DNA how did they know

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DNA how did they know?
h
k
34Å
3.4Å
The human DNA molecule contains about THREE
BILLION base pairs
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From spots to electron density map

• Spot intensity I(hkl) ? F(hkl) ? ?(xyz)
• 
  Fourier transform
• ?(xyz) (1/V) ??? F(hkl)cos? - ?(hkl)
• 
  h k l
• 2?(hx ky lz)

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Calculate Fc and new phases

• F(hkl) F(hkl) exp (-i?hkl) A(hkl) iB(hkl)
• ?(hkl) tan 1 B(hkl)/A(hkl)
• where A(hkl) ?fjcos2?(hxj kyj lzj)
• 
  j
• B(hkl) ?fjsin2?(hxj kyj lzj)
  
• 
  j
• For each reflection hkl and summarizing over
  every jth atom
• (with fj atomic diffraction coefficient) in the
  unit cell

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To find our molecules we use F-observedand
calculated phases ? mapcalculated at certain
gridTo improve the model we find atoms in the
map ? better model (? better Fcalc ? better R)
gives better phases ? better electron density
map etc
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Contribution of different reflections to the map
200
110
210
130
120
14 1 0
420
all above
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Contour the map, look for peaks ? x,y,z
X
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Structure solution and refinement
?
Initial phases, Initial model
Build new model
Maps
x,y,z
Fo
Data reduction
x,y,z,Bj, Fc R ( ? Fo Fc ) / ? Fo
hkl hkl
Refine
new phases
Refine
Verify

• Wheremeans
• Heavy atom derivatives
• MAD
• Molecular replacement
• etc

x,y,z,Bj
PDB deposit
Fo
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Na Cl cubic, a 5.64Åspace group Fm3mZ 4
? 4 Na and 4 Cl-
Reflection conditions General hkl hk,
hl, kl 2n 0kl k,l 2n hhl hl
2n h00 h 2n Positions of molecules If 4
then b m3m 1/2, 1/2, 1/2 or 4
a m3m 0, 0, 0
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Model of NaCl and how to
calculate Z
http//itl.chem.ufl.edu/2045/lectures/lec_h.html
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If map calculated with 0.5Å grid
For cubic cell with edge a 5.6Å, and 20
Fo(hkl) that means 11 points if 1D, 121
points if 2D, 1331 points if 3D XYZ To
calculate ? (XYZ) for this unit cell one has to
do summation over 20 observed reflections for
every XYZ point To calculate Fc(hkl) and new
phase for each reflection (hkl) one has to do
summation over all atoms in the unit cell (4 Na
and 4 Cl) using their coordinates xyz (found in
the map)
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for NaCl . Homework 3/29/05 Extra



credit

• Fm3m is centrosymmetric therefore B(hkl) 0
• ( and phases could be only 0 or multiplication of
  ? )
• and F (hkl) A(hkl) ?fjcos2?(hxj kyj
  lzj)
• 
  j
• Na 1 0 0 0, ½ ½ 0, ½ 0 ½, 0 ½ ½
  
• Cl -1 ½ ½ ½, 0 0 ½ , 0 ½
  0, ½ 0 0
• Calculate F(hkl) for reflections hkl 111,
  222, 333, 120
• Calculate discrepancy factor
• R ( ? Fobs Fcalc )/ ? Fobs
• 
  hkl
  hkl

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Remember? fj is a function of ?, calculate
(sin?)/? for each reflection knowing that
n ? 2d sin ?
(use n1)and that for cubic
system 1/d (h2 k2 l2)/a2 so you can
find appropriate fj coefficient (see table below)
(sin? )/? 0 0.1 0.2 0.3 0.4
0.5 0.6 0.7 0.8 0.9 1.0
1.1 Na 1 10 9.551 8.39 6.925 5.51
4.328 3.424 2.771 2.314 2.001 1.785 1.634 Cl 1
18 16.02 12.2 9.4 8.03 7.28
6.64 5.97 5.27 4.61 4.0 3.47
summation is over all atoms (j) in the unit
cell Compare with observed reflections F(111)
16.1 F(222) 64.3 F(333) 11.2 F(120) 0 ?
explain this
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Application of Bioinformatics, Proteomics, and
Genomics BIPG 640 spring 2005(3)
Protein-protein, protein-ligand interactions,
protein modeling and targeted drug design.How
proteins talk to one another, interactions with
small molecules, steps in drug development.

• Ewa Skrzypczak-Jankun, Medical College of Ohio,
  Urology
• Dowling Hall r.2257, 419-383-5414,
  eskrzypczak_at_mco.edu

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How we visualized prothrombinase complex ( 1985)
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Not only active site
Banner, Nature 2005, 404,449
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Crystal packing of Prethrombin Fragment 1
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Different binding sites of thrombin
Heparin binding site
Lys binding site
Active site
Exosite
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Gustafsson et al., Nature Reviews Drug Discovery
(2004), 3 (Aug) ,649-59.
Melagatran in the active site of human - ? -
thrombin (pink in (c ) ) Benzamidine part
inserted into a specificity pocket blocking
Arg receptor site specificity pocket.
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Discussing drug design examples from

• Gustafsson et al., Nature Reviews Drug Discovery
  2004,
• 3(August),
  649-659.
• Bruncko et al., Bioorganic Medicinal Chemistry
  Letters
• 2005, 15,
  93-98.