Protein structure validation
Seminar series 2
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
From NMR spectra to structure
B
A
If proton A and proton B are close in space, socalled ‘cross peaks appear’ in a spectrum due to
the Nuclear Overhauser Effect (NOE).
The NOE depends on the distance between
proton A and B
From NMR spectra to structure
B
A
H1
A
A
B
C
..
H2
B
C
D
D
..
Distance(Å)
3
4
2
1
..
The NMR data thus contains distance information!
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.
From NMR spectra to structure
The list of distances can be used in a computer
simulation that is reminiscent of protein folding.
From NMR spectra to structure
.. until the protein is ‘folded’.
From NMR spectra to structure
NMR ‘ensemble’
NMR refinement
Multiple
minima
NMR refinement
Green lines:
Distance OK
Red lines:
Protons too far away
from each other
NMR Q-factor
Error = Σ NOE-violations + Energy term2
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.
More about (protein) crystallography and NMR in:
Kristalstructuur
Magnetische Resonantie
Fourier Analyse
and Structuur, Functie, Bioinformatica, of course
Why validation ?
Why have we 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 connecting 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
Little things hurt big
X-ray specific
His, Asn, Gln ‘flips’
Hydrogen bond network
Your best check:
Contact Probability
Contact Probability
Contact probability box
A positive nitrogen around a Phe
X-ray
How bad is bad?
In a normal distribution, 68%
of the points are within 1
standard deviation of the
mean
In a normal distribution, half of the
points are above and half of the
points are below average
ΔG = -RT ln K
95% are within 2 sd
Less than 1 in 10000 points is more than 4 sd away from the mean
One slide about homology modelling
How difficult can it be?
1CBQ
2.2 A
How difficult can it be?
How difficult can it be?
How difficult can it be?
How difficult can it be?
1CBQ
0.33
1.00
0.33 0.33
0.33
2.2 A
How difficult can it be?
This is what standard viewers show:
1CBQ
2.2 A
Even if the oxygen labels would have been reversed,
the so-called ‘asymmetric unit’ could also have been chosen
in a much better way…
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