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TLS MODELLING OF ANISOTROPY IN MACROMOLECULAR REFINEMENT

TLS MODELLING OF ANISOTROPY IN MACROMOLECULAR REFINEMENT. Martyn Winn CCP4, Daresbury Laboratory, U.K. York, April 11th 2002. Aims. Mean square atomic displacements (static and dynamic) are an important part of model of protein.

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TLS MODELLING OF ANISOTROPY IN MACROMOLECULAR REFINEMENT

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  1. TLS MODELLING OF ANISOTROPY IN MACROMOLECULAR REFINEMENT Martyn Winn CCP4, Daresbury Laboratory, U.K. York, April 11th 2002

  2. Aims • Mean square atomic displacements (static and dynamic) are an important part of model of protein. • Atomic displacements are likely anisotropic, but rarely have luxury of refining individual anisotropic Us. Instead isotropic Bs. • Intermediate descriptions??

  3. Contributions to atomic U U = Ucrystal + UTLS + Uinternal + Uatom Ucrystal : overall anisotropic scale factor w.r.t. crystal axes. UTLS : rigid body displacements e.g. of molecules, domains, secondary structure elements, side groups, etc. Uinternal : internal displacements of molecules, e.g. normal modes of vibration, torsions, etc. Uatom : anisotropy of individual atoms

  4. Rigid body model

  5. General displacement of atom (position r w.r.t. origin O) in rigid body: u = t + D.r For small libration : u t +   r Rigid body motion

  6. TLS parameters • Corresponding dyad: uu = tt + t r - rt - r  r • Average over dynamic motion and static disorder gives atomic anisotropic displacement parameter (ADP): UTLS  <uu> = T + ST r - r S - r L r • T,L andS describe mean square translation and libration of rigid body and their correlation. • T  6 parameters, L  6 parameters, S  8 parameters

  7. Use of TLS UTLS  <uu> = T + ST r - r S - r L r • Given refined U’s, fit TLS parameters - analysis • Use TLS as refinement parameters TLS  U’s  structure factor - refinement

  8. TLS in refinement • TLS parameters are contribution to displacement parameters of model • Can specify 1 or more TLS groups to describe contents of asymmetric unit (or part thereof) • 6 + 6 + 8 = 20 parameters per group (trace of S is undetermined) • Number of extra refinement parameters depends on how many groups used!

  9. At what resolution can I use TLS? • Resolution < 1.2 Å - full anisotropic refinement • Resolution ~ 1.5 Å - marginal for full anisotropic refinement. But can do detailed TLS, e.g. Howlin et al, Ribonuclease A, 1.45 Å, 45 side chain groups; Harris et al, papain, 1.6Å, 69 side chain groups. • Resolution 1.5 Å - 2.5 Å model molecules/domains rather than side chains. • Sandalova et al 2001 - thioredoxin reductase at 3.0 Å - TLS group for each of 6 monomers in asu

  10. Implementation in REFMAC Suggested procedure: • Choose TLS groups (currently via TLSIN file). • Use anisotropic scaling. • Set B values to constant value. • Refine TLS parameters (and scaling parameters) against ML residual. • Refine coordinates and residual B factors.

  11. NCS • Different molecules in asymmetric unit may have different overall thermal parameters. • Refine independent overall TLS tensors for each molecule. • REFMAC can then apply NCS restraints to residual B values of NCS-related molecules.

  12. TLSIN TLS Chain O NAD binding RANGE 'O 1.' 'O 137.' ALL RANGE 'O 303.' 'O 340.' ALL TLS Chain O Catalytic RANGE 'O 138.' 'O 302.' ALL TLS Chain Q NAD binding RANGE 'Q 1.' 'Q 137.' ALL RANGE 'Q 303.' 'Q 340.' ALL TLS Chain Q Catalytic RANGE 'Q 138.' 'Q 302.' ALL

  13. Choice of TLS groups • chemical knowledge, e.g. aromatic side groups of amino acids, secondary structure elements, domains, molecules • best fit of TLS to ADPs of test structure, e.g. Holbrook & Kim (1984); rigid-body criterion applied to ADPs, e.g. Schneider (1996) • dynamic domains identified from multiple configurations, e.g. more than one crystal form (DYNDOM), difference distance matrices (ESCET), MD simulations.

  14. TLS - refmac5 interface • Select TLS & restrained refinement at top of interface • Specify TLS in and TLS out files. • TLS Parameters folder: • Number of cycles, e.g. 20 • Fix Bfactors to e.g. 20 (value not crucial, but option is recommended)

  15. What to look for in output • Usual refinement statistics. • Check R_free and TLS parameters in log file for convergence. • Check TLS parameters to see if any dominant displacements. • Pass XYZOUT and TLSOUT through TLSANL for analysis • Consider alternative choices of TLS groups

  16. TLSOUT TLS Chain O NAD binding RANGE 'O 1.' 'O 137.' ALL RANGE 'O 303.' 'O 340.' ALL ORIGIN 94.142 6.943 75.932 T 0.5200 0.5278 0.5031 -0.0049 -0.0040 0.0081 L 0.11 0.08 0.04 -0.03 0.02 0.06 S -0.014 0.020 0.002 0.022 0.000 -0.016 -0.005 0.022 . . .

  17. Equivalent isotropic B factors • TLS tensors  UTLS for atoms in group. • UTLS BTLS and anisotropy A • Also have individually refined Bres • Hence, BTOT = BTLS + Bres

  18. Running TLSANL XYZIN: output coordinates from refmac with residual B factors (BRESID keyword) TLSIN: output TLS parameters from refmac TLSIN: ANISOU records including TLS and residual B contributions ATOM records containing choice of B (ISOOUT keyword) AXES: If AXES keyword set, file of principal axes in molscript format

  19. Output of TLSANL Origins (T and S, but not L, origin-dependent): • Origin of calculation • Centre of Reaction Axial systems for each tensor: • orthogonal • librational “Simplest” description: 3 non-intersecting screw axes + 3 reduced translations

  20. Displaying derived ADPs ORTEP: http://www.ornl.gov/ortep/ortep.html Mapview: http://www.chem.gla.ac.uk/~paule/chart/ xtalview: http://www.scripps.edu/pub/dem-web/toc.html Rastep in RASTER3D : using the script: grep NAD file.pdb | rastep -auto -Bcol 5. 35. > ellipsoids.r3d render -jpeg < ellipsoids.r3d > ellipsoids.jpeg Xfit: http://www.ansto.gov.au/natfac/asrp7_xfit.html povscript: http://people.brandeis.edu/~fenn/povscript

  21. 11 a.a. (8% of TLS group) 30% probability level

  22. Ex. 1 - mannitol dehydrogenase Hörer et al., J.Biol.Chem.276, 27555 (2001) 1.5 Å data 3 tetramers in a.s.u. TLS refinement with 1 group per monomer Free-R 23.6%  20.9% Tetramer B’s before TLS B’s after TLS crystal contacts ABCD 27.1 13.3 38 EFGH 18.0 13.3 50 IJKL 18.6 13.3 49

  23. Ex. 2 - light harvesting complex Complex is nonamer. Each monomer contains:  peptide  peptide 2 x B850 bacteriochlorophyll 1 x B800 bacteriochlorophyll 2 x carotenoids Crystallographic asu = 3 monomers

  25. TLS models a) 1 group for a.s.u. (20 parameters) b) 1 group per NCS unit (3 x 20 pars) c) 1 group per molecule (18 x 20 pars) d) 3 groups per peptide + 1 group per pigment (total 30 x 20 parameters)

  26. Ex. 3 - peptide-MHC complexes Markus Rudolph Class I peptide-MHC complexes (1.7 Å – 1.9 Å) Alpha chain – 1 or 2 TLS groups Beta chain – 1 TLS group Bound peptide – 1 TLS group Free R decrease 22.3 to 21.1 in best case. Eigenvalues of L for peptide: 64.7 0.9 -4.1

  27. Example 4 - GAPDH • Glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus solfataricus (M.N.Isupov et al, JMB, 291, 651 (1999)) • 340 amino acids • 2 chains in asymmetric unit (O and Q), each molecule has NAD-binding and catalytic domains. • P41212, data to 2.05Å

  28. GAPDH: R factors Model TLS groups R factorRfree 1023.8 30.4 2 1* 25.1 29.4 3 1 21.4 25.9 4 2 21.2 26.2 5 4 21.1 25.8 * B factors constant at 20 Å2

  29. TLS values - 1 TLS group Eigenvalues of reduced translation tensor: 0.267 Å2 0.112 Å2 –0.005 Å2 Eigenvalues and pitches of screw axes: 1.403 ()2 0.888Å 1.014 ()2 1.472Å 0.204 ()2 1.214Å

  30. Screw axes - 1 TLS group

  31. Contributions to equivalent isotropic B factor

  32. B’s from NCS-related chains

  33. Correlations between NCS-related B's Model TLS groups R factorRfree CorrCoeff 1023.8 30.4 0.537 2 1* 25.1 29.4 - 3 1 21.4 25.9 0.752 4 2 21.2 26.2 0.812 5 4 21.1 25.8 0.821 CorrCoeff - mean correlation coefficient between (residual) B factors of NCS-related chains

  34. Application of NCS restraints Model TLS groups NCSR factorRfree CorrCoeff 10no 23.8 30.4 0.537 6 0 yes 25.0 30.3 0.989 5 4 no 21.1 25.8 0.821 7 4 yes 22.0 25.7 0.999 NCS - whether NCS restraints applied

  35. Example 5: GerE • transcription regulator from Bacillus subtilis (Ducros et al). • 74 amino acids • six chains A-F in asymmetric unit • C2, data to 2.05Å

  36. GerE

  37. GerE: R factors ModelTLS groups R factorRfree 1 0 21.9 29.3 2 6 21.3 27.1

  38. Contributions to equivalent isotropic B factor

  39. B’s from NCS-related chains

  40. GerE: NCS ModelTLS groups NCSR factorRfree CorrCoeff 1 0 no 21.9 29.3 0.519 3 0 yes 22.5 30.0 0.553 2 6 no 21.3 27.1 0.510 4 6 yes 21.4 27.2 0.816 NCS - whether NCS restraints applied CorrCoeff - mean correlation coefficient between (residual) B factors of NCS-related chains

  41. Choice of TLS groups 6 groups - each group split between 2 chains (for illustration only!) ModelTLS groups R factorRfree 1 0 22.5 30.0 2 6 21.4 27.2 5 6 22.2 28.6

  42. Choice of TLS groups

  43. Choice of TLS groups

  44. Inclusion of bound waters 6 TLS groups, with some waters included, Waters R factorRfree None 21.4 27.2 1 shell, cutoff 2.8 21.5 27.4 1 shell, cutoff 3.2 21.5 27.2 2 shell, cutoff 3.2 21.6 27.2

  45. Acknowledgements • BBSRC (CCP4 grant) • Garib Murshudov • Miroslav Papiz (LH2) • Stefan Hörer (mannitol dehydrogenase) • Markus Rudolph (MHC) • Misha Isupov (GAPDH) • Valerie Ducros, Jim Brannigan, Tony Wilkinson (GerE)

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