
67
File to be opened: m.out
4. Enter Project decsription: 
68
6. Angular units in the input file:
4*pi*sin(theta)/lambda [1/angstrom] (1)
4*pi*sin(theta)/lambda [1/nm
] (2) <
1 >: 
69
7. Portion of the curve to be fitted……<1.000>: 
70
8. Initial DRM (CR for random)……< gasbor .pdb>: 
71
9. Symmetry: P1…19 or Pn2 (n=1,..,12) or P23 or P432 or PIC0….< P1>: 
72
10. number of residues in asymmetric part..< 62>: 
73
11. Fibonacci grid order……<9>: 
74
12. Expected particle shape: <P>rolate, <O>blate, or <U>nknown……<Unknown>: 
75
==== Simulated annealing procedure started ====
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