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Effects of TLS parameters in Macromolecular Refinement. Martyn Winn Daresbury Laboratory, U.K. IUCr99 08/08/99. Overview. Background to the use of TLS tensors. Details of TLS refinement. Implementation in REFMAC: examples. Contributions to atomic U.

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effects of tls parameters in macromolecular refinement
Effects of TLS parameters in Macromolecular Refinement
  • Martyn Winn
  • Daresbury Laboratory, U.K.
  • IUCr99 08/08/99
overview
Overview
  • Background to the use of TLS tensors.
  • Details of TLS refinement.
  • Implementation in REFMAC: examples
contributions to atomic u
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 a.s.u., molecules, domains, secondary structure elements,

aromatic rings of side groups, etc.

Uinternal : internal displacements of molecules, e.g. normal modes of vibration, torsions, etc.

Uatom : anisotropy of individual atoms

tls small molecules
TLS: small molecules
  • D.W.J.Cruikshank (1956) - TL analysis
  • G.S.Pawley (1964, 1966) - TL refinement
  • V.Schomaker & K.N.Trueblood (1968) - introduction of S in analysis of ADPs
  • J.D.Dunitz & D.N.J.White (1973) - inclusion of internal torsional motion of “attached rigid group”
tls macromolecules
TLS: macromolecules
  • S.R.Holbrook et al (1985) - duplex DNA dodecanucleatide, 1.9Å, 70 groups (phosphate, ribose, base), CORELS
  • B.Howlin et al (1989) - bovine Ribonuclease A, 1.45Å, RESTRAIN
  • G.W.Harris et al (1992) - papain, 1.6Å , RESTRAIN
  • Sali et al (1992) - endothiapepsin complex, 1.8Å, RESTRAIN
rigid body motion
Rigid body motion
  • Linearise general displacement u of atom (mean position r) in rigid body: u = t + D.r  t +  x r
  • Corresponding dyad: uu = tt + t x r - r x t - r x  x r
  • Average over dynamic motion and static disorder gives atomic ADP: U  <uu> = T + ST x r - r x S - r x L x r
  • T,L andS describe mean square translation, libration and their correlation of rigid body.
tls in refinement
TLS in refinement
  • Need to specify TLS groups for molecule of interest.
  • 6 + 6 + 8 = 20 parameters per group (trace of S is undetermined).
  • T and S (but not L) origin-dependent. S is symmetric if origin is Centre of Reaction.
  • Gradients of residual w.r.t. TLS parameters follow from gradients w.r.t. U’s via chain rule.
slide8
NCS
  • REFMAC applies restraints to B and U values of NCS-related molecules.
  • But different molecules in a.s.u. may have different overall thermal parameters.
  • Refine independent overall TLS tensors for each molecule before refining restrained individual parameters.
choice of tls groups
Choice of TLS groups
  • Choose TLS groups using:
    • chemical knowledge, e.g. aromatic side groups of amino acids, domains of macromolecules
    • fit to ADPs of test structure, e.g. Holbrook & Kim (1984) compared 7 rigid body models of CMP and used best as basis for partitioning other nucleic acids.
    • dynamic domains identified from similar structures, e.g. from apo and holo forms of alcohol dehydrogenase
implementation in refmac
Implementation in REFMAC
  • Refine TLS parameters against ML residual, using previously refined atomic coordinates and B factors.
  • TLS parameters held in TLSIN/TLSOUT files.
  • Analyse with TLSANL program.

 libration axes, etc and ADPs

To be implemented:

  • Allow TLS refinement prior to or simultaneously with refinement of other parameters.
e g 1 gapdh
E.g. 1 - GAPDH
  • Glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus solfataricus (M.N.Isupov et al, JMB, in press)
  • P41212, 2.0Å, 2 molecules in a.s.u., each molecule has NAD-binding and catalytic domains.
e g 1 gapdh1
E.g. 1 - GAPDH

ScalingBisoTLS groupsR factorRfree

Isotropic Refined 0 23.3 29.6

Anisotropic Refined 0 22.6 28.9

Isotropic Refined 1 21.0 26.4

Isotropic Refined 2 20.9 26.2

Isotropic Refined 4 20.8 26.1

Isotropic 35Ų 0 29.5 34.4

Isotropic 35Ų 1 26.6 32.1

Isotropic 35Ų 2 24.2 29.0

Isotropic 35Ų 4 23.8 28.4

e g 1 axes of libration
E.g.1: axes of libration

Refined Bs.

Blue - chain O, NAD-binding domain

Red - chain O, catalytic domain

Green - chain Q, NAD-binding domain

Yellow - chain Q, catalytic domain

e g 1 axes of libration1
E.g.1: axes of libration

Constant Bs.

Blue - chain O, NAD-binding domain

Red - chain O, catalytic domain

Green - chain Q, NAD-binding domain

Yellow - chain Q, catalytic domain

e g 2 adh
E.g.2: ADH
  • horse liver alcohol dehydrogenase (S.Ramaswamy et al).
  • apo form: C2221, 2.0Å, single chain in a.s.u.
  • DYNDOM results from apo vs. holo forms.

ScalingBisoTLS groupsR factorRfree

Isotropic Refined 0 27.9 32.5

Anisotropic Refined 0 23.6 29.1

Isotropic Refined 1 22.5 27.4

Isotropic Refined 4 22.3 27.4

Isotropic Refined 6 22.2 27.6

e g 2 dynamic domains
E.g.2: dynamic domains

Results from DYNDOM.

Blue - first domain

Red - second domain

Green - hinge region

e g 2 axes of libration
E.g.2: axes of libration

TLS groups:

Blue - first dynamic domain

Red - second dynamic domain

Green - hinge region

Yellow - flexible loop

e g 3 lysozyme complex
E.g. 3: lysozyme complex
  • Hen egg white lysozyme complexed withcamelid single-chain antibody (K. Decanniere et al).
  • C2, 2.1Å, single copy in a.s.u.

ScalingBisoTLS groupsR factorRfree

Isotropic Refined 0 20.2 24.3

Anisotropic Refined 0 20.0 24.0

Isotropic Refined 4 19.9 23.7

Isotropic Refined 4* 19.9 23.7

e g 3 axes of libration
E.g.3: axes of libration

Simple minimisation.

Blue - antibody

Red - CRD3 loop of antibody

Green - lysozyme

Yellow - lysozyme

e g 3 axes of libration1
E.g.3: axes of libration

Minimisation with TLS constrained to be positive semi-definite

Blue - antibody

Red - CRD3 loop of antibody

Green - lysozyme

Yellow - lysozyme

acknowledgements
Acknowledgements
  • CCP4
  • BBSRC
  • Garib Murshudov (REFMAC)
  • Misha Isupov (GAPDH)
  • S Ramaswamy (ADH)
  • Klaas Decanniere (lysozyme complex)