SASREF CV/MX manual

Written by M.V. Petoukhov
Post all your questions about SASREF to the ATSAS Forum.

© ATSAS Team, 2006-2013

Table of Contents

• Manual
  • Introduction
  • Running SASREF CV / MX
    • Interactive Configuration
    • Runtime Output
  • SASREF CV / MX Input Files
  • SASREF CV / MX Ouput Files
• Examples
  • Building a Complex against X-ray and Contrast Variation SANS Data Sets
  • Quaternary Structure Analysis of a Weak Tetramer
  • Modelling of a Transient Heterodimer
  • Complex with Excess of a Component in Solution
  • Presence of a Sub-Complex in the Mixture

Manual

The following describes the special versions of the rigid body modelling program SASREF, SASREFCV (formerly known as SASREF7) for fitting of SANS contrast variation series (also combinable with SAXS) and SASREFMX for the structural analysis of transient complexes and weak oligomers from polydisperse data. In the latter case, the rigid body modelling is coupled with mixture analysis, whereby the volume fractions of the dissociation products are estimated. The two approaches have very much in common, so this manual provides details of the dialog prompt as well as the required configuration / input files as well as the produced output for both programs.

If you use results from SASREFCV in your own publication, please cite:
Petoukhov, M.V. & Svergun, D.I. (2006) Joint use of small-angle X-ray and neutron scattering to study biological macromolecules in solution. Eur. Biophys. J. 35(7), 567-576 (© EBSA 2006 Eur. Biophys. J. 35(7), 567-576)

If you use results from SASREFMX in your own publication, please cite:
Petoukhov, M.V., Franke, D., Shkumatov, A.V., Tria, G., Kikhney, A.G., Gajda, M., Gorba, C., Mertens, H.D.T., Konarev, P.V. and Svergun, D.I. (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Cryst. 45, 342-350 © International Union of Crystallography DOI

Introduction

SASREF CV / MX perform quaternary structure modeling of a complex particle formed by subunits with known atomic structure against the SAS data set in case of contrast variation series and a polydisperse system, respectively. Multiple data sets can be fitted simultaneously, e.g. different D2O content and/or perdeuteration (in SASREFCV) or profiles recorded at different conditions (concentration, temperature, pH, ionic strength) yielding different affinity of the complex particle (in SASREFMX). Both algorithms are capable to account for the symmetry (which can be subunit- and data-specific).

A simulated annealing protocol is employed to construct an interconnected ensemble of subunits without steric clashes, while minimizing the discrepancy between the experimental scattering data and the predicted curves from the appropriate subunits assemblies. In case of SASREFMX, the experimental data is fitted by the linear combination of the profiles calculated from the intact particle and from the dissociation products.

For futher details of the rigid body modelling approach please refer to SASREF manual and to the papers cited above.

Running SASREF CV / MX

Interactive Configuration

SASREF CV / MX can only be run in the dialog mode, no command line arguments are accepted. Similarly to MONSA, significant amount of the user input is provided using configuration files. There are two modes, EXPERT and USER. In the former mode, the user have the options to adjust more parameters. In the latter mode, fewer questions are asked as the default values are used for the most of the program parameters. The default settings are the same in both modes.

SASREF CV / MX interactive prompt:

Runtime Output

On runtime, two lines of output will be generated for each temperature step:

j:   4 T: 0.729E+01 Suc:  1000 Eva:    12497 CPU:  0.208E+03 F:99.4301 Pen: 13.803
The best chi values:11.64871 5.96331

The fields can be interpreted as follows, top-left to bottom-right:

SASREFMX additionally outputs the volume fraction of the intact construct for each of the fitted curves:

 Associated volume fractions: 0.42157 0.64983 

SASREF CV / MX Input Files

Three compulsory configuration files are to be created containing the information about:

• (a) - the scattering data,
• (b) - the subunits (rigid bodies) and
• (c) - the contribution of each subunit to each scattering curve.

Data control file (data.con in the following examples) has the following format:

 - The first line contains one integer K (total number of scattering curves
   for SASREFCV and total number of scattering curves+1 for SASREFMX)
 - K lines, each containing 8 parameters related to the scattering data set:

Last line in case of SASREFMX describes the dissociation products and contains 
all dummy values except for the symmetry.

Subunits control file (subs.con in the examples) describes the rigid bodies. Its format is the following:

 - The first line contains one integer M (total number of subunits)
 - M lines, each containing 4 parameters related to the subunit:

Cross-correlation file (table.con in the examples) contains a table which sets the relationship between the subunits and the scattering profiles. The number of its columns equals to the total number of the subunits (M) and the number of its rows equals to the total number of the scattering curves (K). The value in the i-th column and j-th row gives the contribution of the i-th subunit in the j-th scattering data set. For SANS curve this value ([0.0-1.0 or -1.0]) is the subunit perdeuteration (D₂O content in solution where the protein is expressed), whereby -1.0 means that the given subunit is not present in the corresponding construct. For X-ray scattering curve, 0.0 is to be used, if the subunit is present.

In case of SASREFMX, the last row describes the dissociation products which are mixed to all the curves. Here, an integer number, 0 or -1 are allowed, whereby -1 means that the subunit is not among the dissociation products, 0 means that the subunit is a part of a sub-complex which is a dissociation product of a larger assembly (there could be not more than just one such sub-complex) and an integer means the molar ratio (stoichiometry) of the subunit in the original (fully dissociated) sample.

Distance restraints may be imposed on the model using contacts conditions file (optional) in the following format:

      dist 7.0
      1 0 0 2 1 1
      dist 5.0
      2 0 0 3 1 1
      dist 7.0
      1 342 342 2 25 25
      1 350 350 2 17 17
      dist 6.0
      1 290 297 2  64 79
      dist 7.0
      1 1 0 3 1 0

"dist 7.0" means that the minimum distance between CA atoms of the residues (or P atoms in the nucleotides) specified in the following lines should not exceed 7 Å. The first and the fourth numbers in the line not containing keyword "dist" mean the ordial numbers of the 1st and the 2nd subunits having the contact by any residue/nucleotide of the 1st subunit in the range from second number to third number with any residue of the 2nd subunit in the range from fifth number to sixth number. 0 means the last residue/nucleotide of the subunit.

If two (or more) alternatives are given after the line with the keyword "dist", the program compares the better (smaller) distance among them with the specified one.

Please refer to SASREF manual for more details. Important: there is a small difference here in the numbering of the subunits due to the possibility of distinct symmetries applied to individual subunits in SASREF CV / MX. First, all subunits in the asymmetric part appear (as in conventional SASREF version) followed by all symmetry mates of the first subunit, then of the second and so on until the last one.

SASREF CV / MX Output Files

After each simulated annealing step, SASREF CV /MX creates a set of output files, each filename starts with a customizable prefix that gets an extension appended. If a prefix has been used before, existing files will be overwritten without further note.

Examples

Building a Complex against X-ray and Contrast Variation SANS Data Sets

A simulated exampe of T7 DNA Polymerase Ternary Complex with DCTP (PDB entry 1t8e). PDB files containing the atomic coordinates of the three subunits are:

phsave1.pdb   -   Polymerase
phsave2.pdb   -   DCTP
phsave3.pdb   -   DNA

Simulated SAS data contain 17 curves in total: 2 X-ray profiles (from the entire complex and from the binary construct without DNA) + 15 neutron scattering curves from the complex [(series of D₂O content: 0, 40, 55, 70 and 100% D₂O)* (3 perduterations of DCTP: 0, 50 and 100%)]:

x-prot.dat       X-ray protein complex
x-compl.dat      X-ray, ternary complex
complh_0.dat     ternary complex with protonated DCTP in 0%D2O
complh_40.dat                                         in 40%D2O
complh_55.dat                                         in 55%D2O
complh_70.dat                                         in 70%D2O
complh_100.dat                                        in 100%D2O
compl50d_0.dat                    50% deuterated DCTP in 0%D2O
compl50d_40.dat                                       in 40%D2O
compl50d_55.dat                                       in 55%D2O
compl50d_70.dat                                       in 70%D2O
compl50d_100.dat                                      in 100%D2O
compl100d_0.dat                 fully deuterated DCTP in 0%D2O
compl100d_40.dat                                      in 40%D2O
compl100d_55.dat                                      in 55%D2O
compl100d_70.dat                                      in 70%D2O
compl100d_100.dat                                     in 100%D2O

Content of the curves.con file:

17
x-prot.dat       -1.00 P1 1 1.0 0 1.0 y
x-compl.dat      -1.00 P1 1 1.0 0 1.0 y
complh_0.dat      0.00 P1 1 1.0 0 1.0 y
complh_40.dat     0.40 P1 1 1.0 0 1.0 y
complh_55.dat     0.55 P1 1 1.0 0 1.0 y
complh_70.dat     0.70 P1 1 1.0 0 1.0 y
complh_100.dat    1.00 P1 1 1.0 0 1.0 y
compl50d_0.dat    0.00 P1 1 1.0 0 1.0 y
compl50d_40.dat   0.40 P1 1 1.0 0 1.0 y
compl50d_55.dat   0.55 P1 1 1.0 0 1.0 y
compl50d_70.dat   0.70 P1 1 1.0 0 1.0 y
compl50d_100.dat  1.00 P1 1 1.0 0 1.0 y
compl100d_0.dat   0.00 P1 1 1.0 0 1.0 y
compl100d_40.dat  0.40 P1 1 1.0 0 1.0 y
compl100d_55.dat  0.55 P1 1 1.0 0 1.0 y
compl100d_70.dat  0.70 P1 1 1.0 0 1.0 y
compl100d_100.dat 1.00 P1 1 1.0 0 1.0 y

Content of the subunits.con file:

3
phsave1.pdb Y F P1
phsave2.pdb Y N P1
phsave3.pdb Y N P1

Content of the table.con file:

0.0  0.0 -1.0 
0.0  0.0  0.0 
0.0  0.0  0.0 
0.0  0.0  0.0 
0.0  0.0  0.0 
0.0  0.0  0.0 
0.0  0.0  0.0 
0.0  0.5  0.0 
0.0  0.5  0.0 
0.0  0.5  0.0 
0.0  0.5  0.0 
0.0  0.5  0.0 
0.0  1.0  0.0 
0.0  1.0  0.0 
0.0  1.0  0.0 
0.0  1.0  0.0 
0.0  1.0  0.0 

A listing of questions/answers for a sample run in the USER mode is as follows:

 Computation mode (User or Expert) ...... <         User >:
 Log file name .......................... <         .log >: test1
 Enter project description .............. : T7 DNA POLYMERASE WITH DCTP, 17 curves
 Symmetry: Pn(2) (n=2-6) ................ <           P1 >:
 File name with curves info ............. <         .con >: curves
 File name with smearing parameters ..... <         .res >:
 File name with subunits info ........... <         .con >: subunits
 File name with cross-dependencies ...... <         .con >: table
 File name, contacts conditions, CR for none <         .cnd >:
 Expected particle shape: <P>rolate, <O>blate,
  or <U>nknown .......................... <      Unknown >:
 ...

Quaternary Structure Analysis of a Weak Tetramer

Let weak_tetramer.dat be a SAXS profile from a polydisperse sample containing a tetramer with P222 symmetry and its monomer (i.e. oligomeric equilibrium). The atomic structure of the latter is contained in monomer.pdb. Content of the curves.con file is then:

2
weak_tetramer.dat -1.00 P222 1 1.0 0 1.0 y
dummy.dat         -1.00 P1   1 1.0 0 1.0 y

Content of the subunits.con file:

1
monomer.pdb Y N P222

Content of the table.con file:

0.0   
0.0   

Modelling of a Transient Heterodimer

Equimolar mixture of proteins A and B yields an equilibrium between the AB complex and its components in unbound state. Concentration series with distinct volume fracions of the intact complex can be fitted simultaneously. Content of the configuration files in this case is as follows.

curves.con:

4
transient_c1.dat -1.00 P1 1 1.0 0 1.0 y
transient_c2.dat -1.00 P1 1 1.0 0 1.0 y
transient_c3.dat -1.00 P1 1 1.0 0 1.0 y
dummy.dat        -1.00 P1 1 1.0 0 1.0 y

subunits.con:

2
a.pdb Y N P1
b.pdb Y N P1

table.con:

0.0 0.0  
0.0 0.0  
0.0 0.0  
1.0 1.0  

Complex with Excess of a Component in Solution

If an excess of subunit C is needed for formation of a stable complex ABC, resulting sample will be polydisperse. Content of the configuration files in this case is as follows.

curves.con:

2
mixture.dat -1.00 P1 1 1.0 0 1.0 y
dummy.dat   -1.00 P1 1 1.0 0 1.0 y

subunits.con:

3
a.pdb Y N P1
b.pdb Y N P1
c.pdb Y N P1

table.con:

0.0 0.0 0.0 
-1.0 -1.0 0.0 

Presence of a Sub-Complex in the Mixture

By lack of subunit C (e.g. in a stoichiometric study), some amount of sub-complex AB migh be present in the mixture with the ternary ABC. Content of the configuration files is then as follows.

curves.con:

2
mixture.dat -1.00 P1 1 1.0 0 1.0 y
dummy.dat   -1.00 P1 1 1.0 0 1.0 y

subunits.con:

3
a.pdb Y N P1
b.pdb Y N P1
c.pdb Y N P1

table.con:

0.0 0.0 0.0 
0.0 0.0 -1.0