Sidechains

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Sidechains
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A protein without sidechains would not fold,
would not function
Poly-G has no structure. Unique 3D structures are
the result of (1) hydrophobic effect, (2)
H-bonds, (3) geometric contraints. Geometric
contraints describes the effect of covalent
geometry on the conformation.
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Geometric constraints on the backbone
C
C
N
CA
f
CA
CA
Ramachandran plot. Some combinations of angles
are geometrically impossible.
C
N
w
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PROCHECK Ramachandran Plot
Out of bounds residues. Possible errors in the
model.
Note modifications, especially in positive phi
region.
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Sidechain geometric constraints
Sidechain angles Atoms c1 C-CA-CB-xG c2 CA-CB-C
G-xD c3 CB-CG-CD-xE c4 CG-CD-CE-xZ
(If there is more than one xGn, use xG1.)
A rotamer is a set of typical chi angles.
Some combinations of chi angles are impossible.
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Reminder positive torsion angle is right-handed
Newman projection
This is a 45 angle. Place thumb of right hand
along bond. Fingers now point in the positive
direction. 0 is defined as eclipsed (e.g. N
over N)
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1-4 interactions define rotamers
Subtle energetics define preferred rotamers
1-4 interactions are interactions (van der Waals
attraction/repulsion mostly) between atoms
separated by exactly 3 bonds, when the middle one
can rotate.
Some conformations that are possible are more
preferred than others. Why?
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Extracting energetic quantities from the database

• High energy angles should occur rarely.
• Low energy angles should occur frequently.
• Counting the occurence of rotamers tells us their
  energyif we view protein structures as a
  representative sample of all rotamers.

DG -RT log Keq DG -RT log (P(x)/P(y))
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Energies of chi2 angles depend on chi1 angles.
r1
c1 rotamers
r2
c2 energy
r3
Different sidechains have different energy
profiles.
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In class exercise rotamers of Phe
Using Rasmol, load a molecule of your choice
(your project protein?) restrict phe wireframe
80 stereo -7 Measure the chi1 and chi2 angles by
eye. Write them down. Note 180 c1, c2
180 What chi1,chi2 rotamers are most common?
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Subtleties about deriving energies from data...
The PDB database is not evenly
sampled. Solution non-redundant database (1
representative from each sequence family.
http//www.cmbi.kun.nl/gv/pdbsel/) Systematic
dependence. Covariance. Solution conditional
probabilities (Dunbracks solution) Lower
probability, larger the error bars. Solution ??
high energy is not well defined.
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Interaction of the sidechain with the mainchain
Preferred rotamers are backbone dependent.
Dunbracks rotamer library is broken down into
10 bins in f and y. c angle probabilities are
conditional on f and y
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How a rotamer library is used

• Procheck find sidechains that might be wrong.
• Adding sidechains to a backbone structure.
• Predicting protein structure, using homology.

• Dead-end elimination algorithm
• Select two sidechains that are touching. Freeze
  all other atoms.
• Try every pair of rotamers. Calculate the energy.
• If a rotamer is never lower in energy, eliminate
  that rotamer from the list.
• Freeze those two. Choose another pair. Cycle
  until no more rotamers can be eliminated.

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Something about valine...
A valine sidechain might be off by 180 in chi1
if the B-factors for the sidechain are high.
This also happens with Ile, Thr, Leu (chi2).
O
C
CA
N
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In class exercise find bad geometry.

• Run validation on your project protein. Read
  procheck.out
• Identify the filenames containing your results.
• Display the postscript files (xxxx_nn.ps) using
  showps
• Identify the outliers.
• Find the outliers in Rasmol. Are they probably
  right? probably wrong? (consider the resolution,
  B-factors, restraints) Are they in an important
  region of the molecule?

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A sentence for your term paper
PROCHECK analysis of molecule name found that
no, 1,2,3.., many residues had backbone angles
in the Ramachandran excluded regions. None, 1,
2, .. All of these occur in the active site,
interface, binding site,... and this may
therefore cast doubt on, re-affirm the quality
of the model in this region. Rare sidechain
rotamers are seen in no,1,2,..many cases, and
this too leads us to believe that...
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Topics of discussion for your term paper
Xtallization conditions. Data collection
(wavelength, method, resolution, completeness).
Phasing (heavy atoms? seleno-Met? molecular
replacement?). Refinement (data/params ratio,
restraints, waters, free R). Errors in the
model according to PROCHECK (validation). What
was discovered by solving this xtal structure?
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Suggestions for term paper
Make a TOPS diagram. Find your SCOP superfamily.
Is function conserved? Use CE to superimpose two
structures. Compare them.