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MRC software for 2d crystal structure determination

MRC software for 2d crystal structure determination. Introduction Basic Functions and Principles. Henderson & Unwin (1975) Nature 257, 28-32. History. Developed over 30 yrs by a number of people Written in Fortran image2010 image format compatible with ccp4 plots in postscript format.

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MRC software for 2d crystal structure determination

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  1. MRC software for 2d crystal structure determination • Introduction • Basic Functions and Principles Henderson & Unwin (1975) Nature 257, 28-32

  2. History • Developed over 30 yrs by a number of people • Written in Fortran • image2010 • image format compatible with ccp4 • plots in postscript format http://www.tonh.net/museum/plotter.html

  3. Documentation • Crowther, Henderson and Smith (1996) J. Struct. Biol.116, 9-16 • Docs in the package: image2010/doc/ • Docs on 2dx.org • Help Docs in 2dx applications C LATLINPRESCAL : program to correct image amplitudes for effects of CTF and C to calculate appropriate weights for output to LATLINE. C******************************************************************************** C Control cards: C C Card 1: NSER,ZMIN,ZMAX (*) C serial number on ORIGTILT input C and ZSTAR limits to be passed on to LATLINE. C Card 2: IQMAX (*) C maximum value of IQ for spots to be used C C INPUT file is raw file straight from ORIGTILT : on unit FOR001 C OUTPUT has the format required by LATLINE : on unit FOR003 C C*******************************************************************************

  4. 3D reconstruction from 2D TEM images Unwin, Scientific America

  5. Single Image Processing Unbending MMBOX Boxing Fourier Transform

  6. Unbending is possiblebecause distortion can be tracked along the lattices QUADSERCHK The distortion is plotted at 10x the actual values

  7. Correlation against perfect crystalline structure gives distortion map Reference from rough 3D reconstruction MAKETRAN Cross Correlation Reference from best region in the image

  8. Determine Amplitudes and Phases of Diffraction spots MMBOX / TTBOX + CTFAPPLY Amos, Henderson & Unwin (1982) Prog. Biophys. molec. Biol., 39, 183-231 h k Amp Phase IQ Background 0 1 434.9 34.2 3 156.8 0 2 7924.5 44.6 1 148.5 0 3 1373.7 210.6 1 158.3 0 4 1411.4 228.4 1 124.3 0 5 710.5 206.4 2 106.3 0 6 1870.8 126.0 1 121.1 . . .

  9. Relationship between Amplitude and Phase Error IQ values • determined by amplitudes • Used as measure of phase error Im IQ~2 IQ~4 Re image2000/source/mmboxa.for

  10. Merging information and Refinement ORIGTILT LATLINE Unwin, Scientific America

  11. ORIGTILT program • Move the projection images (i.e., phase origins) to a common center and compatible orientation • Reference • Symmetry special position • Last set of merged image • Last calculated 3D model • Refine • Phase origin • Tilt geometry • Beam Tilt • Standardize a, baxes • Rotation • Flip crystal upside down • Defocus inversion

  12. Lattice line fitting (LATLINEK) • model fitting in the reciprocal space of the continuous line in the non-repeating (z*) direction based on the assumption that the density in the real space is limited by a window function Agard (1983) JMB 167,849-852 a=D D a = 1 Stahlberg

  13. ALAT = c in 3D xtal lattice parameter LATLINEK Parameters RCUT DELPRO PFACT protein density • AK, IWF, IWP for weighting of amp error vs. phase error of the fit. D=RMAX-RMIN RMAX REAL SPACE RMIN BINSIZE FOURIER SPACE 1/ALAT DELPLT 1/D GENERATE CURVE, FIT STATS, and FIT SAMPLE GUESS SAMPLE CURVE for PLOT

  14. y=a0+a1x y=a0+a1x+a2x2 Importance of Defining Correct Window Function Width D=100 A D=260 A

  15. Avoid mistakes Good Bad • Unbending • Reference should look like perfect crystalline structure • Exacting Phase/Amplitude • Real signal should become significant when added up 5-3.5 A 777 7 7 8 7 6 7 7 777 7 6 7 8 7 7 7 777 7 7 8-6 A 789 9 8 7 10 13 11 7 8 8 11 11 8 7 7 10 11 9 7 79 9 8 • Merging/Refinement • Projected symmetry should be obvious in untilted image and its Fourier transform • Choose window function width according to property of the specimen • 8 IQ8 spot does not make the result as reliable as 1 IQ1 spot 5Å 7Å 10Å

  16. What MRC package can not do Make your first crystal Merging results from different crystal form Untwist Rotational disorder in the crystal Correct for bending perpendicular to the membrane Merging information from unknown and variable number layers of stacked crystals ….

  17. Take this home: • Computers have changed but the principle is the same. • Keep a critical eye during the process while enjoying the express ride of 2dx and IPLT. A 1947 or prior photograph (cropped) of IBM (Hollerith) operators at the Caltech Computing Center machine room performing crystallographic computation using the IBM card punch systems as described in Shaffer et al. (1946) and briefly summarized in Swift (1947). http://journals.iucr.org/a/issues/2008/01/00/sc5015/sc5015bdy.html

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