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

Contents Refinement program – Refmac Simple refinement: Selection of weights Automatic twin refinement – Rfactor warnings 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

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 using. 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 matrix

“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 Domain 2 Twinning operator - 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 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

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 higher

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

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 improvement

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 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, 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 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.

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.

External (reference structure restraints) The program will be available from ccp4. Currently if you want to try it you should ask Rob Nicholls at ran105@york.ac.uk 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.

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.

Add external keywords file in refmac interface Browse files

Add external keywords file in refmac interface Select keywords file

Add external keywords file in refmac interface

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 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.

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.

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

Acknowledgment 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: www.ysbl.york.ac.uk/refmac/latest_refmac.html Balbes and other programs: www.ysbl.york.ac.uk/refmac/YSBLPrograms/index.jsp This and other presentations can be found on: www.ysbl.york.ac.uk/refmac/Presentations/