Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography

Comolli, Luis R.; Downing, Kenneth H.

doi:10.1016/j.jsb.2005.08.004

Cited by 46 publications

(41 citation statements)

References 14 publications

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“…Our initial trials with single-particle specimens revealed that observation at a higher temperature provided images with better contrast. This is in agreement with recent investigations on the influence of specimen temperature on data quality (Comolli and Downing, 2005;Iancu et al, 2006). All data presented in this report was collected by allowing the liquid helium to evaporate, and then waiting for the specimen to warm up to 50-60 K. The temperature then stabilizes through the cooling from the outer liquid nitrogen shield, and imaging can be carried out without noticeable specimen drift.…”

Section: Electron Microscopysupporting

confidence: 89%

Single particle analysis based on Zernike phase contrast transmission electron microscopy

Danev

Nagayama

2008

Journal of Structural Biology

107

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Section: Electron Microscopysupporting

confidence: 89%

Single particle analysis based on Zernike phase contrast transmission electron microscopy

Danev

Nagayama

2008

Journal of Structural Biology

107

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“…Cell thickness is a limiting factor in such investigations, and studies have used small cells (16,17), the edge of frozen-hydrated cells (18-20), or vitreous sections (21, 22). Here we report electron cryomicroscopy of whole-mount human umbilical vein endothelial cells (HUVECs) that are vitrified by rapid plunge-freezing, providing an outstanding view of WPBs and their surrounding architecture at the thin edge of the cell without the fixation, dehydration, embedding, staining, and sectioning used in earlier studies employing conventional plastic sections (23) or high pressure freezing/freeze substitution (24).…”

mentioning

confidence: 99%

“…Overexposure during a dose series (Fig. S2) or a tilt series (Ͼ50 e Ϫ /Å 2 ) (Movie S1), caused a selective bubbling (16,17) of the internal contents of WPBs compared to other organelles and cell structures. Because some carbohydrates have been shown to promote bubbling (29), we suggest that this radiation damage effect may be attributed to the extensive glycosylation of the VWF glycoprotein (Ͼ15% by weight) (30) combined with its density in the granule.…”

mentioning

confidence: 99%

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

Berriman

Hewlett³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

140

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In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.electron cryomicroscopy ͉ paracrystal ͉ von Willebrand factor ͉ tomography E ndothelial cells line the inner surfaces of blood vessels and play important roles in hemostasis, thrombosis, and inflammation. Some of these roles are achieved by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release functions as an adhesive protein to bind platelets to sites of vascular injury. VWF circulating in the bloodstream also functions as a carrier for coagulation Factor VIII, increasing its lifetime. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease (1).VWF is synthesized as a 350-kDa precursor (proVWF) that forms disulfide-linked dimers in the ER through its C-terminal cysteine knot domain. Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2, 3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4). Secretagogues stimulate WPB exocytosis, releasing VWF and other low molecular weight molecules such as cytokines and chemokines into the bloodstream (5), although mature VWF and its proregion account for greater than 95% of the protein in the granule (6). On release, VWF multimers are able to unfurl to strings up to 100 m long and associate with multiple ligands on platelet and endothelial cell surfaces at the site of vascular injury to help form a platelet plug. Mechanical shear exposes ligand binding sites on VWF as well as sites for cleavage by the protease ADAMTS13, which regulates the length of VWF multimers in the bloodstream (7).Like most other secretory granules, WPBs are thought to form at the trans-Golgi network (TGN) in a pH-dependent process. P-selectin is also recru...

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“…The issues in tomography have to do with alignment of successive images, discussed in the paragraph above; the phenomenon of ''bubbling"; and other processes that destroy even the coherent, particle-like distribution of mass of an entire protein molecule. Prior to the onset of bubbling, it appears that there is no improvement in the dose at which ordered protein structures such as bacterial S-layers are destroyed (Comolli and Downing, 2005). Comparisons at higher electron exposures (Comolli and Downing, 2005;Iancu et al, 2006) indicate that smaller bubbles are produced at helium temperature than at nitrogen temperature.…”

Section: Radiation-induced Movement Has Emerged As Another Limitationmentioning

confidence: 99%

Retrospective: Radiation damage and its associated “Information Limitations”

Glaeser

2008

Journal of Structural Biology

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Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography

Cited by 46 publications

References 14 publications

Single particle analysis based on Zernike phase contrast transmission electron microscopy

Single particle analysis based on Zernike phase contrast transmission electron microscopy

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

Retrospective: Radiation damage and its associated “Information Limitations”

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