EMBL Virus Structure Resource EMBL Virus Structure Resource
This server provides images of virus structures determined by our
group
in the Structural Biology Programme at EMBL in Heidelberg. It also includes documentation for a number
of the program packages used in for the determination of icosahedral structures and the processing of
cryo-electron micrographs.
The icosahedral programs are based on the method of common lines introduced by R.A.Crowther (1971- Phil Trans Royal Soc. London B261 : 221-230). The set of programs used here is a modification of these to incorporate features necessary for dealing with the lower contrast images obtained by cryoelectron microscopy (Fuller - 1987 Cell 48:221-230). In particular, the original set of programs was based on the premise that the user would determine by eye the orientation of the particles used for reconstruction, whereas the new programs use the common lines residual to find the orientations and origins. Other added features include the ability to compare and refine images against each other and the inclusion of scaling to compensate for the transfer function. An important improvement is the addition of the pft method for model based orientation search.
A more complete description of the theoretical details of the reconstruction process with links to descriptions of the individual programs.
Pick out "good" particles (ie particles which are relatively homogeneous in size, show now obvious defects, have no dirt or debris blocking the image, etc...) from scanned negatives with an appropriate box size. Subtract background ramp. Center the particle by cross- correlation. mask each particle with a circular mask.
(If necessary, convert image files to MRC format.) Pad images to appropriate size for Fourier Transformation (usually 256X256 or 512X512) with BOXIM. Fourier Transformation is done with FFTRANS. All subsequent processing steps are done with on Fourier transforms.
A first run of EMICOFV (FV - findview) can now be made. All orientations (theta, phi, omega) in the asymmetric unit are searched in one degree steps for the lowest common-lines residual after weighting for degeneracy. The program computes various residuals and outputs the ten best orientations (those giving lowest residuals) as well as a residual map for each. By inspection of these, pick the best orientation which is not affected by artefacts such as degeneracy.
EMICOORG1 minimizes the common-lines residual to determine the phase origin (X,Y) for the particle in the orientation (theta, phi, omega) given by FV.
After this, it is necessary to feed the new origin back into FV, and search again for the best orientation and repeat this process until (hopefully ) the process converges on the best orientation. The orientation and origin searches are sensitive to the input parameters, i.e. resolution limits for search (MINR, MAXR), and cutoff level (FMIN), which may have to be experimented with.
To check the orientations, single-particle reconstructions can be made, in which case the same procedure as in point 7 below is followed.
SIMPLEX takes a set of particles and refines them against each other by finding the set of orientations and/or origins that give the best cross-common lines correspondence between the particles. It can be run in either gradient or simplex mode. Gradient refinement is used for most runs, simplex mode is used only for the "final touch". Theta, phi, omega, x and y can be refined separately or in any combination. The "flip" option will also allow handedness refinement to determine which combination of hands that gives the best residual.
Simplex is run in two ways: 1) Refining a set of particles against each other, and 2) Refining a set of higher resolution images by comparing to a set of low resolution images. In the latter case, only the orientations of the high resolution set are made to move, keeping the low resolution orientations (Which have been previously refined together) fixed; only the overlapping resolution range is used.
The reconstruction is generated by these four programs:
EMICOMAT applies the icosahedral symmetry and samples the Fourier transforms in cylindrical coordinates, generating the equations F = B G
EMICOBG solves the linear equations for G(R,Z)
EMICOLG inverts the set of coefficients to yield gn(r,Z).
EMICOFB applies the inverse Fourier transform to the gn's and generates three-dimensional maps of the averaged particle in various settings.
In generating the final reconstruction, EMICOFB only imposes the D5 symmetry to the map. To apply the full icosahedral symmetry , the program SYMMETRIZE is used, and applies the three-fold symmetry missing from the map. SYMMETRIZE also allows interpolation and rotation of the map.
PTONE takes MRC format image files and generates half tone output in postscript format printable on the laserwriters.
TWCONT is a contouring program from MRC format files which produces postscript output.
SCONT makes a surface representations (shadowed and lighted) of an MRC three-dimensional volume and outputs it as a 2D MRC file.
EMICOPROJ takes a 3D reconstruction and makes a projection through it in an icosahedral orientation givin by theta,phi,omega. EMICOPROJ_FULL does the same thing with more options, but is slower.
SPIDER: WEB (the graphics side of SPIDER) allows viewing of all the sections of the reconstruction as well as viewing the surface of the reconstruction.
AVS: A sophisticated program system for representation and manipulation of 3D images available on the Stardent GS2000. The MRC format reconstructions must be reformatted with MRC2VOL and transferred with FTP.
The initial structure can be used to check the orientations of particles and to increase the number of particles included in the data set. The algorithm is the polar Fourier transform method developed by Drs. T.S. Baker and R. H. Cheng (Purdue University) and compares the projections of the model with the original images. Once a model is available, this approach is usually more efficient than the common lines method of orientation search.
Steps 5 through 9 are repeated so that larger numbers of particles can be included and the signal to noise increased.
Author: Stephen Fuller
Last Updated: 12sep00
