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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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Tetragonal lysozyme crystals. The long axis [001] of the large crystal on the left is greater than 500 μm.
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fig1: Tetragonal lysozyme crystals. The long axis [001] of the large crystal on the left is greater than 500 μm.

Mentions: Large lysozyme crystals (>0.5 mm) were grown in one week and the shape of these was typical of the tetragonal form (Fig. 1). All of the glycerol-embedded crystals diffracted X-rays to better than 2.1 Å, an example of which is shown in Fig. 2. The lysozyme space group determined by MOSFLM [12] was primitive tetragonal (P4) with unit cell dimensions of a=b=79.03 Å, c=37.04 Å. This diffraction pattern is representative of the average quality of more than 12 that were collected from different crystals.


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)

Tetragonal lysozyme crystals. The long axis [001] of the large crystal on the left is greater than 500 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Tetragonal lysozyme crystals. The long axis [001] of the large crystal on the left is greater than 500 μm.
Mentions: Large lysozyme crystals (>0.5 mm) were grown in one week and the shape of these was typical of the tetragonal form (Fig. 1). All of the glycerol-embedded crystals diffracted X-rays to better than 2.1 Å, an example of which is shown in Fig. 2. The lysozyme space group determined by MOSFLM [12] was primitive tetragonal (P4) with unit cell dimensions of a=b=79.03 Å, c=37.04 Å. This diffraction pattern is representative of the average quality of more than 12 that were collected from different crystals.

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