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