Download NMR structure validation

Protein structure validation
ECCB 2020 Gent
Introduction to protein structure validation
(and improvement)
Gert Vriend
Protein structure validation
The plan for today:
Gert Vriend:
Robbie Joosten:
Jurgen Doreleijers:
Bas Vroling (and you):
All (and you):
Split-up in groups:
At the end:
Introduction to validation
X-ray structure validation and improvement
NMR structure validation (and improvement )
YASARA
General validation practicals
General validation issues
X-ray specific issues
NMR specific issues
Continuation of validation practicals
Overview of validation and related facilities
And in-between we have coffee, lunch, tea, and whatever else they
throw at us at any moment that anybody feels like it.
Structure validation
Everything that can go wrong, will go wrong, especially with
things as complicated as protein structures.
What is real?
What is real?
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
1
2
3
4
5
6
7
8
N
CA
C
O
CB
CG
CD1
CD2
LEU
LEU
LEU
LEU
LEU
LEU
LEU
LEU
1
1
1
1
1
1
1
1
-15.159
-14.294
-14.694
-14.350
-12.829
-11.745
-11.895
-10.378
11.595
10.672
9.210
8.577
10.836
10.348
11.027
10.636
27.068
26.323
26.499
27.502
26.772
25.834
24.495
26.402
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
18.46
9.92
12.20
13.43
13.48
15.93
13.12
15.12
X-ray
X-ray
X-ray
‘FFT-inv’
FFT-inv
And now move the atoms
around till the calculated
reflections best match the
observed ones.
X-ray refinement / multiple minima
Multiple
minima
X-ray R-factor
Error = Σ w.(obs-calc)
R-factor = Σ w.|obs-calc|
2
X-ray resolution
NMR data collection
NMR data
NMR data consists of short inter-atomic distances
between atoms. We call these NOEs.
Most NOEs are between close neighbours in the
sequence. Those hold little information.
The ‘good’ NOEs are between atoms far away in
the sequence. There are few of those, normally.
NOEs are known with low precision. E.g. NOEs
are binned 2.5-4.0, 4.0-5.5, and 5.5-7.0.
NMR can also measure some angles, and relative
orientations. The latter, called RDCs are powerful.
NMR Q-factor
Error = Σ NOE/RDC-violations + Energy term
2
NMR versus X-ray
With X-ray you measure reflections. Each reflection holds information
about each atom.
With NMR you measure pair-wise distances, angles, and orientations.
These all hold local information.
X-ray requires crystals, and crystals cause/are artefacts.
NMR is in solution, but provides much less precision.
NMR versus X-ray
‘Error’
Mobility
Crystal artefacts
Material needed
Cost of hardware
Drug design
NMR
1-2 Å
yes
no
20 mg
4 M Euro
no
X-ray
0.1-0.5 Å
not really
yes
1 mg
near infinite (share)
almost
Better combine and use the best of both worlds.
Why validation ?
Why does a sane (?) human being spend
twenty years to search for millions of errors in
the PDB?
Validation because:
Everything we know about proteins comes from
PDB files.
Errors become less dangerous when you know
about them.
And, going back to the red thread through this
series, if a template is wrong the model will be
wrong.
What kind of errors can the software find?
Administrative errors.
Crystal-specific errors.
NMR-specific errors.
Really wrong things.
Improbable things.
Things worth looking at.
Ad hoc things.
Smile or cry?
A
B
C
D
5RXN
7GPB
1DLP
1BIW
1.2
2.9
3.3
2.5
Why? Simple, proteins are very complex.
X-ray specific
Little things hurt big
X-ray
How bad is bad?
Check with force fields
A force field is a set of parameters together with a set of rules to
use those parameters to see how normal something is. Most
force fields are designed to score events, or to predict the future.
In structure validation we often look at structures and count
‘things’ For example, we count that the number of buried
hydrogen bond donors that do not make a hydrogen bond is
4.6+/-1.2 per 100 amino acids in well-solved proteins. So we call
that normal, and now, using ΔG=-RTln(K), we can calculate the
energy penalty for proteins with more than 4.6 unsatisfied buried
unsatisfied hydrogen bonds.
Contact Probability
Contact Probability
Contact probability box
One slide about homology modelling
His, Asn, Gln ‘flips’
Hydrogen bond network
Your best check:
How difficult can it be?
1CBQ
2.2 A
How difficult can it be?
Errors or discoveries?
Buried histidine.
Warning for buried
histidine triggered
biochemical follow -up
and new mechanism for
KH-module of Vigilin.
(A. Pastore, 1VIG).
Acknowledgements:
Rob Hooft
Robbie Joosten
Elmar Krieger
Sander Nabuurs
Chris Spronk
Maarten Hekkelman