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Seminar series 2
Protein structure validation
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 1 N LEU 1 -15.159 11.595 27.068 1.00 18.46ATOM 2 CA LEU 1 -14.294 10.672 26.323 1.00 9.92ATOM 3 C LEU 1 -14.694 9.210 26.499 1.00 12.20ATOM 4 O LEU 1 -14.350 8.577 27.502 1.00 13.43ATOM 5 CB LEU 1 -12.829 10.836 26.772 1.00 13.48ATOM 6 CG LEU 1 -11.745 10.348 25.834 1.00 15.93ATOM 7 CD1 LEU 1 -11.895 11.027 24.495 1.00 13.12ATOM 8 CD2 LEU 1 -10.378 10.636 26.402 1.00 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.
Multiple minima
X-ray refinement / multiple minima
X-ray R-factor
Error = Σ w.(obs-calc)2
R-factor = Σ w.|obs-calc|
X-ray resolution
NMR data collection
From NMR spectra to structure
A
B
If proton A and proton B are close in space, so-called ‘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
A
B
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.
H1 H2 Distance(Å)A B 3A C 4B D 2C D 1.. .. ..
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’
Multiple minima
NMR refinement
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
NMR X-ray‘Error’ 1-2 Å 0.1-0.5 ÅMobility yes not reallyCrystal artefacts no yesMaterial needed 20 mg 1 mgCost of hardware 4 M Euro near infinite (share)Drug design no almost
Better combine and use the best of both worlds.
More about (protein) crystallography and NMR in:
KristalstructuurMagnetische ResonantieFourier 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 5RXN 1.2 B 7GPB 2.9 C 1DLP 3.3 D 1BIW 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
How bad is bad?
X-ray
In a normal distribution, half of the points are above and half of the points are below average
In a normal distribution, 68% of the points are within 1 standard deviation of the mean
Less than 1 in 10000 points is more than 4 sd away from the mean
95% are within 2 sd
ΔG = -RT ln K
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
2.2 A0.33
1.000.33
0.33
0.33
How difficult can it be?
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…
This is what standard viewers show:
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:
Elmar Krieger Sander Nabuurs Chris SpronkMaarten Hekkelman
Rob Hooft
Robbie Joosten
