**PURPOSE**- To simulate a bright field electron microscope image of a structure by using complex atomic scattering amplitudes. Atomic information of the structure needs to be provided in the form of a PDB file. Further info on CTF related operations in SPIDER.

**SEE ALSO****TF**[Transfer Function - Generate image showing effect of defocus on CTF]

**USAGE**- .OPERATION: TF SIM

- .OUTPUT FILE: TFSIM001

[Enter the name of the output file that will store the computed image.].OPERATING VOLTAGE [KV]: 100

[Enter the operating voltage of the microscope in KVOLTS.].PDB INPUT FILE: ADP.PDB

[Enter the name of PDB file with extension.].VOXEL SIZE [A]: 1.47

[Enter the voxel size of SPIDER file in Angstroms.].VOLUME NX, NY, & NZ: 100, 102, 85

[Enter size of the SPIDER file (number of voxels in each dimension).].CS [MM]: 2.0

[Enter the spherical aberration constant.].DEFOCUS [A]: 2000

[Enter the amount of defocus, in Angstroms. Positive values correspond to underfocus (the preferred region); negative values correspond to overfocus.].SOURCE SIZE [1/A], DEFOCUS SPREAD [A]: 0.005, 250

[Enter the size of the illumination source in reciprocal Angstroms. This is the size of the source as it appears in the back focal plane of the objective lens. A small value results in high coherence; a large value, low coherence. Enter the estimated magnitude of the defocus variations corresponding to energy spread and lens current fluctuations.].ASTIGMATISM [A], AZIMUTH [DEG]: 400, 30

[Enter the defocus range due to axial astigmatism. The value given indicates a defocus range of +/- 400A around the nominal value as the azimuth is varied. Then, enter the angle, in degrees, that characterizes the direction of astigmatism. The angle defines the origin direction where the astigmatism has no effect.].GAUSSIAN ENVELOPE HALFWIDTH [1/A]: 0.34

[Enter GEH. The envelope parameter specifies the 2 sigma level of the Gaussian (see note 2 for details).].IMAGE CREATED BY (A)AMPLITUDE ONLY, (P)PHASE ONLY, OR (B)BOTH: A

[Enter the choice for type of output image].IMAGE INTENSITY MULTIPLIED BY (E)ENVELOPE FUNCTION,

(B)BOTH ENVELOPE & GAUSSIAN FUNCTIONS, (N)NEITHER: N [Enter the desired choice (see Note 2 for details).]

**NOTES**

- Theory and all definitions of electron optical parameters
are according to:

Frank, J. (1973). The envelope of electron microscopic transfer functions for partially coherent illumination. Optik, 38(5), 519-536.

and

Wade, R. H., & Frank, J. (1977). Electron microscope transfer functions for partially coherent axial illumination and chromatic defocus spread. Optik, 49(2), 81-92.

Internally, the program uses the generalized coordinates defined in these papers. - The expression used for image intensity is:

If image intensity multiplied by both Envelope function and Gaussian function:

I(K) = [(OR(K)*sin(GAMMA) - OI(K)*cos(GAMMA)]*ENV(K)*exp[-(K/GEH)^2

If image intensity multiplied by Envelope function only:

I(K) = [(OR(K)*sin(GAMMA) - OI(K)*cos(GAMMA)]*ENV(K)

If image intensity multiplied by neither:

I(K) = [(OR(K)*sin(GAMMA) - OI(K)*cos(GAMMA)]

References:

(i) J. Frank (1996), Three-dimensional Electron Microscopy of Macromolecular Assemblies. Academic Press, San Diego.

(ii)J. Haase (1970), Zusammenstellung der Koeffizienten fur die Anpassung komplexer Streufaktoren fur schnelle Elektronen durch Polynome, Zeitschrift fur Naturforschung, 1219-1236.

**SUBROUTINES**- TRAFSIM

**CALLER**- UTIL1

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