1 Acknowledge EMBL Hamburg BioSAXS team memebers effort in ATSAS software package. The data analysis software, ATSAS software package, are downloaded from.

1

BL23A BioSAXS check list

Acknowledge EMBL Hamburg BioSAXS team memebers’ effort in ATSAS software package. The data analysis software, ATSAS software package, are downloaded from http://www.embl-hamburg.de/biosaxs/atsas-online/download.phpIf one use some programs in the work, please cite the corresponding papers.

0.110-4

10-3

10-2

10-1

I(q)

(cm

-1)

q (Å-1)

fitted solution envelope of native cyt. c

0.05 0.10 0.15 0.200.000

0.003

0.006

0.009

Cytc-10mg/ml Cytc-5mg/ml Cytc-2mg/ml

I(q

)/c

(c

m-1m

g-1m

l)

q (Å-1)

Bio-SAXS Check list

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

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

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

1. Check the radiation damage

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

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

7. Dammin / Gasbor ProgramAn ensemble of dummy atom/residues model simulation with P(r) output by GNOM.

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

) (c

m-1)

q (Å-1)

3

1. Check the radiation damage

Instruction: (1) Plot each frame and combined data.(2) Check the low-q region in the all profiles.

• If all profiles are overlapped well at low-q region, no radiation damage occurs.• If some frames of them appear up-turn at low-q region, radiation damage may occur.The radiation damage might result from long data requisition time or aggregation sample. Resolving ❶ 1. combine the frames without up-turn and out a low-q reliable curve. 2. combine all frames and output high-q reliable curve. 3. fine-tune and merge the two curves and output into one profile.Resolving ❷ 1. Run HPLC

1

2

well-overlapped

0.01 0.1 11E-6

1E-5

1E-4

1E-3

0.01

0.1

1

G86_0 G86_1 G86_2 G86_3 G86_4 G86_5 G86_6 G86_7 G86_8 G86_9 combined

I(q

) (c

m-1)

q (Å-1)

0.02 0.04 0.06 0.080.02

0.04

0.06

0.08

0.1

G86_0 G86_1 G86_2 G86_3 G86_4 G86_5 G86_6 G86_7 G86_8 G86_9 combined

I(q

) (c

m-1)

q (Å-1)

4

Instruction: (1) Start program: Primus -> AutoRg; input file type: txt, dat.(2) In Plot tab, see the raw data.(3) In Guinier tab, check the aggregation and define the qmin before unreliable range.(4) In Info tab, read the sRg limits (Guinier region), I0, and Rg values.

1

3 4

2. Check aggregation by AutoRg program.

2

Define qmin before unreliable range.

5

Instruction: (1) Fill in protein amino acids numbers in AQ column.(2) Fill in protein concentration in B18 cell.(3) Fill in the aggregation number in B(13) cell.(4) Check the molecular weight calculated in B(17).(5) Check the calculated I(0) at B(19).(6) Input measured I(0) in B(27) to check the aggregation no. of oligomers.

Cytochrome c (PDB ID: 1HRC), MW~12368e.g.

1

2

3

6

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

4

5

6

0.05 0.10 0.15 0.200.000

0.003

0.006

0.009

Cytc-10mg/ml Cytc-5mg/ml Cytc-2mg/ml

I(q

)/c

(c

m-1m

g-1m

l)

q (Å-1)

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

Instruction: (1) Plot all different concentration curves.(2) Confirm if concentration dependence is distinguishable at low-q region.

e.g. There is concentration dependence behavior in below figure. The high conc. profile displays lower I0/c value due to the interference from inter-particle interaction.

inter-particle interaction

Attention!Need to measure low concentration sample as low as possible.Make sure that more than two I0/c curves are overlapped well.

7

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

Instruction: (1) Start program: Primus(2) Click “Tools” to select the low and high concentration data for merging.(3) Click “plot” to plot the two curves.

2

3*nBeg valueFirst 45 points in #1 and #2 are cut because of beamstop.

**

8

4

(4) According to the q-cut range (see ① and ②) , input the parameters into nBeg (begin point #) and nEnd (end point #) of Data Processing.

(5) Click “Plot Scale ” and make sure that the curves are superimposable.

0.05 0.10 0.15 0.20 0.25

0.000

0.003

0.006

0.009

q(Å-1)

cytc-5mg/ml cytc-10mg/ml

I(q)/c

(cm

-1m

g-1m

l)

① remove concentration dependence ② remove divergence region

The high-q endpoints of low conc. profile do not meet the other curve.

q2-cut range(low conc. profile)

q1-cut range(high conc. Profile)

The scale multiplier is based on the intensity of the first file (#1)

Input q-cut region based on ① and ②

5

9

Attention!• Need to measure low concentration sample as low as possible. • Need to check concentration dependence before model simulation.• Make sure that all different concentration curves are superimposable after q-cut.

Each Merge-click can output one merged file.Filename of 1st profile-merging: Merge00.datFilename of 2nd profile-merging: Merge01.dat

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

6

Instruction: (1) Enter your option <0>: (2) Select the PDB file (protein data bank)(3) Maximum order of harmonics <15>: (4) Order of Fibonacci grid <17>: (5) Maximum s value <0.5>: (6) Number of points <51>: (7) Account for explicit hydrogens <no>: (8) Fit the experimental curve <yes>: (9) Enter data file (experimental dat file)(10) Subtract constant <no>: (11) Angular units in the input file <1>: (12) Electron density of the solvent <0.334>: (13) Plot the fit <yes>: (14) Another set of parameters <no>: (15) Press “ ” to terminate the program

2

9

10

Cytochrome c (1HRC), MW~12368

e.g.

5. Compare (fit) solution structure with PDB crystal structure by Crysol Program (in ATSAS package).

Experimental SAXS data

0.01 0.11E-4

1E-3

0.01

0.1

Cyt c

Crysol fit, 2=2.576

I(q

) (c

m-1)

q (Å-1)

(16) Plot the 1st (experimental scattering vector) and 2nd (theoretical intensity in solution) columns of fit file.(17) Open the log file to read the experimental and theoretical Rg values.

Refer to http://www.embl-hamburg.de/biosaxs/manual_crysol.html#un

fitted solution envelope of native cyt. c

12

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

Instruction:(1) Printer type [ postscr ] : (2) Input data, first file : (select the file)(3) Output file [ gnom.out ] : filename.out (4) No of start points to skip [ 0 ] :(5) Input data, second file [ none ] : (6) No of end points to omit [ 0 ] : (7) Angular scale (1/2/3/4) [ 1 ] : (8) Plot input data (Y/N) [ Yes ] : (9) File containing expert parameters [ none ] : (10) Kernel already calculated (Y/N) [ No ] : (11) Type of system (0/1/2/3/4/5/6) [ 0 ] : (12) Zero condition at r=rmin (Y/N) [ Yes ] : (13) Zero condition at r=rmax (Y/N) [ Yes ] : (14) Rmax for evaluating p(r) : (input a proper value) (15) Number of points in real space [ 101 ] : (16) Kernel-storage file name [ kern.bin ] : (17) Experimental setup (0/1/2) [ 0 ] : (18) Initial ALPHA [ 0.0 ] : (19) Plot results (Y/N) [ Yes ] : (20) Press CR to continue

probability distribution function P(r)

In this case, Rmax (diameter of the object) = 40 Å

13

7. Dammin Program Dummy atom model simulation with P(r) output by GNOM program.

(1) Mode: <[F]>ast, [S]low, [J]ag, [K]eep, [E]xpert < Fast >: (2) Log file name < .log >: filename .log(PLEASE SELECT THE INPUT FILE NAME)(3) Input data, GNOM output file name < .out >: filename File to be opened: m.out Project identificator : m(4) Enter project description : 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 >: Maximum s value [1/angstrom] : 0.3924 Number of Shannon channels . : 4.996 Portion of the curve to be fitted < 1.000 >: 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(6) Initial DAM: type S for sphere [default], E for ellipsoid, C for cylinder, P for parallelepiped or start file name <.pdb >: (select one type) (7) Symmetry: P1...19 or Pn2 (n=1,..,12) or P23 or P432 or PICO < P1 >: (8) Expected particle shape: <P>rolate, <O>blate, or <U>nknown < unknown >: Start running simulation

Blue-colored text come from out file read by Dammin

Dammin Output Files:1. log file: contain the same information as the screen output.2. -0.pdb file: contain the beads of the solvent inside the search volume3. -1.pdb: represent the modeled particle. 4. fir file: Fit of the simulated scattering curve versus the experimental data.

Columns in the output file are: 's', 'Iexp', 'Errexp'and 'Isim'.

χ2=1.179

14

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

(1) Computation mode (User or expert)……<User>: (2) Log file name <.log>:filename .log***Please Select The Input Filename***File to be opened: m.out(3) Enter Project decsription: (4) Angular units in the input file: 4*pi*sin(theta)/lambda [1/angstrom] (1) 4*pi*sin(theta)/lambda [1/nm ] (2) < 1 >: (5) Portion of the curve to be fitted……<1.000>: (6) Initial DRM (CR for random)……< .pdb>: (7) Symmetry: P1…19 or Pn2 (n=1,..,12) or P23 or P432 or PIC0….< P1>: (8) number of residues in asymmetric part..< 62>: (9) Fibonacci grid order……<9>: (10) Expected particle shape: <P>rolate, <O>blate, or <U>nknown……<Unknown>:

Cytochrome c (1HRC)

GASBOR Output Files:1. log file: contain the same information as the screen output.2. fir file: fit to the raw experimental data3. pdb file: resulting model in PDB-like format that can be viewed

with MASSHA (in ATSAS package) or VMD (molecular visualization program)

Displayed by VMD

View along x

View along y

View along z

χ2=2.286

Start running simulation

15

FoXS: Fast SAXS Profile Computation with Debye Formula

On-line fit solution structure (experimental profile) with crystal structure (PDB or upload file).

Appendix.

Instruction: (1) Open the FoXS website

http://modbase.compbio.ucsf.edu/foxs/index.html(2) Input the PDB ID or upload the PDB file.(3) Upload the experimental profile.(4) Click “Submit Form”.