Not just a pretty picture
2011. Jul. 14
Hyunwook Lee
Visualization and Representation
• Which program do you want to use?
• At which density (contour) level are you
going to present your structure?
• How will you show people what you
see?
105 softwares!
http://molvis.sdsc.edu/visres/molvisfw/titles.jsp#C
Density (contour) level
• Volume is very sensitive to small
changes in contour level, which in turn
is sensitive to scaling and CTF
correction.
• As a guide, workers typically need to
contour at about 120% of the expected
volume in order to obtain a surface that
makes biological sense.
EV71(+Fab)
0.5
1.0
2.0
CVB3-CAR
0.5
1.0
2.0
How to show them?
• We should present the structure in a
way which makes people can see what
we see.
Human Rhinovirus 14 & Fab
QuickTime™ and a
decompressor
are needed to see this picture.
Hansong Liu et.al (1994) JMB
Different color for chemically distinct groups
Rotavirus & Fab
QuickTime™ and a
decompressor
are needed to see this picture.
Prasad et al. 1990 Nature
Cut-open views
T. maritima nanocompartment
http://schaechter.asmblog.org/schaechter/2011/04/beyond-the-bacterial-microcompartment.html
Radially cut surface : Simian
virus 40
QuickTime™ and a
decompressor
are needed to see this picture.
TS Baker. 1994 PNAS
Icosahedrally cut surface
Infectious Bursal Disease Virus
QuickTime™ and a
decompressor
are needed to see this picture.
B. Bottcher et al.
1997 J of Virol
Polar sections
Semliki Forest Virus
 =  60
r = 356, 296, 288, 272,
216, 196 A
SD Fuller et al. 1995 Cell
QuickTime™ and a
decompressor
are needed to see this picture.
Radial-Depth
Cueing
Reovirus
S.M. Spencer et al.,
1997
J of Struc. Biol.
QuickTime™ and a
decompressor
are needed to see this picture.
Bluetongue
Virus
QuickTime™ and a
decompressor
are needed to see this picture.
J.M.Grimes et al. 1997 Structure
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
Reliability of Difference Imaging
• Two maps must be calculated to the same
resolution and scaled in such a way that the
differences are minimized.
• Double-check with another difference-map
from independent reconstructions for same
structure.
• Results around symmetry axes and low
radius region can mislead your interpretation.
Tips :
Two files in the same folder &
Try to move the folder location
Modeling and Comparison with
X-Ray Structures
Constrained fitting of an atomic model to low-resolution
electron microscopic images can yield “pseudo-atomic
precision” in which model atoms could, it was proposed, be
placed with an accuracy of 4- to 5-fold better than the
nominal experimental resolution, i.e. 4 Å detail could be
interpreted from a map at 20 Å resolution.
F. Fabiola et al. 2005 Structure
are needed to see this picture.
decompressor
QuickTime™ and a
HRV14 with Fab
QuickTime™ and a
decompressor
are needed to see this picture.
General outline
1.
Absolute magnification of the reconstruction
•
2.
accurate pixel size, comparison with X-ray
Matching variation in density through the
reconstruction to that in the X-ray structure
•
•
•
•
Convolve x-ray structure with CTF and match resolution
If possible, mask out extra part of EM reconstruction and
adjust two maps by comparing Fourier transforms of the
projections of those maps
Normalize EM map for positive and same range of density
values as the corresponding X-ray map
Maximum-entropy approach can be used for the treatment of
CTF effects
3. Interactive fit between the EM density and
the X-ray structure
•
•
Quality of the fit : hand of the structure
Single rigid body vs multiple domains
4. Assessment of the quality and uniqueness
of the fit
•
R-factor : a measure of the agreement between
two maps
5. Refinement in reciprocal space and in real
space by objective method
Popular search and refinement methods
• Global search for initial configuration
– SITUS, COAN, and DOCKEM
• Final refinement
– URO, NMFF-EM, and RSRef
• Methods bridging between search and refinement
– EMFIT, SITUS, and CHARMM
Refinement of the E. coli Ribosome in Its Initiation-like State
with RSRef Real-Space Refinement
QuickTime™ and a
decompressor
are needed to see this picture.
Gao et al. 2003 Cell
Refinement of the Myosin 10S Complex
with RSRef Real-Space Refinement
QuickTime™ and a
decompressor
are needed to see this picture.
Liu et al. 2003 JMB
Phasing of X-Ray Data with EM Data
• The Phase Problem of X-ray crystallography
• Classic technique to solve the Phase Problem
– MIR, multiple isomorphous replacement
– Useless if the crystal is not isomorphous
• EM map can be used to help solve the phase
problem by applying molecular replacement
– Similar molecule's phases are grafted onto the
intensities which are experimentally determined