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

Sader, Kasim; Studer, Daniel; Zuber, Benoît; Gnaegi, Helmut; Trinick, John

doi:10.1016/j.ultramic.2009.09.004

Cited by 17 publications

(10 citation statements)

References 25 publications

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“…Improved resolution should be possible. Using lysozyme crystals, Sader et al (35) showed that vitreous cryosections retrieve structural information down to 7.9 Å, meaning that a resolution of <1 nm can be expected in the cellular context. The field of high-resolution membrane analysis is now largely open to further progress by the combination of high-resolution CEMOVIS and CET.…”

Section: Discussionmentioning

confidence: 99%

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Leforestier

Lemercier

Livolant

2012

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

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Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure-function relationships.thylakoid | transmembrane proteins | multilamellar body | Golgi | cortical alveoli B iological membranes are highly specialized dynamic entities that limit structural and functional compartments and control the exchange of matter and energy. They may exhibit a wide diversity of shapes with variable curvature, including spherical, tubular, planar, sponge, or cubic bicontinuous organizations (1). They are composed of hundreds of lipid and protein species, in some cases modified by sugars. Their structure and composition vary among species, cell types, and organelles and, even within a given membrane type, in time and space. This structural and chemical variability is directly related to their functional properties (2). Lipids (mostly phospholipids) represent more than 50% of membrane components. Their amphiphilic nature leads to the formation of bilayers. The hydrophobic chains are buried inside the membrane, and the polar heads are exposed to the outer, hydrated environment. Proteins may be bound to the lipid bilayer through electrostatic interactions or partially embedded within the bilayer.Transmission electron microscopy provides various techniques for exploring cell ultrastructure. Among these, cryoelectron microscopy of vitreous sections (CEMOVIS) provides snapshots of unstained frozen-hydrated cells and tissues in their native state. The analysis of ordered fibrillar objects like DNA in sperm cells (3), bacteria (4), and dinoflagellates (5) has benefited from this approach. Nevertheless, cryoelectron tomography (CET) is often preferred over CEMOVIS to overcome limitations arising from superimpositions on 2D projections and to obtain 3D views. CET has been used to successfully image entire small cells, organelles, and thin cellular extensions (6, 7), as well as thicker objects sliced into cryosections (8-11). Spectacular results have been obtained with CET in combination with image analysis; for example, the Ccadherin atomic structure has been fit into averaged subtomograms of epidermal desmosomes (8). However, the relatively low resolution of CET (generally between 5 and 8 nm; 3 nm in the optimum cases) remains insufficient to address many biological questions, especially those dealing with membrane structure.Fine structural details, particularly the bilayer nature of membranes, have been...

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Section: Discussionmentioning

confidence: 99%

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Leforestier

Lemercier

Livolant

2012

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

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“…We propose that large, tightly ordered macromolecular complexes, like the 80S ribosome, can withstand this specific type of mechanically induced deformation, because of its relatively small size (compared to the whole cell) and its structural order. In a similar study, it has been shown that electron diffraction patterns of high-pressure frozen vitreous cryo-sectioned lysozyme crystals can extend to 7.9 Å (Sader et al, 2009). Both studies indicate that after vitreous cryo-sectioning (and all associated sectioninduced artifacts) structural information can be retained up to the given resolution limit.…”

Section: Discussionmentioning

confidence: 79%

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Pierson

Ziese

Sani

et al. 2011

Journal of Structural Biology

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“…The methods used to maintain crystal quality while diffraction data are collected include sugar-embedding (e.g. using trehalose; Schebor et al, 2010), Paratone-N oil coverage (Hunter et al, 2014), vitrification (Sader et al, 2009), 'sandwich' enclosure (Coquelle et al, 2015) and wrapping in watertight graphene sheets (Wierman et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

et al. 2017

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A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the 'diffract-before-destroy' regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si 3 N 4 windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution.

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

Cited by 17 publications

References 25 publications

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

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