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