SASREF 6 manual

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

© ATSAS Team, 2003-2009

Table of Contents

• Manual
  • Introduction
  • Running sasref
    • Interactive Configuration
    • Runtime Output
  • sasref Input Files
  • sasref Ouput Files
• Examples
  • Constructing a Complex

Manual

The following describes the method implemented in SASREF, details of the dialog prompt as well as the required input and the produced output files.

If you use results from SASREF in your own publication, please cite:

Petoukhov, M.V. & Svergun, D.I. (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J. 89, 1237-1250.

Introduction

SASREF performs quaternary structure modeling of a complex formed by subunits with known atomic structure against the SAXS data set. Further, it can simultaneously fit multiple SAXS data sets from the subcomplexes, if available, and account for the particle symmetry.

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 curves calculated from the appropriate subunits assemblies.

The theoretical scattering patterns I(s) are expressed in terms of spherical harmonics from the partial scattering amplitudes of the subunits A_lm(s) in their given positions and orientations. The subunit's amplitudes in arbitrary arrangement depend on its scattering amplitudes in the reference position and on three rotational and three translational parameters. The reference partial scattering amplitudes of the subunits have to be precomputed by the program CRYSOL (recommended values are lm=15, ns=51).

Eventual symmetry (must be the same for all subcomplexes) can be taken into account, whereby SASREF searches for the subunits arrangement inside the asymmetic part and the rest is generated according to symmetry rules.

Please refer to the paper cited above for further details about the implemented algorithm.

Running sasref

Interactive Configuration

SASREF can only be run in the dialog mode, no command line arguments are accepted. There are two modes, EXPERT and USER. In the former mode, the user have the options to adjust any program parameters. In the latter mode, fewer questions are asked as the default values are used for the most of program parameters, the user only needs to provide basic input. The default settings are the same in both modes.

SASREF 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:

sasref Input Files

SASREF uses the SAXS experimental data files (*.dat) in ascii format containing 3 columns: (1) experimental scattering vector, (2) experimental intensity and (3) experimental errors; binary files with partial scattering amplitudes computed by CRYSOL; and optional contacts conditions file 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 ordinal 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.

Important: here, residue/nucleotide number is the ordinal number of CA (or P) atom in the PDB file, i.e. in the following file, Pro32 will have residue number equal to 2.

ATOM 1  N   GLY A 31 -6.047 33.786  1.442
ATOM 2  CA  GLY A 31 -5.711 33.334  0.066
ATOM 3  C   GLY A 31 -4.332 32.718  0.000
ATOM 4  O   GLY A 31 -3.676 32.483  0.995
ATOM 5  N   PRO A 32 -3.874 32.485 -1.215
ATOM 6  CA  PRO A 32 -2.562 31.874 -1.416
ATOM 7  C   PRO A 32 -1.444 32.754 -0.866
ATOM 8  O   PRO A 32 -1.566 33.990 -0.808
ATOM 9  CB  PRO A 32 -2.464 31.760 -2.936
ATOM 10 CG  PRO A 32 -3.446 32.698 -3.473
ATOM 11 CD  PRO A 32 -4.564 32.799 -2.483
ATOM 12 N   LEU A 33 -0.348 32.111 -0.506
ATOM 13 CA  LEU A 33  0.834 32.815 -0.070
ATOM 14 C   LEU A 33  1.392 33.614 -1.230
ATOM 15 O   LEU A 33  1.470 33.154 -2.364
ATOM 16 CB  LEU A 33  1.900 31.869  0.390
ATOM 17 CG  LEU A 33  1.537 31.036  1.611
ATOM 18 CD1 LEU A 33  2.576 29.958  1.797
ATOM 19 CD2 LEU A 33  1.490 31.984  2.815

If for instance 3 domains form one polypeptide chain and nothing is missing between C- and N-termini of subsequent pdb files, the simplest view of the contacts conditions file would be:

     dist 4.0
     1 0 0 2 1 1
     dist 4.0
     2 0 0 3 1 1

sasref Output Files

After each simulated annealing step, SASREF 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

Constructing a Complex

A simulated complex constructed using crystallographic coordinates of two proximal monomers of glutamil-tRNA synthetase complexed with tRNA (PDB entry 1g59). The entire dimer has molecular weight of 156 kDa and contains 468 amino acids and 75 bases per monomer, the monomers are related by two-fold symmetry axis. The theoretical scattering curves of the dimeric tRNA and the entire complex computed by CRYSOL are stored in the files trnadim.dat and complex.dat, respectively.

trna.pdb and prot.pdb are the structures of monomeric tRNA and protein in arbitrary orientations, both centered at the origin. The files trna.alm and prot.alm contain the scattering amplitudes of the above monomers calculated using CRYSOL.

Additional information on the contacts between tRNA and protein (U513 with Pro303 and A573 with Gly121 ) is given in the file contacts.cnd

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 >: nucpro
 Project identificator .................................. : nucpro
 Enter project description .............. : dimeric protein-RNA complex
 Random sequence initialized from ....................... : 164152
 Input total number of subunits ......... <            1 >: 2
 Symmetry: P1...19 or Pn2 (n=1,..,12) ... <           P1 >: p2
 Input total number of scattering curves  <            1 >: 2
 Input first & last subunits in 1-st construct <            1,           2 >: 1,1
 Enter file name, 1-st experimental data  <         .dat >: trnadim
 Number of experimental points found .................... : 201
 Angular units in the input file :
 4*pi*sin(theta)/lambda [1/angstrom] (1)
 4*pi*sin(theta)/lambda [1/nm      ] (2)
 2*   sin(theta)/lambda [1/angstrom] (3)
 2*   sin(theta)/lambda [1/nm      ] (4)  <            1 >:
 Fitting range in fractions of Smax ..... <        1.000 >:
 Experimental radius of gyration ........................ : 29.40
 Number of points in the Guinier Plot ................... : 29
 Input first & last subunits in 2-nd construct <            1,           2 >:
 Enter file name, 2-nd experimental data  <         .dat >: complex
 Number of experimental points found .................... : 201
 Angular units in the input file :
 4*pi*sin(theta)/lambda [1/angstrom] (1)
 4*pi*sin(theta)/lambda [1/nm      ] (2)
 2*   sin(theta)/lambda [1/angstrom] (3)
 2*   sin(theta)/lambda [1/nm      ] (4)  <            1 >:
 Fitting range in fractions of Smax ..... <        1.000 >:
 Experimental radius of gyration ........................ : 42.51
 Number of points in the Guinier Plot ................... : 21
 Amplitudes, 1-st subunit ............... <         .alm >: trna
 Maximum order of harmonics ............................. : 15
 Number of points in partial amplitudes ................. : 51
  SASREF --W- Lm reduced to compute cross term
 Current subunit:   1597 atoms read, center at     0.00    0.00    0.00
 Initial rotation by alpha .............. <          0.0 >:
 Initial rotation by beta ............... <          0.0 >:
 Initial rotation by gamma .............. <          0.0 >:
 Initial shift along X .................. <     8.140e-6 >: 0
 Initial shift along Y .................. <     1.209e-4 >: 0
 Initial shift along Z .................. <    -3.757e-6 >: 0
 Fix the subunit at this position? [ Y / N ] <           No >:
 ALMGRZ --- :   91800 summation coefficients used
 Amplitudes, 2-nd subunit ............... <         .alm >: prot
  SASREF --W- Lm reduced to compute cross term
 Current subunit:   3813 atoms read, center at     0.00    0.00    0.00
 Initial rotation by alpha .............. <          0.0 >:
 Initial rotation by beta ............... <          0.0 >:
 Initial rotation by gamma .............. <          0.0 >:
 Initial shift along X .................. <    -1.668e-4 >: 0
 Initial shift along Y .................. <     2.098e-5 >: 0
 Initial shift along Z .................. <    -6.819e-6 >: 0
 Fix the subunit at this position? [ Y / N ] <           No >:
 Cross value ............................................ : 14.17
 Discontiguity value .................................... : 0.0
 File name, contacts conditions, CR for none <         .cnd >: contacts
 Condition #  1: Distance   5.000
    Between subunit #  1, Residues from  P     U A 513  to  P     U A 513
        and subunit #  2, Residues from  CA  PRO B 303  to  CA  PRO B 303
 Condition #  2: Distance   5.500
    Between subunit #  1, Residues from  P     A A 573  to  P     A A 573
        and subunit #  2, Residues from  CA  GLY B 121  to  CA  GLY B 121
 Contacts conditions penalty ............................ : 42.36
 Expected particle shape: Prolate, Oblate,
  or Unknown .......................... <      Unknown >:
 Shift penalty is normalized by ......................... : 30.48
 Shift penalty .......................................... : 0.0
 Shift penalty weight ................................... : 1.000
 Total penalty .......................................... : 565.3
 1-st curve:
 NEXP reduced to ........................................ : 200
 Theoretical  points from            1  to           51  used
 2-nd curve:
 NEXP reduced to ........................................ : 200
 Theoretical  points from            1  to           51  used
 The best chi values:11.1205510.89429
 Initial fVal ........................................... : 686.5
 Initial annealing temperature .......................... : 10.00
 Annealing schedule factor .............................. : 0.9000
 Max # of iterations at each T .......................... : 10000
 Max # of successes at each T ........................... : 1000
 Min # of successes to continue ......................... : 100
 Max # of annealing steps ............................... : 100
  ====  Simulated annealing procedure started  ====
 j:   1 T: 0.100E+02 Suc:  1000 Eva:     2884 CPU:  0.747E+02 F: 7.7639 Pen:  0.7008
 The best chi values: 2.32386 2.95396
 j:   2 T: 0.900E+01 Suc:  1000 Eva:     6153 CPU:  0.159E+03 F: 7.7639 Pen:  0.7008
 The best chi values: 2.32386 2.95396
 ...