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Title: Application of Bioinformatics, Proteomics, and Genomics BIPG 640 spring 2006 2Xray analysis of prote


1
Application of Bioinformatics, Proteomics, and
GenomicsBIPG 640 spring 2006(2)X-ray analysis
of proteins and enzymesProtein ? crystal ? data
? interpretation, evaluation and results ? 3D
structure.
Ewa Skrzypczak-Jankun, MUO, Urology, DH 2257,
x5414 eskrzypczak_at_meduohio.edu
2
Crystals big (small molecules) and small (big
molecules)
Potassium dihydrogenn phosphate from Lawrence
Livermore Lab grown for National Ignition
Facility to help maintain safety. Plates cut
from these crystal should convert lasers light
into VIS before beam strikes the laser target.
Protein crystals 0.? mm
3
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4
PDB statistic
a 1MBN myoglobin b 2DHB hemoglobin c 2LYZ
lysozyme d 4TNA 6TNA transfer RNA e 1FC1
1MCP antibodies f 2STV virus g 1ATN actin h
1AOI nucleosome i 1DFK myosin j 1FFK 1FKA
1J5E ribosomal subunits k 1IWO calcium
pump
5
Journals
Some journals reporting X-ray structures are not
listed by PubMed For journals of Acta
Crystallographica look at http//www.iucr.org
This site has many useful information
including job oppenings
6
X-ray analysis flow chart of action
Operation Information obtained Time
frame   Crystallization Crystal
morphology hours ? years Solubility,
storage, dm, etc. Crystal selection hours?mon
ths Preliminary data Unit cell
a,b,c,?,?,? Space group, Z, dc, molecular
symmetry Measure intensity data list of h,k,l,
I, ?(I) hours?days Data reductions Solve the
structure Preliminary model of the
crystallographic minutes?months, molecule
years? Complete the
structure Find all atoms (if possible) minutes?mo
nths years? Refine the structure
model x,y,z,B, R-factor minutes?months 3D
molecular architecture,
years? i.e. configuration and
geometry Interpret the results Correlation with
physicochemical and ???? biological data,
explanation of mechanism, etc. Deposition of
results All crystallographic results go to the
public database weeks Publish months
7
Modern, necessary resources
Crystallization ? robots for screening
thousands of conditions X-ray source ?
rotating anode 100mA 50kV or
synchrotron radiation Collimation ? focusing
mirrors Detection ? area detectors Data
reduction ? software written for modern
instrumentation
and techniques Calculations ? software working
under Linux, Unix or
Windows, utilizing parallel processing and
vectorization,
virtual machines Graphical representation ?
3D display, simultaneous manipulation
of many objects (maps,
models, etc), tracking
changes and performing calculations on a
fly Databases ? sequences, structures,
references, etc.
8
Hampton Research company specializing in
crystallographic supplies
9
Crystallization screen factorials probing
different conditions
10
Robotic crystallization
Honeybee System www.genomicsolutions.com 20 s to
fill 1536 wells with 500nL
SYRRX, Inc quick screening 400/plate/386
conditions www.nvxtal.com
11
Methods of protein crystallization
Hanging drop
Sandwich drop
or sitting drop
Dialysis via gel
Drop under oil
Microdialysis
12
Cryo technique
  • Find proper cryoprotectant
  • Try to introduce cryoprotectant into the
    crystallization
  • protocol to avoid extra steps
  • Dont blow your crystal away when putting into
    cryostream
  • Check diffraction any extra rings? Spots from
    ice?
  • Does the liquid in the loop forms glassy
    material?
  • Is crystal still transparent? Any cracks?

13
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14
DNA how did they know?
h
k
34Å
3.4Å
The human DNA molecule contains about THREE
BILLION base pairs
15
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16
From spots to electron density map
  • Spot intensity I(hkl) ? Fo(hkl) ? ?(xyz)

  • Fourier transform
  • ?(xyz) (1/V) ??? F(hkl)cos? - ?(hkl)

  • h k l
  • 2?(hx ky lz)

17
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

18
hkl Miller indices
n? 2dsin? ? 2?(hx ky lz)
19
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
20
Contribution of different reflections to the map
hkl 200
110
210
130
120
14 1 0
420
S over all above
21
Contour the map, look for peaks ? x,y,z
X
22
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
23
Area detector (PROTEUM from Bruker)
24
Making every X-ray photon count ? CCD
Phosphors are designed for a given ?
Protein crystallography requires very high CCD
image sensor performance. Improvements in the
quantum efficiency, dynamic range and noise floor
of CCDs are helping to increase system accuracy
and throughput so that researchers can more
quickly unlock the secrets of human physiology.
Biophonics International Feb 2005
25
Area detector imaging plate (Rigaku)
  • Exposure ? readout
  • erasing
  • Dual plates speed the process

26
Confocal focusing mirrors (Osmic, Inc)
27
Data processing? results
h k l Fo ?
0 0 18 37080.2 2278.1 0 0 19 7993.2
503.8 0 0 20 5868.4 383.9 0 0 21
6665.3 556.6 0 0 22 422.8 131.4 0
0 23 178.6 177.2 0 0 24 4591.4
528.9 0 0 25 1205.2 224.7 0 0 26
2849.8 556.3 0 0 27 1700.6
343.1/.../ 55 9 -3 178.3 204.1 55 11
-6 12.4 127.1 55 11 -5 -106.8 173.7
55 11 -4 191.3 185.7 55 11 -3 78.4
270.4 55 11 0 301.9 225.1 55 13 -3
-23.3 188.4 55 13 -2 254.5 213.9 56
0 -4 -95.4 196.2 56 0 -3 534.3
292.4 
Soy lipoxygenase-3 97 kDa a112, b 137, c 61Å,
?96º Space group C2, Z 4 0 lt h lt 56 0 lt k lt
67 -30 lt l lt30 107,634 measured
data 41,035 unique reflections
within 40 2.0Å resolution i.e. I had 41,035
equations for 7249x4 28,996
variables
28
Map only
  • Map only ?
  • knowing the sequence
  • and looking at the shape of
  • the electron density
  • one can build a protein
  • Starting with
  • main chain

29
Add side chains according to sequence and shape
of density
30
Add side chains according to sequence and shape
of density
31
Map (2Fo Fc)
32
Model overall
details
33
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
  • Wheremeans
  • Heavy atom derivatives
  • MAD
  • Molecular replacement

Verify
x,y,z,Bj
PDB deposit
Fo
34
Homework due 3/23/06
  • Chose an entry from the Protein Data Bank 1VZQ
    and retrieve information about this structure
    (? www.pdb.org )
  • Describe
  • Provide reference to publication.
  • Method of crystallization?
  • How data were collected, to what resolution, on
    what instrument,
  • what radiation?
  • Describe the molecule (classification of
    structure and biological
  • function) and provide a drawing to illustrate
    its features
  • List hydrophilic and hydrophobic interactions of
    SHY inhibitor

35
Materials utilized in the lectures
The Collected Works of Dorothy Crowfoot Hodgkin.
Ed. GG Dodson, JP Glusker, S Ramaseshan, K
Venkatesan. Indian Academy of Sciences1994 Crystal
Structure Analysis for Chemists and Biologists.
JP Glusker, M Lewis, M Rossi Wiley-VCH
1994 International Union of Crystallography
Newsletters American Crystallographic Association
Newsletters PDB Annual Report 2003 Crystallizatio
n Research Tools, Hampton Research CA Different
publicly available web resources Authors own
roentgenograms, photographs and results.
Laboratory real crystallization robot and X-ray
detectors when Friday 3/24/06, 2
330 pm where at the University of Toledo, at
Bancroft campus Instrumentation Center at
Bowman-Oddy (basement) meet me in front of
r.227
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