Macromolecular refinementwith

REFMAC5 and SKETCHERof the

CCP4 suite

Roberto A. Steiner – University of York

Organization

1General aspects of refinement and overview of REFMAC5

• TLS• Dictionary

2Demo

• TLS refinement in REFMAC5 • SKETCHER

3Future

1General aspects of refinement

and overview of REFMAC5

A common problem in physical sciences

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)

R

Model fitting

Experiment Mathematical model

Generation of additional data

Inference

Analysis

Model fitting in crystallography

experimental (I,I ) (F, F)

model (heavy atoms, protein, ..)

FC

Best model

R

Key aspects in model fitting

• Parameterization of the model• Type of residual• Type of minimization• Prior information

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.Jayneshttp://bayes.wustl.edu

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)=0.005179P(D;P)=(0.0002)(0.9)/(0.005179)=0.0348Less than 3.5% of persons diagnosed to have the disease

do actually have it.

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

Maximum likelihood refinement programs

•REFMAC5

•CNS/CNX

•BUSTER-TNT

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)

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.

Decomposition of ADPs

U = Ucryst+UTLS+Uint+Uatom

Ucryst : overall anisotropy of the crystalUTLS : TLS motions of pseudo-rigidy bodiesUint : collective torsional librations or

internal normal modesUcryst : individual atomic motions

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

TLS parameters

Dyad product:

uuT = ttT + tTrT -rtT -rTrT

ADPs are the time and space average

UTLS = uuTT + STrT -rS -rLrT

T = ttT6 parameters, TRANSLATIONL = T6 parameters, LIBRATIONS = tT8 parameters, SCREW-ROTATION

Use of TLS

UTLS = uuTT + STrT -rS -rLrT

• analysis: given inidividual aniso-ADPs fit TLS parameters Harata et al., (2002) Proteins, 48, 53-62Harata et al., (1999) J. Mol. Biol., 30, 232-43

• refinement: TLS as refinement parameters Howlin et al., (1989) Acta Cryst., A45, 851-61Winn et al., (2001) Acta Cryst., D57, 122-33

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)

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

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

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

GAPDH before and after TLS

TLS R Rfree

0 22.9 29.5

1 21.4 25.94 21.1 25.84/NCS 22.0 25.7

Refinement GAPDH

Model TLS R Rfree

iso/rB 0 23.6 30.3ani/rB 0 22.9 29.5ani/rB 1 21.3 26.8ani/rB 4 21.1 26.5iso/20 0 30.0 35.7ani/20 0 29.5 35.2ani/20 1 25.1 29.4ani/20 4 24.4 28.8

iso = isotropic scaling; ani = anisotropic scalingrB = TLS refinement starting from refined Bs; 20 = TLS refinement starting from Bs fixed to 20 Å2

Contributions to equivalent isotropic Bs

Screw axis

Three translations together with three screw-displacements along three mutually perpendicular non-intersecting axes

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

Refinement GerE

ModelTLS NCS R Rfree ccB

1 0 No 21.9 29.3 0.5192 0 Yes 22.5 30.0 0.5533 6 No 21.3 27.1 0.5104 6 Yes 21.4 27.2 0.816

Contribution to equivalent isotropic Bs

Bs from NCS related chains

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

Selected topic 2: dictionary

The use of prior knowledge requires its organized storage.

$CCP4/html/mon_lib.htmlwww.ysbl.york.ac.uk/~alexei/dictionary.html

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

Monomer library

$CCP4/lib/data/monomers/

ener_lib.cif -definition of atom typesmon_lib_com.cif -definition of links and

modificationsmon_lib_list.html -missing file in version 4.20/,1/,... -definition of various

monomers

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

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

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

What happens when you run REFMAC5

• You have a monomer for which there is a complete descriptionThe program carries on and takes everything from the dictionary

• You have a monomer for which there is only a minimal description or no descriptionThe program tries to generate a complete library

description and then STOPS for you to check it.

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

2Demo

IF YOU WERE NOT IN SAN ANTONIO

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FOR THE NEXT OCCASION

FOR THE NEXT OCCASION

3Future (near and far)

• Fast calculation of complete Hessian matrix• Refinement along internal degrees of freedom

• Refinement using anomalous data• Bayesian refinement of twinned data• Lots more

Future

• 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