MRC software for 2d crystal structure determination Henderson & Unwin (1975) Nature 257, Introduction Basic Functions and Principles

History Developed over 30 yrs by a number of people Developed over 30 yrs by a number of people Written in Fortran Written in Fortran image2010 image2010 image format compatible with ccp4 image format compatible with ccp4 plots in postscript format plots in postscript format

Documentation Crowther, Henderson and Smith (1996) J. Struct. Biol.116, 9-16 Docs in the package: image2010/doc/ Docs on 2dx.org Docs on 2dx.org Help Docs in 2dx applications 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*******************************************************************************

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

Single Image Processing Unbending Boxing MMBOX Fourier Transform

Unbending is possible because distortion can be tracked along the lattices The distortion is plotted at 10x the actual values QUADSERCHK

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

Determine Amplitudes and Phases of Diffraction spots MMBOX / TTBOX + CTFAPPLY h k Amp Phase IQ Background Amos, Henderson & Unwin (1982) Prog. Biophys. molec. Biol., 39,

IQ values determined by amplitudes determined by amplitudes Used as measure of phase error Used as measure of phase error Relationship between Amplitude and Phase Error Re ImIQ Amp/(RMS background) cutoff cutoff Phase Error image2000/source/mmboxa.for IQ~4 IQ~2

Merging information and Refinement Unwin, Scientific America ORIGTILT LATLINE

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

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 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, a=D = D 1 a Stahlberg

LATLINEK Parameters RMIN RMAX RCUT PFACT protein density ALAT = c in 3D xtal lattice parameter DELPRO BINSIZE D=RMAX-RMIN GUESS 1/D DELPLT 1/ALAT AK, IWF, IWP for weighting of amp error vs. phase error of the fit.

Importance of Defining Correct Window Function Width D=260 A D=100 A y=a 0 +a 1 x y=a 0 +a 1 x+a 2 x 2

Avoid mistakes Unbending Unbending Reference should look like perfect crystalline structure Reference should look like perfect crystalline structure Good Bad Exacting Phase/Amplitude Exacting Phase/Amplitude Real signal should become significant when added up Real signal should become significant when added up Merging/Refinement Merging/Refinement Projected symmetry should be obvious in untilted image and its Fourier transform Projected symmetry should be obvious in untilted image and its Fourier transform Choose window function width according to property of the specimen Choose window function width according to property of the specimen 8 IQ8 spot does not make the result as reliable as 1 IQ1 spot 8 IQ8 spot does not make the result as reliable as 1 IQ1 spot 8-6 A A Å5 Å 7 Å7 Å 10 Å

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

Take this home: Computers have changed but the principle is the same. 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. 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). c5015/sc5015bdy.html