1. Macromolecular refinement with REFMAC5 and SKETCHER of the CCP4 suite Roberto A. Steiner – University of York

2. Organization 1 General aspects of refinement and overview of REFMAC5 TLS Dictionary 2 Demo TLS refinement in REFMAC5 SKETCHER 3 Future

3. 1 General aspects of refinement and overview of REFMAC5

4. A common problem in physical sciences Given Set experimental values of quantity q (q E,  E ) Model M(a I,b I,c I )   q I C Estimate Best model, i.e. M(a B,b B,c B ) which is most consistent with the data The accuracy of (a B,b B,c B ) R

5. Model fitting Experiment Mathematical model Generation of additional data Inference Analysis

6. Model fitting in crystallography experimental (I,  I )  (F,  F ) model (heavy atoms, protein,..) F C Best model R

7. Key aspects in model fitting Parameterization of the model Type of residual Type of minimization Prior information

8. Bayesian approach The best model is the one which has highest probability given a set of observations and a certain prior knowledge. BAYES' THEOREM P(M;O)=P(M)P(O;M)/P(O) Probability Theory: The Logic of Science by E.T.Jaynes http://bayes.wustl.edu

9. 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? P(D;P)=P(D)P(P;D)/P(P) P(P)=P(P;D)P(D)+P(P;notD)P(notD)=(0.9)(0.0002)+(0.005)(1- 0.0002)=0.005179 P(D;P)=(0.0002)(0.9)/(0.005179)=0.0348 Less than 3.5% of persons diagnosed to have the disease do actually have it.

10. 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

11. Maximum likelihood refinement programs REFMAC5 CNS/CNX BUSTER-TNT

12. 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 minimization) Extensive built-in dictionary (LIBCHECK) Graphical control (CCP4i) Flexible parameterization (iso-,aniso-,mixed-ADPs, TLS, bulk solvent) Easy to use (coordinate and reflection files, straightforward inclusion of alternate conformations)

13. 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.

14. Decomposition of ADPs U = U cryst +U TLS +U int +U atom U cryst : overall anisotropy of the crystal U TLS : TLS motions of pseudo-rigidy bodies U int : collective torsional librations or internal normal modes U cryst : individual atomic motions

15. 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

16. TLS parameters Dyad product: uu T = tt T + t T  r T -r  t T -r  T  r T ADPs are the time and space average U TLS =  uu T  T + S T  r T -r  S -r  L  r T T =  tt T  6 parameters, TRANSLATION L =  T  6 parameters, LIBRATION S =  t T  8 parameters, SCREW-ROTATION

17. Use of TLS U TLS =  uu T  T + S T  r T -r  S -r  L  r T 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

18. 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, 9533-8) 6 TLS groups (1 for each of 6 monomers in asu)

19. 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

20. What to do with output Check R free and TLS parameters for convergence Check TLS parameters to see if there is any dominant displacement Pass XYZOUT and TLSOUT through TLSANL for analysis

21. Example GAPDH ● Glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus solfataricus (Isupov et al., (1999) J. Mol. Biol., 291, 651- 60) ● 340 amino acids ● 2 chains in asymmetric unit (O and Q), each molecule has NAD-binding and catalytic domains. ● P4 1 2 1 2, data to 2.05Å

22. GAPDH before and after TLS TLSR R free 022.929.5 121.425.9 421.125.8 4/NCS22.025.7

23. Refinement GAPDH ModelTLSR R free iso/rB023.630.3 ani/rB022.929.5 ani/rB121.326.8 ani/rB421.126.5 iso/20030.035.7 ani/20029.535.2 ani/20125.129.4 ani/20424.428.8 iso = isotropic scaling; ani = anisotropic scaling rB = TLS refinement starting from refined Bs; 20 = TLS refinement starting from Bs fixed to 20 Å 2

24. Contributions to equivalent isotropic Bs

25. Screw axis Three translations together with three screw-displacements along three mutually perpendicular non-intersecting axes

26. 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

27. Refinement GerE ModelTLSNCSR R free cc B 10No21.929.30.519 20Yes22.530.00.553 36No21.327.10.510 46Yes21.427.20.816

28. Contribution to equivalent isotropic Bs

29. Bs from NCS related chains

30. 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 percent TLS refinement in REFMAC5 is fast and therefore can be used routinely

31. Selected topic 2: dictionary The use of prior knowledge requires its organized storage. $CCP4/html/mon_lib.html www.ysbl.york.ac.uk/~alexei/dictionary.html

32. 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

33. Monomer library $CCP4/lib/data/monomers/ ener_lib.cif-definition of atom types mon_lib_com.cif-definition of links and modifications mon_lib_list.html -missing file in version 4.2 0/,1/,... -definition of various monomers

34. Description of monomers In the files: */###.cif For every monomer contain catagories: _chem_comp_atom _chem_comp_bond _chem_comp_angle _chem_comp_tor _chem_comp_chir _chem_comp_plane_atom

35. Monomer library (_chem_comp_atom) loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.type_symbol _chem_comp_atom.type_energy _chem_comp_atom.partial_charge 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

36. Monomer library (_chem_comp_bond) loop_ _chem_comp_bond.comp_id _chem_comp_bond.atom_id_1 _chem_comp_bond.atom_id_2 _chem_comp_bond.type _chem_comp_bond.value_dist _chem_comp_bond.value_dist_esd 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

37. 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.

38. SKETCHER 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

39. 2 Demo IF YOU WERE NOT IN SAN ANTONIO (ACA2002) I'M AFRAID YOU'LL HAVE TO WAIT FOR THE NEXT OCCASION

40. 3 Future (near and far)

41. Fast calculation of complete Hessian matrix Refinement along internal degrees of freedom Refinement using anomalous data Bayesian refinement of twinned data Lots more Future

42. 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 People