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Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections.

Sader K, Studer D, Zuber B, Gnaegi H, Trinick J - Ultramicroscopy (2009)

Bottom Line: In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained.The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife.In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, University of Leeds, LS2 9JT, UK. k.sader@leeds.ac.uk

ABSTRACT
We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

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Related in: MedlinePlus

Defocussed electron diffraction pattern showing the area of a vitreous cryo-section of lysozyme from which diffraction patterns in Figs. 4 and 5 were recorded. The denser L-shaped area corresponds to the edge of the supporting square mesh.
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fig3: Defocussed electron diffraction pattern showing the area of a vitreous cryo-section of lysozyme from which diffraction patterns in Figs. 4 and 5 were recorded. The denser L-shaped area corresponds to the edge of the supporting square mesh.

Mentions: Electron diffraction patterns were recorded from the area shown in the defocussed diffraction pattern in Fig. 3, which is approximately 5 μm in diameter. Focussed diffraction patterns were recorded with exposure times of 2 and 5 s at the calibrated camera length of 4.5 m and are shown displayed with different contrast levels in Figs. 4 and 5. The first diffuse diffraction ring from vitreous water was observed with shorter camera lengths, and no reflections from crystalline ice. In Fig. 5, the furthest diffraction spot with a SNR>5 lies at 593 pixels from the undiffracted beam, corresponding to 7.9 Å. Of the four best electron diffraction patterns from different sections (from three ribbons on two grids), the mean resolution was 9.0±1.3 Å.


Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections.

Sader K, Studer D, Zuber B, Gnaegi H, Trinick J - Ultramicroscopy (2009)

Defocussed electron diffraction pattern showing the area of a vitreous cryo-section of lysozyme from which diffraction patterns in Figs. 4 and 5 were recorded. The denser L-shaped area corresponds to the edge of the supporting square mesh.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2824107&req=5

fig3: Defocussed electron diffraction pattern showing the area of a vitreous cryo-section of lysozyme from which diffraction patterns in Figs. 4 and 5 were recorded. The denser L-shaped area corresponds to the edge of the supporting square mesh.
Mentions: Electron diffraction patterns were recorded from the area shown in the defocussed diffraction pattern in Fig. 3, which is approximately 5 μm in diameter. Focussed diffraction patterns were recorded with exposure times of 2 and 5 s at the calibrated camera length of 4.5 m and are shown displayed with different contrast levels in Figs. 4 and 5. The first diffuse diffraction ring from vitreous water was observed with shorter camera lengths, and no reflections from crystalline ice. In Fig. 5, the furthest diffraction spot with a SNR>5 lies at 593 pixels from the undiffracted beam, corresponding to 7.9 Å. Of the four best electron diffraction patterns from different sections (from three ribbons on two grids), the mean resolution was 9.0±1.3 Å.

Bottom Line: In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained.The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife.In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, University of Leeds, LS2 9JT, UK. k.sader@leeds.ac.uk

ABSTRACT
We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

Show MeSH
Related in: MedlinePlus