Not just a pretty picture

1. Not just a pretty picture 2011. Jul. 14 Hyunwook Lee

2. 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?

3. 105 softwares! http://molvis.sdsc.edu/visres/molvisfw/titles.jsp#C

5. 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.

6. EV71(+Fab) 2.0 1.0 0.5

7. CVB3-CAR 1.0 0.5 2.0

8. How to show them? • We should present the structure in a way which makes people can see what we see.

9. Human Rhinovirus 14 & Fab Hansong Liu et.al (1994) JMB

10. Different color for chemically distinct groups Rotavirus & Fab Prasad et al. 1990 Nature

11. Cut-open views T. maritima nanocompartment http://schaechter.asmblog.org/schaechter/2011/04/beyond-the-bacterial-microcompartment.html

12. Radially cut surface : Simian virus 40 TS Baker. 1994 PNAS

13. Icosahedrally cut surface Infectious Bursal Disease Virus B. Bottcher et al. 1997 J of Virol

14. Polar sections Semliki Forest Virus =  60r = 356, 296, 288, 272, 216, 196 ASD Fuller et al. 1995 Cell

15. Radial-Depth Cueing Reovirus S.M. Spencer et al., 1997 J of Struc. Biol.

16. Bluetongue Virus J.M.Grimes et al. 1997 Structure

18. 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.

19. Tips : Two files in the same folder & Try to move the folder location

20. 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

21. HRV14 with Fab

22. General outline • Absolute magnification of the reconstruction • 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

23. 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 • Assessment of the quality and uniqueness of the fit • R-factor : a measure of the agreement between two maps • Refinement in reciprocal space and in real space by objective method

24. 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

25. Refinement of the E. coli Ribosome in Its Initiation-like State with RSRef Real-Space Refinement Gao et al. 2003 Cell

26. Refinement of the Myosin 10S Complex with RSRef Real-Space Refinement Liu et al. 2003 JMB

27. 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