1. Refinement with REFMAC
Garib N Murshudov
York Structural Laboratory
Chemistry Department
University of York

4. Contents Refinement program – Refmac
Simple refinement: Selection of weights
Automatic twin refinement – Rfactor warnings
Low resolution refinement tools

5. 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 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

6. Simple refinement

7. Simple refinement

8. “Optimisation” of weights

9. “Optimisation” of weights
After refinement final statistics are:
Initial Final
R factor
R free
Rms BondLength
Rms BondAngle
Rms ChirVolume
RMSD of bond lengths is too large.

10. “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 using.
Weight matrix
Actual weight is applied to the X-ray term
If rmsd is large then you can use half of currently used weight matrix (around 2.2).

11. “Optimisation” of weights
Change weight matrix

12. “Optimisation” of weights
With new weights RMSD is reasonable.
Initial Final
R factor
R free
Rms BondLength
Rms BondAngle
Rms ChirVolume

13. Twin refinement

14. merohedral and pseudo-merohedral twinning
Crystal symmetry: P3 P P2
Constrain: β = 90º
Lattice symmetry *: P P P2
(rotations only)
Possible twinning: merohedral pseudo-merohedral
Domain 1
Domain 2
Twinning operator -
Crystal lattice is invariant with respect to twinning operator.
The crystal is NOT invariant with respect to twinning operator.

15. Twin refinement (it works with older version also

16. Twin refinement
Twin refinement in REFMAC is carried out in several stages
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 operators
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

17. Twin refinement: Group/subgroup

18. 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 > 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 higher

19. Twin refinement: Effect of twin on Rmerge
R merges without experimental error
No twinning %
Along non twinned axes with another axis than twin 37.5%
Non twin
Twin

20. Twin refinement
3) Stage 3: Estimate twin fractions and remove small twin domains
Filtering out small twin domains, step 2
Twin domains with fraction < E-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

21. Twin refinement
3) Stage 4: Perform twin refinement with all survived twin operators (in this example all four operators survive):
Twin fractions =
Rfactors look very good:
Initial Final
R factor
R free
Rms BondLength
Rms BondAngle
Rms ChirVolume

22. Rfactors from non-twinned refinement
Initial Final
R factor
R free
Rms BondLength
Rms BondAngle
Rms ChirVolume

23. 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 improvement

24. 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.

25. Low resolution refinement

26. Low resolution refinement tools
Jelly body (implicit normal modes) refinement
NCS: local and global restraints
NCS constraints
Restraints to reference structures
Regularised map sharpening
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,

27. 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
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

28. Restraints to current distances
The term is added to the target function:
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.
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

29. 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.
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

30. Auto NCS: local and global
Align all chains with all chains using Needleman-Wunsh method
If alignment score is higher than predefined (e.g.80%) value then consider them as similar
Find local RMS and if average local RMS is less than predefined value then consider them aligned
Find correspondence between atoms
If global restraints (i.e. restraints based on RMS between atoms of aligned chains) then identify domains
For local NCS make the list of corresponding interatomic distances (remove bond and angle related atom pairs)
Design weights
The list of interatomic distance pairs is calculated at every cycle
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

31. Add external keywords file in refmac interface
Browse files

32. Add external keywords file in refmac interface
Select keywords file

33. Add external keywords file in refmac interface

35. Instructions you may want to play with
# Jelly body Ridge dist sigma 0.01 ridge dist dmax # 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
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

36. 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
Should be able to well-align similar proteins, but should also be able to align dissimilar proteins, so that a meaningful score can be given in all cases.

37. Conclusions
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 refinement

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