Refinement of Macromolecular Structures (PowerPoint)

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					Refinement with REFMAC

   Garib N Murshudov
York Structural Laboratory
  Chemistry Department
    University of York

1)   Refinement program – Refmac
2)   Simple refinement: Selection of weights
3)   Automatic twin refinement – Rfactor warnings
4)   Low resolution refinement tools

               What can REFMAC do?
•   Simple maximum likelihood restrained refinement
•   Twin refinement
•   Phased refinement (with Hendrickson-Lattmann coefficients)
•   SAD/SIRAS refinement
•   Structure idealisation
•   Library for more than 10000 ligands (from the next version)
•   Covalent links between ligands and ligand-protein
•   Rigid body refinement
•   Low res: NCS local, restraints to external structures, jelly body
•   TLS refinement
•   Map sharpening
•   Occupancy refinement
•   etc                                                   5
Simple refinement

Simple refinement

“Optimisation” of weights

             “Optimisation” of weights
After refinement final statistics are:

                                     Initial   Final
     R factor 0.2783 0.1831
      R free 0.2668 0.2030
   Rms BondLength 0.0284 0.0327
   Rms BondAngle 4.5704 2.3083
   Rms ChirVolume 0.1696 0.1645

RMSD of bond lengths is too large.

             “Optimisation” of weights

• If rmsd of bond lengths is too large (>0.022) or too tight (<0.01)
  then you may want to change weights. It can be done using
  weight matrix on the interface.
• Look at the log file. Refmac prints out current weights it is

Weight matrix 4.4438701
Actual weight 10.000000       is applied to the X-ray term

If rmsd is large then you can use half of currently used weight
   matrix (around 2.2).

“Optimisation” of weights

                        Change weight

           “Optimisation” of weights
With new weights RMSD is reasonable.

                               Initial   Final
    R factor 0.2783 0.1876
     R free 0.2668 0.2052
  Rms BondLength 0.0284 0.0201
  Rms BondAngle 4.5704 1.6554
  Rms ChirVolume 0.1696 0.1063

Twin refinement

       merohedral and pseudo-merohedral twinning
Crystal symmetry:        P3                 P2                         P2
Constrain:               -                  β = 90º                    -
Lattice symmetry *:      P622               P222                       P2
(rotations only)
Possible twinning:       merohedral         pseudo-merohedral              -

   Domain 1

  Twinning operator

   Domain 2                                                                    -

    Crystal lattice is invariant with respect to twinning operator.

    The crystal is NOT invariant with respect to twinning operator.
Twin refinement (it works with older version also

                           Twin refinement
Twin refinement in REFMAC is carried out in several stages

1) Stage 1: Identify potential twin operators. It is done by analysis
   of lattice and crystal symmetry.
In this case space group is P31 and there are four potential twin

Potential twin domain 1 with operator: H, K, L, metric score 0.000
Potential twin domain 2 with operator: -K, -H, -L, metric score 0.000
Potential twin domain 3 with operator: -H, -K, L, metric score 0.000
Potential twin domain 4 with operator: K, H, -L, metric score 0.000

Twin refinement: Group/subgroup

                   Twin refinement
2) Stage 2: Filter using agreement between “twin” related
   reflections (using Rmerge)

  Filtering out small twin domains, step 1
Twin ops with Rm > 0.44 will be removed

SymOp= -K,-H,-L:R_m=0.248:twin is probable
SymOp= -H,-K, L:R_m=0.237:twin is probable
SymOp= K, H,-L:R_m=0.027:twin or higher symm

At this stage REFMAC may suggest that space group could be
   Twin refinement: Effect of twin on Rmerge

R merges without experimental error
No twinning                                               50%
Along non twinned axes with another axis than twin        37.5%

                                                          Non twin

                  Twin refinement
3) Stage 3: Estimate twin fractions and remove small twin domains

             Filtering out small twin domains, step 2
Twin domains with fraction < 7.00000003E-02 are removed
        Twin operators with estimated twin fractions

Twin op: H, K, L: Fr = 0.391; Eq ops: K, -H-K, L; -H-K, H, L
Twin op: -K, -H, -L:Fr = 0.112; Eq ops: -H, H+K, -L; H+K, -K, -L
Twin op: -H, -K, L:Fr = 0.108; Eq ops: -K, H+K, L; H+K, -H, L
Twin op: K, H, -L:Fr = 0.390; Eq ops: H, -H-K, -L; -H-K, K, -L

                   Twin refinement
3) Stage 4: Perform twin refinement with all survived twin
   operators (in this example all four operators survive):

Twin fractions     =   0.3773 0.1246 0.1206 0.3775

Rfactors look very good:
                                  Initial   Final
     R factor 0.1912 0.1566
      R free 0.1796 0.2047
   Rms BondLength 0.0088 0.0235
   Rms BondAngle 1.4825 2.1812
   Rms ChirVolume 0.1077 0.1336
Rfactors from non-twinned refinement

                        Initial Final
    R factor 0.3103 0.2779
      R free 0.3184 0.3496
  Rms BondLength 0.0088 0.0129
  Rms BondAngle 1.4825 1.5648
  Rms ChirVolume 0.1077 0.1034

Twin refinement: Rfactors – be careful
                Cyan – perfect twin and twin modelled
                Black – no twin and not modelled
                Red – perfect twin and not modelled
                Blue – no twin and perfect twin modelled

                       Rfactor drop can be as large
                       as 15% without atomic model

  Twin refinement: Alternative indexing
If crystal can be twinned then there may be more than one
   indexing of hkl. Different indexing are related with the
   symmetry operator of lattice but not the crystal.
Best way of dealing with indexing “problem” is to use the
   program pointless by Phil Evans. You can either give a
   reference mtz file or a reference structure. Then all
   subsequent data will be indexed in consistent manner.

Low resolution refinement

             Low resolution refinement tools
1.   Jelly body (implicit normal modes) refinement
2.   NCS: local and global restraints
3.   NCS constraints
4.   Restraints to reference structures
5.   Regularised map sharpening
6.   Long range B value restraints based on Kullback-Liebler distances

Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA,
  Nicholls RA, Winn MD, Long F, Vagin AA “REFMAC5 for the
  Refinement of Macromolecular Crystal Structures” Acta Cryst: ,
  D67, 355-367

         External (reference structure restraints)

Restraints to external structures are generated by the program ProSmart:
1) Aligns structure in the presence of conformational changes. Sequence is not used
2)           Gernates           restraints         for          aligned           atoms
3) Identifies secondary structures (at the moment helix and strand, but the approach is
general        and       can         be      extended       to        any        motif).
4)         Generates          restraints       for        secondary           structures

Note 1: ProSmart has been written by Rob Nocholls and available from him (now). It
will be distributed by ccp4 (hopefully from the next release)

Note 2: Robust estimator functions are used for restraints. I.e. if differences between
target and model is very large then their contributions are downweighted
                   Restraints to current distances

The term is added to the target function:

                       w(| d |  | d   current   |)2

Summation is over all pairs in the same chain and within given distance (default
4.2A). dcurrent is recalculated at every cycle. This function does not contribute to
gradients. It only contributes to the second derivative matrix.

It is equivalent to adding springs between atom pairs. During refinement inter-atomic
distances are not changed very much. If all pairs would be used and weights would
be very large then it would be equivalent to rigid body refinement.

It could be called “implicit normal modes”, “soft” body or “jelly” body refinement.

      External (reference structure restraints)

The program will be available from ccp4. Currently if you want to
try it you should ask Rob Nicholls at

Once you have downloaded you can run using this command

prosmart –p1 refined_structure.pdb –p2 reference_structure.pdb

It will generate many useful info including restraints to the
reference structure.

               Auto NCS: local and global
1. Align all chains with all chains using Needleman-Wunsh method
2. If alignment score is higher than predefined (e.g.80%) value then consider
them as similar
3.Find local RMS and if average local RMS is less than predefined value then
consider them aligned
4. Find correspondence between atoms
5. If global restraints (i.e. restraints based on RMS between atoms of aligned
chains) then identify domains
6.For local NCS make the list of corresponding interatomic distances (remove
bond and angle related atom pairs)
7.Design weights

The list of interatomic distance pairs is calculated at every cycle

Add external keywords file in refmac

                                Browse files

Add external keywords file in refmac

                               Select keywords
                               file 32
Add external keywords file in refmac

                               Keywords file

        Instructions you may want to play with

#                                     Jelly                 body
Ridge                 dist               sigma               0.01
ridge                 dist                dmax                4.2

#                                                              ncs
ncsr                                                         local

#    to     control    restraints    to    reference   structures.
#      Restraints       are       generated       by     prosmart
external                          dmax                         4.2
external                weight                scale              4
external cut 10                                        35
         Low resolution refinement: Some results

        Initial   Simple   Jelly    NCS local   Jelly/NCS   Reference structure

R       0.3605    0.2218   0.2533   0.2232      0.2535      0.2557
Rfree   0.3563    0.3116   2961     0.3124      0.2955      0.2907

If you want to use current version then you may need to run several
time to get parameters right. In this case maximum radius for reference
structure restraint was 4.0, maximum radius for NCS local was 4.2, if
deviation between reference distance and current distance was more
than 10 sigma then it was excluded, sigmas for reference structures
were 0.07.

At lower resolution (5-7Å) radius may need to be 5.5 and sigma 0.02
• Auto weight works fine for large class of cases, however you may
  need to change weights
• Twin is automatic but Rfactors are poor indicators
• Use of available information may improve low resolution

York                                        Leiden
Alexei Vagin                                Pavol Skubak
Andrey Lebedev                              Raj Pannu
Rob Nocholls
Fei Long
                 CCP4, YSBL people
REFMAC is available from CCP4 or from York’s ftp site:
Balbes and other programs:
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