Download Refinement

Automated Refinement
(distinct from manual building)
Two TERMS:
Etotal = Edata(wdata)+ Estereochemistry
Edata describes the difference between observed and calculated data.
wdata is a weight chosen to balance the gradients arising from the two
terms.
Estereochemistry comprises empirical information about chemical
interactions between atoms in the model. It is a function of all atomic
positions and
includes information about both covalent and non-bonded interactions.
Edata (R-factor)
Move atoms to minimize the
R-factor. Discrepancy
between Fobs and Fcalc.
positive negative
density density
Specifically, minimize E
E=S w(Fobs-Fcalc)2
Over all hkl. Least squares
refinement.
Atoms shift toward positive
density in a difference Fourier
electron density map.
r(x)=1/V*S|Fobs-Fcalc|e-2pi(hx-fcalc)
Radius of
convergence
is limited
Estereochemistry (Geometry)
–BOND LENGTHS & ANGLES
have standard values. Engh &
Huber dictionary.
- CHIRALITY of a-carbons
–PLANARITY of peptide bonds
and aromatic side chains
–NONBONDED CONTACTS -two
atoms cannot occupy the same
space at the same time
-TORSION ANGLE
PREFERENCES side chains have
preferred rotamers.
–some values of f and y are
forbidden. -Ramachandran. Not
restrained- used for validation.
Jeopardy clue:
The appearance of the atomic model when
stereochemical restraints are not included in
crystallographic refinement.
Etotal =Estereochemistry + wdataEdata
What is spaghetti, Alex?
Importance of supplementing the
Data to Parameter Ratio
in crystallographic refinement.
PARAMETERS
Each atom has 4 parameters
(variables) to refine:
x coordinate
y coordinate
z coordinate
B factor
In proteinase K there are
approximately 2000 atoms to refine.
This corresponds to
2000*4= 8000
variables.
DATA
At 2.5 A resolution we have 8400
observations (data points) (Fobs).
When # of observations= # of variables
A perfect fit can be obtained irrespective of
the accuracy of the model.
At 1.7 A resolution we have 25,000
observations.
About 3 observations
per variable. The reliability of the model is
still questionable.
Adding stereochemical restraints is equivalent to adding observations
2nd Jeopardy clue:
The value of the R-factor resulting when
stereochemical restraints are not included in
crystallographic refinement.
Etotal =Estereochemistry + wdataEdata
What is zero, Alex?
therefore
An atomic model should be validated
by several unbiased indicators
Rfree is an unbiased indicator of the discrepancy between the model and the data.
The data used in this R-factor calculation were not used in determining atomic shifts
in the refinement process.
Ramachandran plot is unbiased because phi and psi torsion angles are not
restrained in the refinement process.
BAD
BACKBONE AMIDE
O
N
H
2.8 Å
H
N
O
H
Asn
GOOD
BACKBONE AMIDE
O
N
H
2.8 Å
H
O
N
H
Asn
ERRAT plot examines the geometric relationship between
non-bonded atoms. Looks at the fraction of non-bonded
contacts with C, N, O as a function of distance.
Verify 3D plot –Gives an indication if the sequence has been improperly
threaded through the backbones. Each of the 20 amino acid types has a
characteristic (1) Surface area buried (2) fraction of side-chain area covered by
polar atoms (3) local secondary structure. Verify 3D plots correlation between
ideal and your model. Compatibility of a model with its sequence.
RuBisCo chain traced backwards
Plan for today
1) The refinement of native proteinase K is assumed to be
complete thanks to ARP/wARP.
2) You will refine the structure of the proteinase K- PMSF
complex using the |Fobs| data measured earlier in the
course.
3) The starting model for the refinement will be the native
proteinase K structure.
4) Begin 5 cycles of automated refinement. This will only
move atoms. It will not add new atoms.
5) Then manually build the PMSF inhibitor into an Fo-Fc
difference Fourier map. Refinement process typically iterates
between automated and manual building. Automated refinement has a
limited radius of convergence. For example- automated refinement cannot
jump between rotamers or flip between cis and trans peptides.
6) Validate structure. Fill out Refinement Statistics table.
Difference Fourier map
r(x)=1/V*S|Fobs-Fcalc|e-2pi(hx-fcalc)
Here, Fobs will correspond to the ProteinaseK-PMSF complex.
Fcalc will correspond to the model of Proteinase K by itself after a few
cycles of automated refinement.
Positive electron density will correspond to features present in the PMSF
Complex that are not in the native structure.
Negative electron density will correspond to features present in the native
structure that should be removed in the inhibitor complex.
After model building, do more automated refinement and then validate.
Cis
O
O
peptide plane
C
Ca
vs.
peptide
Trans
peptide plane
C
N
Ca
Ca
N
Ca
Cis OK with glycine or proline
O
O
peptide plane
C
Ca
peptide plane
N
C
Ca
Ca
N
Ca
Steric hindrance equivalent
for cis or trans.
Steric hindrance equivalent
for cis or trans proline
O
peptide plane
C
O
Ca
peptide plane
Cb
N
C
Cg
Ca
Cd
Cd
Cg
N
Cb
Ca
Ca
.
Name _______________________
Proteinase K –PMSF complex