Image page: Elucidation of the High Resolution Structure of Crotoxin Complex:

Jaap Brink, Ph.D.

W.K. Keck Center for Computational Biology and Verna and Marrs McLean Department for Biochemistry, Rm. 326B
Baylor College of Medicine, 1 Baylor Plaza, Houston TX 77030, U.S.A.

For a description of my project, click here.

The source of venom:

Image (218 Kb) of Crotalus durissus terrificus found in the Brazilian rain forest. Venom is collected from the snake at the Institut de Butantan in Sao Paolo, Brazil. It is separated using a 2-step column chromatography procedure from convulxin and crotamin and obtained at a 60-70% yield.

Diffraction data:

A low-magnification image (79 Kb) of a crotoxin complex crystal recorded in the JEOL4000EX at 400 kV on a Gatan 1024 x 1024 slow-scan CCD camera (model 679). This CCD camera was purchased with financial support from the W.M. Keck Foundation. This glucose-embedded crystal measures over 3 micron on edge and is ~128 Å thick. Crystals like these are prepared on grids (200-400 holes/inch) covered with a perforated carbon film, and a thin continuous carbon film.

An electron diffraction pattern (92 Kb) of a crotoxin complex crystal like seen above showing reflections extending to 3 Å. The projected unit cell of the crystal is 38.8 x 38.8 Å with an included angle of 90^o. This diffraction pattern was recorded on our slow-scan CCD camera. The electron dose used for this pattern was 0.02 electron/Ų.

Montage (73 Kb) of the 1st and the 128th electron diffraction pattern from a tilt series consisting of 128 patterns. These patterns were acquired from a single crystal of crotoxin complex embedded in glucose on the slow-scan CCD camera. The tilt angle was decremented for each subsequent pattern from +43^o to finally -53.8^o. The crystal was 128 Å thick.

Click here to see a MPEG movie (1.2 Mb) of a tilt series of 125 diffraction patterns recorded from a single crystal 256 Å thick, that was tilted from +50^o to -52^o. The individual diffraction patterns were computer processed to remove the background due to inelastic scattering, and to include a tilt angle indicator. The unique set of images was duplicated in the computer in reverse order to generate a set of images from -52^o to +50^o.

Image (40 Kb) of the merged intensity showing the 3D sampling by 353 patterns. Six tilt series were merged out of more than 15 series that were recorded. This data set contains diffraction data from 54,868 reflections that are present to 3.3 Å resolution isotropically; the completeness is 88.2 %, and the Rmerg is 26.9% for the 3D merging.

Postscript file (596 Kb) and image (11 Kb) of the merged intensity showing the intensity distribution after merging the 353 patterns. The data was projected onto the plane made by the z*-axis and the combined a*b*-axis. The diameter of the dots is proportional to the reflections' intensity all scaled by the highest intensity.

Postscript file (421 Kb) and image (20 Kb) of four intensity profiles of lattice rod after merging. Plotted are the fitted intensity (continuous line), the observed intensities (small triangles) and the error of the fit (very small dots) for the (8,1) (top-left), the (7,6) (top-right), the (1,8) (bottom-left) and the (2,2) (bottom-right). The rods are at 4.8 Å to 14 Å resolution. The 3D resolution, however, for the (8,1) and (1,8) is 3.2 Å.

Image data:

Postscript file (33 Kb) of the summed intensity at all lattice points within shells of resolution for crotoxin complex crystals embedded in vitreous ice as well as glucose.

Image (265 Kb) showing two spots from a spot-scan micrograph of crotoxin complex embedded in vitreous ice. Owing to a difference in density between the vitreous ice and the protein, the lattice shows up quite clearly. The picture was recorded on film at 40,000x using 6-7 electrons/Ų.