Macromolecular refinement with REFMAC5 and SKETCHER of the CCP4 suite

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

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

3. 1 General aspects of refinement and overview of REFMAC5

4. Given Set experimental values of quantity q (qE,E) Model M(aI,bI,cI)  qIC Estimate Best model, i.e. M(aB,bB,cB) which is most consistent with the data The accuracy of (aB,bB,cB) A common problem in physical sciences R

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

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

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

8. 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) Bayesian approach Probability Theory: The Logic of Science by E.T.Jaynes http://bayes.wustl.edu

9. 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. Application of Bayes theorem

10. 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)] Maximum likelihood residual Murshudov et al., Acta Cryst. (1997) D53, 240-255

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

12. 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) Essential features of REFMAC5

13. 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. Selected topic 1: TLS

14. 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 Decomposition of ADPs

15. 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 Rigid-body motion

16. 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

17. 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

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 Rfree 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. • P41212, data to 2.05Å

22. 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

23. 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

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 Model TLS NCS R Rfree ccB 1 0 No 21.9 29.3 0.519 2 0 Yes 22.5 30.0 0.553 3 6 No 21.3 27.1 0.510 4 6 Yes 21.4 27.2 0.816

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

32. 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 description

33. $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 Monomer library

34. 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 Description of monomers

35. 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 Monomer library (_chem_comp_atom)

36. 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 Monomer library (_chem_comp_bond)

37. 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. What happens when you run REFMAC5

38. 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 SKETCHER

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

42. People • 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