Not just a pretty picture
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Transcript of Not just a pretty picture
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?
http://molvis.sdsc.edu/visres/molvisfw/titles.jsp#C
105 softwares!
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
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Hansong Liu et.al (1994) JMB
Different color for chemically distinct groups Rotavirus & Fab
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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
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TS Baker. 1994 PNAS
Icosahedrally cut surface Infectious Bursal Disease Virus
B. Bottcher et al. 1997 J of Virol
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Polar sections Semliki Forest Virus
= 60r = 356, 296, 288, 272,
216, 196 A
SD Fuller et al. 1995 Cell
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Radial-Depth Cueing
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Reovirus
S.M. Spencer et al.,1997J of Struc. Biol.
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J.M.Grimes et al. 1997 Structure
Bluetongue Virus
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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
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HRV14 with Fab
General outline
1. Absolute magnification of the reconstruction• accurate pixel size, comparison with X-ray
2. 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
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Refinement of the E. coli Ribosome in Its Initiation-like State with RSRef Real-Space Refinement
Gao et al. 2003 Cell
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Refinement of the Myosin 10S Complex with RSRef Real-Space Refinement
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