# Macromolecular refinement with REFMAC5 and SKETCHER of the CCP4 suite by sdfwerte

VIEWS: 10 PAGES: 42

• pg 1
```									Macromolecular refinement
with
REFMAC5 and SKETCHER
of the
CCP4 suite

Roberto A. Steiner – University of York
Organization

1
General aspects of refinement and overview of
REFMAC5
•   TLS
•   Dictionary

2
Demo
•   TLS refinement in REFMAC5
•   SKETCHER

3
Future
1
General aspects of refinement
and
overview of REFMAC5
A common problem in physical sciences

Given

•   Set experimental values of quantity q (qE,E)
RC
•   Model M(aI,bI,cI)  qI

Estimate

•   Best model, i.e. M(aB,bB,cB) which is most consistent with
the data
•   The accuracy of (aB,bB,cB)
Model fitting

Generation of additional data

Experiment   Mathematical model                        Inference

Analysis
Model fitting in crystallography

experimental (I,I )  (F, F)

model (heavy atoms, protein, ..)
R

FC

Best model
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.Jaynes
http://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)=0.005179
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.
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 crystal
UTLS       : TLS motions of pseudo-rigidy bodies
Uint       : collective torsional librations or
internal normal modes
Ucryst : 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 + 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
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
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       25.9
4                21.1       25.8
4/NCS            22.0       25.7
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
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

Model TLS    NCS     R       Rfree   ccB
1      0     No      21.929.30.519
2      0     Yes 22.530.00.553
3      6     No      21.327.10.510
4      6     Yes 21.427.20.816
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.html
www.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 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
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 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.
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
2
Demo
3
Future (near and far)
Future

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

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

```
To top