Macromolecular refinement with REFMAC5 and SKETCHER of the CCP4 suite by sdfwerte


									Macromolecular refinement
         of the
      CCP4 suite

  Roberto A. Steiner – University of York

General aspects of refinement and overview of
 •   TLS
 •   Dictionary

 •   TLS refinement in REFMAC5

General aspects of refinement
  overview of REFMAC5
        A common problem in physical sciences


•   Set experimental values of quantity q (qE,E)
•   Model M(aI,bI,cI)  qI


•   Best model, i.e. M(aB,bB,cB) which is most consistent with
    the data
•   The accuracy of (aB,bB,cB)
                    Model fitting

                       Generation of additional data

Experiment   Mathematical model                        Inference

Model fitting in crystallography

  experimental (I,I )  (F, F)

 model (heavy atoms, protein, ..)


         Best model
      Key aspects in model fitting

• Parameterization of the model
• Type of residual
• Type of minimization
• Prior information
                    Bayesian approach

The best model is the one which has highest probability
 given a set of observations and a certain prior

                    BAYES' THEOREM


  Probability Theory: The Logic of Science by E.T.Jaynes
                Application of Bayes theorem

Screening for disease D.

On average 1 person in 5000 dies because of D. P(D)=0.0002
Let P be the event of a positive test for D.
P(P;D)=0.9, i.e. 90% of the times the screening identifies the disease.
P(P;notD)=0.005 (5 in 1000 persons) false positives.

What is the probability of having the desease if the test says it is positive?

Less than 3.5% of persons diagnosed to have the disease do actually have it.
          Maximum likelihood residual

  P(M;O) = P(M)P(O;M)/P(O) = P(M)L(M;O)

  max P(M;O)  min [-logP(M) -logL(M;O)]

Murshudov et al., Acta Cryst. (1997) D53, 240-255
Maximum likelihood refinement programs

             Essential features of REFMAC5

REFMAC5 is a ML FFT program for the refinement
 of macromolecular structures

•   Multiple tasks (phased and non-phased restrained,
    unrestrained, rigid-body refinement, idealization)
•   Fast convergence (approximate 2nd-order diagonal
•   Extensive built-in dictionary (LIBCHECK)
•   Graphical control (CCP4i)
•   Flexible parameterization (iso-,aniso-,mixed-ADPs, TLS, bulk
•   Easy to use (coordinate and reflection files, straightforward
    inclusion of alternate conformations)
                  Selected topic 1: TLS

ADPs are an important component of a macromolecule
 • Proper parameterization
 • Biological significance

Displacements are likely anisotropic, but rarely we have the
 luxury of refinining individual aniso-U. Instead iso-B are used.

TLS parameterization allows an intermediate description.
                Decomposition of ADPs

           U = Ucryst+UTLS+Uint+Uatom

Ucryst : overall anisotropy of the crystal
UTLS       : TLS motions of pseudo-rigidy bodies
Uint       : collective torsional librations or
  internal normal modes
Ucryst : individual atomic motions
Rigid-body motion
      General displacement of a rigid-body
      point can be described as a rotation
      along an axis passing through a fixed
      point together with a translation of that
      fixed point.
                   u = t + Dr

      for small librations
                  u  t + r

      D = rotation matrix
      = vector along the rotation axis of
      magnitude equal to the angle of rotation
                         TLS parameters

Dyad product:
           uuT = ttT + tTrT -rtT -rTrT

ADPs are the time and space average

           UTLS = uuTT + STrT -rS -rLrT
T = ttT 6 parameters, TRANSLATION
L = T 6 parameters, LIBRATION
S = tT 8 parameters, SCREW-ROTATION
                            Use of TLS

       UTLS = uuTT + STrT -rS -rLrT
•   analysis: given inidividual aniso-ADPs fit TLS parameters
Harata et al., (2002) Proteins, 48, 53-62
Harata et al., (1999) J. Mol. Biol., 30, 232-43

•   refinement: TLS as refinement parameters
Howlin et al., (1989) Acta Cryst., A45, 851-61
Winn et al., (2001) Acta Cryst., D57, 122-33
         Choice of TLS groups and resolution

Choice: chains, domains, secondary structure elements,..more
 complex MD,...

Resolution: you have only 20 more parameters per TLS group.
    Thioredoxin reductase 3 Å (Sandalova et al., (2001) PNAS, 98,
    6 TLS groups (1 for each of 6 monomers in asu)
                What to do in REFMAC5

Suggested procedure:

• Choose TLS groups (TLSIN file)
• Use anisotropic scaling
• Set B to a constant value
• Refine TLS parameters against ML residual
• Refine coordinates and residual B factors
• NCS restraints can be applied to residual B values
              What to do with output

• Check Rfree and TLS parameters for convergence
• Check TLS parameters to see if there is any dominant
• Pass XYZOUT and TLSOUT through TLSANL for analysis
                     Example GAPDH

●Glyceraldehyde-3-phosphate            dehydrogenase from
Sulfolobus solfataricus (Isupov et al., (1999) J. Mol. Biol., 291, 651-
● 340 amino acids
● 2 chains in asymmetric unit (O and Q), each molecule

has NAD-binding and catalytic domains.
● P41212, data to 2.05Å
        GAPDH before and after TLS

    TLS                 R          Rfree
0                22.9       29.5

1                21.4       25.9
4                21.1       25.8
4/NCS            22.0       25.7
                               Refinement GAPDH

     Model                       TLS                     R                  Rfree
iso/rB                   0                       23.6             30.3
ani/rB                   0                       22.9             29.5
ani/rB                   1                       21.3             26.8
ani/rB                   4                       21.1             26.5
iso/20                   0                       30.0             35.7
ani/20                   0                       29.5             35.2
ani/20                   1                       25.1             29.4
ani/20                   4                       24.4             28.8

iso = isotropic scaling; ani = anisotropic scaling
rB = TLS refinement starting from refined Bs; 20 = TLS refinement starting from Bs fixed to 20 Å2
Contributions to equivalent isotropic Bs
                               Screw axis

Three translations together with three screw-displacements along three mutually
perpendicular non-intersecting axes
                    Example GerE

● Transcription regulator from Bacillus subtilis
(Ducrois et al., (2001) J. Mol. Biol., 306, 759-71).
● 74 amino acids

● Six chains A-F in asymmetric unit

● C2, data to 2.05Å
                  Refinement GerE

    Model TLS    NCS     R       Rfree   ccB
1      0     No      21.929.30.519
2      0     Yes 22.530.00.553
3      6     No      21.327.10.510
4      6     Yes 21.427.20.816
Contribution to equivalent isotropic Bs
Bs from NCS related chains
                     Summary TLS

• TLS parameterization allows to partly take into account
anisotropic motions at modest resolution (> 3.5 Å)
• TLS refinement might improve refinement statistics of several
• TLS refinement in REFMAC5 is fast and therefore can be used
           Selected topic 2: dictionary

The use of prior knowledge requires its organized

                     Monomer description

REFMAC5 requires a complete chemical description of all
 monomers (any molecular entity) present in the input
 coordinate file

About 2000 common monomers are already          present
 ($CLIBD_MON = $CCP4/lib/data/monomers)

•   Monomer and atoms identifier
•   Element symbol
•   Energy type
•   Partial charge
•   Covalent bonds (target values and SDs)
•   Torsion angles (target values and SDs)
•   Chiral centers
•   Planes
                   Monomer library


ener_lib.cif      -definition of atom types
mon_lib_com.cif   -definition of links and
mon_lib_list.html -missing file in version 4.2
0/,1/,...         -definition of various monomers
                Description of monomers

In the files:

For every monomer contain catagories:

     Monomer library (_chem_comp_atom)
 ALA      N    N    NH1      -0.204
 ALA      H    H    HNH1      0.204
 ALA      CA   C    CH1       0.058
 ALA      HA   H    HCH1      0.046
 ALA      CB   C    CH3      -0.120
 ALA      HB1 H     HCH3      0.040
 ALA      HB2 H     HCH3      0.040
 ALA      HB3 H     HCH3      0.040
 ALA      C    C    C         0.318
 ALA      O    O    O        -0.422
      Monomer library (_chem_comp_bond)
 ALA      N    H       single    0.860    0.020
 ALA      N    CA      single    1.458    0.019
 ALA      CA   HA      single    0.980    0.020
 ALA      CA   CB      single    1.521    0.033
 ALA      CB   HB1     single    0.960    0.020
 ALA      CB   HB2     single    0.960    0.020
 ALA      CB   HB3     single    0.960    0.020
 ALA      CA   C       single    1.525    0.021
 ALA      C    O       double    1.231    0.020
        What happens when you run REFMAC5

• You have a monomer for which there is a complete description
 The program carries on and takes everything from the dictionary
• You have a monomer for which there is only a minimal description

  or no description
 The program tries to generate a complete library     description
  and then STOPS for you to check it.

If a monomer is not in the library then SKETCHER can be used

SKETCHER is a graphical interface to LIBCHECK which creates
 new monomer library description
Future (near and far)

• Fast calculation of complete Hessian matrix
• Refinement along internal degrees of freedom

• Refinement using anomalous data
• Bayesian refinement of twinned data
• Lots more

• Garib N. Murshudov, York
• Alexei Vaguine, York
• Martyn Winn*, CCP4
• Liz Potterton*, York
• Eleanor Dodson, York
• Kim Hendrik, EBI Cambridge
• people who gave their data
* kindly provided many of the slides presented here

Financial support
• CCP4
• Wellcome Trust

To top