Molecular Modelling in Structure Determination: Structure Calculation From NMR Data

Overview
Molecular modelling in structure

determination
Structure calculation from NMR data 1. Introduction: relating data to structure
2. Hybrid energy and treatment of errors
Michael Nilges 3. Minimisation of hybrid energy
Unit de Bio-Informatique Structurale 4. Relation to probability theory
Institut Pasteur 5. Problems with the minimisation approach: an alternative
nilges@pasteur.fr
6. Automated structure calculation
Michael Nilges. Structure calculation from NMR data.
(1) NMR structure determination steps

3 4
NMR experiment
1. Introduction: relating data to structure

Resonance assignment
3. Minimisation of a hybrid energy
Structural restraints
4. Relation to probability theory distances
5. Problems with the minimisation approach: an alternative NOE assignment
6. Automated structure calculation torsion angles, orientation
Structure calculation
Structure validation
Structure calculation Structural data from NMR experiments

5 6
chemical shifts
Have molecule and some data on conformation...
short distances
Objectives:
internal angles
find conformation(s) satisfying experimental data
maintain likely (local) conformation orientation angles
Michael Nilges. Structure calculation from NMR data. Michael Nilges. Structure calculation from NMR data.
Compare conformation to data Distance information from NOE
7 8
Basis of structure calculation: forward model to

approximately calculate data from a structure N OEij rij (x)6
example for data: X-ray map, NMR data 1
0
example for model: Isolated Spin Pair Approximation for NOE
rij (Ccal N OEij ) 6
calculate measurement (NOE) from structure (the interproton distance)
NOE between two protons depends on distance (< 4 )

ISPA: approximate, power neglects
internal dynamics
molecular conformation NOE peak size
spin diffusion
N OEij rij
6 calibration factor Ccal unknown (not measurable)
estimate from reference distances
Extension for ambiguous NOE Penalty function

9 10
! N
# 16
derive a penalty function to measure difference between
"
r (x) = ra6 (x)
calculated and measured data: Edata
a=1
e.g., harmonic potential for distances (... not a good choice)
16
r0 (Ccal N OE)
N!
N OE
#2
ri (x) ri0
"
EN OE
N : number of possible assignments of peak i=1
Ambiguous NOE can be approximately calculated from a

structure with 6th power law
Angular information: coupling constants The angle

11 12
General dependency of J on angle:

Karplus-relationship
J = A + Bcos() + Ccos(2)
= 60
3-bond coupling depends on torsion angle
Parametrisation of from structures Residual dipolar couplings
13 14
Proteins:
direction of bond vectors (e.g., N-
H) can be determined
The parameters A B C need to be parametrized with known relative to coordinate system
(X-ray) structures attached to molecule
A B C tensor parameters
Typical value for 3JHNH
6.51 -1.76 1.60
6.41 -1.46 1.90 ! "
i j 3
6.98 -1.38 1.72 D res
3 Dax (3 cos () 1) + Drh sin () cos(2)
2 2
rij 2
Data summary (2)

15 16
Theory (forward model) to calculate data from structure

Forward models contain non-measurable parameters that 1. Introduction: relating data to structure
are necessary for the modelling
calibration factor
3. Minimisation of hybrid energy
Karplus parameters for scalar couplings
4. Relation to probability theory
tensor parameters
5. Problems with the minimisation approach: an alternative
Data contain noise
Forward models contain approximations - no ideal
agreement possible
Maintain local conformation NMR structure calculation:

17 18
simplified force field
Use force field Ephys covalent interactions: rigid,

uniform force constants
compilation of ideal values (bonds, angles, ...) ideal values from Engh & Huber
compilation of strength of interaction
probability of distortion vdW interaction: quartic
potential, no attractive part
Often simplifications and approximations of MD force field no electrostatics
Hybrid Energy Hybrid energy function
19 20
{
combine data and physical model of molecule force field
into one function (target function, hybrid energy function)
(Levitt)
force field
Ehybrid = Ephys + wdata Edata

guess wdata
minimise this function data

{
Minimisation of hybrid energy How to use this in structure determination

21 22
make random proposal for structure

repeat until minimum is reached:
1. calculate data (approximately) from structure
All data contain errors (experimental noise)
2. compare with experimental bounds
All forward models contain approximations
3. modify structure to improve agreement
No ideal agreement between calculated and measured data
possible
Pseudopotential including data needs to contain way to
include noise
Standard NOE distance restraint potential Other ways to treat noise

23 24
2
N
!noe (ri (X) Li ) if r(X) < Li
Edata 0 if Li r(X) Ui
2
(ri (X) Ui ) if r(X) > Ui

i
potential form
sources of error weight in hybrid energy function
measurement
spin diffusion, internal dynamics
loose upper and lower bounds
FBWH potential
flat bottom harmonic walls
no force between L and U
How to determine the weights Hybrid energy summary
25 26
{
Data potential needs to include parameter to treat
(unknown) noise
potential shape
force field
weights
The weight in the hybrid energy needs to be set by
{
(empirical) means
cross-validation
data experience
Bayesian analysis
(3) 3D structure calculation

27 28

2. Hybrid energy and treatment of errors convert data (1D, 2D)
3. Minimisation of hybrid energy + forcefield
4. Relation to probability theory into 3D model
Minimisation algorithms Multiple minimum problem

29 30
Energy minimisation
High energy
Simulated annealing barriers
Molecular dynamics to fold protein
Torsion angle dynamics
Standard
(Monte Carlo) minimisation
Distance geometry only "downhill"
Genetic algorithm
...
Distance Geometry Minimisation by molecular dynamics
31 32
r
d 2 ri c
2 = rE
dt mi ri hybrid
Molecular dynamics solves Newton's equations of motion

Avoids "folding problem": Molecular dynamics can overcome local energy barriers
Direct conversion distance to (approximate) coordinates
Mostly historical importance
Newton dynamics Temperature control and variation:

33 34
"MD-simulated annealing"
Direction of motion depends on

force (derived from force field and experimental restraints)
momentum
Energy scaling
35 36
more flexible annealing

schemes
different variation of
different energy terms
equivalence:
r mass/ energy/ temperature
d 2 ri c scaling
= rE
dt 2 mi ri hybrid
Structure calculation with MD Structure calculation with MD
37 38
NMR data: distances 100 atoms:

1988:
Start: random structure 20000 s per structure on
mainframe (DISGEO, Havel)
Difficult search
problem: many now:
degrees of freedom
20 s per structure on PC
Torsion angle dynamics Calculation of structure ensembles

39 40
dynamics time step dictated

by bond stretching: waste with identical data/ restraints:
of CPU repeat calculation (20-100 times)
important motions are random variation of initial
around torsions conditions (starting structure/
velocities)
~ 3 degrees of freedom per poor mans probability
AA (cf 3Natom for Newton distribution
dynamics) obtain information on
uniqueness / different fold
Available in X-PLOR,
CYANA, CNS, X-PLOR-NIH
Structure ensembles (4)

41 42
This has very little to do

with dynamics 1. Introduction: relating data to structure
Distribution depends on 2. Hybrid energy and treatment of errors
data
data representation
algorithm 4. Relation to probability theory
forcefield 5. Problems with the minimisation approach: an alternative
algorithm parameters
you
Probability and energy
43 44
Ehybrid = Ephys (X) + wdata Edata (D, X)
Where do potential forms come from

force field Ephys probability (Boltzmann)
Where do all the parameters come from
bounds probability of distortion of molecule
weights force field: background information I

! "
Ephys (X)
P (X|I) = exp
kT
Probability and energy Likelihood

45 46
Example:
Gaussian distribution of error for r,
similar: Edata probability

standard deviation ,
probability is
probability that data is correct, given structure X:
(r r(X))2
! "
likelihood
P (D|X, ) exp
2 2
Likelihood and restraint potential Distance errors are not Gaussian

47 48
Inversely, if we know probability distribution, we can derive

potential
Edata log [P (D|X, )] Log-normal

distributions
For Gaussian error, harmonic potential (least squares)
1 2
Edata (r r(X))
2 2
The weight is related to the error in the data 1 1 2
LN(x0 , x, ) exp[ (log[x0 ] log[x]) ]
2 2 x0 2 2
Lognormal distribution ...Life is LogNormal
49 50
... there are a lot of data with

only positive values
Gaussian distribution examples on
of logarithms
http://stat.ethz.ch/~stahel/
lognormal/
no theoretical derivation
Gaussian distribution
...why do we still use bounds?
Rieping, Habeck, Nilges, JACS 2005
Joined probability from prior and likelihood 51

Hybrid energy revisited 52
To calculate the joined probability from single probabilities, The hybrid energy function is negative logarithm of joint
multiply: probability
Minimum energy corresponds to maximum probability
Probability of a structure
Relative weight should depend on data quality
Posterior Probability likelihood story is incomplete (what about wdata?)
P (X|D, I) P (X|I)P (D|X, , I)
...
Probability of a structure likelihood
prior distribution P (X|D, I) P (X|I)P (D|X, , I)
prior distribution
Bayesian determination of data weight comparison to X-ray for LogNormal

53 54
Data weight has influence on structure quality GB1, 0.5
Bayesian analysis:
kB T
wdata =
2RM S
BPTI, 0.62
Update iteratively during structure calculation
weight overall data quality IL4, 1.14
only possible for least squares-type potential IL8, 1.44
Habeck M, Rieping W, Nilges M (2006). PNAS 103:1756

Summary (5)
55 56
Minimizing hybrid energy corresponds to maximizing the 1. Introduction: relating data to structure
probability of a structure, given data and force field
...if one knows the data quality, scale factors, ...
Relative weights
usually set by empirically (trial and error, experience, cross validation)
Bayesian determination of weight possible
Relationship of error distribution and restraint potentials 6. Automated structure calculation
The problems ...more problems

57 58
(1) solution is not unique inversion not possible:

(3) Many unknown parameters are necessary
same data calculated from distinct conformations
calibration factors
data are incomplete
parameters for functional forms (e.g. Karplus)
(2) data are inconsistent no solution exists: data quality weights; lower and upper bounds
experimental noise how many structures to calculate/ choose?
approximate theory ... local dynamics?
need to massage data to make things consistent
...some more Example NOE

59 60
incompleteness: assignments, NMR-visibility

inconsistency: approximate theory, noise
(4) Figures of merit for structures
unknown quantities: calibration, data consistency
depend critically on auxiliary parameters:
RMSDs around average structure
fit to bounds derived from data basic question: how well do my data determine the structure
remains unanswered, need of heuristics:
(5) No consistent concepts of data quality evaluation
cross validation
independent validation
Structure by deduction: Inference instead of deduction
61 62
inversion of data inference:

assign a probability to each molecular conformation
use probability theory:
prior probability from physical model (force field)
likelihood from forward model
Insufficient/ conflicting data:

inversion is inappropriate to obtain structure from data posterior prior likelihood
P (, , |D, I) P (, , |I)P (D|, , , I)
Sampling Program ISD

63 64
SH3 (Campbell):
150 NOEs from perdeuterated domain
sparse data set; standard structure calculation does not produce unique
fold
Posterior P(X|D) is extremely complex for realistic problem

too many degrees of freedom to do integration
Take representative samples (Markov Chain Monte Carlo) Rieping, Habeck, Nilges, Science (2005)
(6) Automated structure calculation with ARIA

65 66
First ARIA version in 1995

First completely automated NOESY assignment in 1997
Basis of HADDOCK (Bonvin et al.)
ARIA annealing protocols used in RECOORD
ARIA water refinement used in RECOORD
Current version 2.2
(1.2 still available but not developed)
ARIA2.0 written by M. Habeck, W. Rieping Python, XML
currently developed by Benjamin Bardiaux, Aymeric Bernard

Assignment of ambiguous NOEs Error tolerance: soft potential
54 67 68
finite force for large

violations
important for automated
removal of noise peaks
(ARIA)
same as violation
confinement in CYANA
Network anchoring
similar effect as restraint
Ambiguous distance restraints (ADRs) combination
Iterative assignment rules
Experimental terms Final refinement in H2O

69 70
All that is implemented in CNS:

Fixed distance restraints (e.g., hbonds) Improvement of:
NOE-derived distance restraints validation results
Torsion angles
Q-factors
Scalar coupling constants
RDCs X-ray molecular
Relaxation anisotropy (1.2) replacement
Support for solid state (SOLARIA)
soon: SAXS,
Ubiquitin Q-factors Current release: ARIA2.2

71 72
no water with water

WWW site: http://aria.pasteur.fr Registration form to download
73 74
Restraint violation in ARIA Validation results from WhatIf in ARIA

75 76
77
Calculation of symmetric multimeric complexes
Symmetry restraint method to calculate

dimeric proteins
without specification of symmetry axis
successful for simple topologies
Full integration in ARIA2.2
reports in GUI

Molecular Modelling in Structure Determination: Structure Calculation From NMR Data

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Overview

Molecular modelling in structure

Michael Nilges. Structure calculation from NMR data.

(1) NMR structure determination steps

1. Introduction: relating data to structure

Structure calculation Structural data from NMR experiments

Basis of structure calculation: forward model to

NOE between two protons depends on distance (< 4 )

Extension for ambiguous NOE Penalty function

Ambiguous NOE can be approximately calculated from a

Angular information: coupling constants The angle

General dependency of J on angle:

Data summary (2)

Theory (forward model) to calculate data from structure

Maintain local conformation NMR structure calculation:

simplified force field

Use force field Ephys covalent interactions: rigid,

Often simplifications and approximations of MD force field no electrostatics

minimise this function data

Minimisation of hybrid energy How to use this in structure determination

make random proposal for structure

Michael Nilges. Structure calculation from NMR data.

Standard NOE distance restraint potential Other ways to treat noise

(3) 3D structure calculation

1. Introduction: relating data to structure

Minimisation algorithms Multiple minimum problem

Molecular dynamics solves Newton's equations of motion

Newton dynamics Temperature control and variation:

Direction of motion depends on

more flexible annealing

NMR data: distances 100 atoms:

Torsion angle dynamics Calculation of structure ensembles

dynamics time step dictated

Michael Nilges. Structure calculation from NMR data.

Structure ensembles (4)

This has very little to do

Ehybrid = Ephys (X) + wdata Edata (D, X)

Where do potential forms come from

Probability and energy Likelihood

similar: Edata probability

Likelihood and restraint potential Distance errors are not Gaussian

Inversely, if we know probability distribution, we can derive

Edata log [P (D|X, )] Log-normal

... there are a lot of data with

...why do we still use bounds?

Rieping, Habeck, Nilges, JACS 2005

Joined probability from prior and likelihood 51

Ehybrid = Ephys (X) + wdata Edata (D, X)

Bayesian determination of data weight comparison to X-ray for LogNormal

Ehybrid = Ephys + wdata Edata

Data weight has influence on structure quality GB1, 0.5

Habeck M, Rieping W, Nilges M (2006). PNAS 103:1756

The problems ...more problems

(1) solution is not unique inversion not possible:

...some more Example NOE

incompleteness: assignments, NMR-visibility

inversion of data inference:

Insufficient/ conflicting data:

P (, , |D, I) P (, , |I)P (D|, , , I)

Sampling Program ISD

Posterior P(X|D) is extremely complex for realistic problem

(6) Automated structure calculation with ARIA