Antibody-based affinity cryo-EM grid

Yu, Guimei; Li, Kunpeng; Jiang, Wen

doi:10.1016/j.ymeth.2016.01.010

Cited by 70 publications

(66 citation statements)

References 43 publications

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“…Provided that the binding of the target protein would be tight enough, their large molecular mass (∼5 MDa) would allow relatively straightforward separation of the target protein from smaller contaminants through the use of, for example, gel filtration of sucrose gradients. Alternatively, one could try to fix the DNA supports directly on the grid surface and use the grids themselves for on-grid affinity purification in a similar manner as was done with nitrilotriacetic acid-modified lipid monolayers (54,55) or antibodies attached to carbon films (56,57).…”

Section: Discussionmentioning

confidence: 99%

Design of a molecular support for cryo-EM structure determination

Martin

Bharat

Joerger

et al. 2016

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

100

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Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.cryo-EM | DNA-origami | single particle analysis | structural biology | p53 C ryo-electron microscopy (cryo-EM) structure determination of biological macromolecules is undergoing rapid progress. With the advent of efficient direct electron detectors and the development of powerful algorithms for image processing, numerous structures to near-atomic resolution have been reported in the past few years (1, 2). In cryo-EM single-particle analysis, solutions of purified protein and/or nucleic acid complexes are typically applied to a thin, amorphous carbon film with micrometer-sized holes in it that is held in place by a metal grid. Excess liquid is then blotted away with filter paper, and the sample is rapidly plunged in liquid ethane (3,4). This procedure ideally results in the formation of a film of vitreous ice that is only slightly thicker than the macromolecular complex of interest. Keeping the frozen sample at liquid nitrogen temperatures allows its insertion into the high vacuum of a transmission electron microscope and limits the effects of radiation damage by the electrons that are used for imaging (5). Images taken through the holes of the carbon film ideally contain 2D projections of many, assumedly identical copies of the macromolecular complex of interest, which are often called particles. Projections from different viewing directions can then be combined in a 3D reconstruction of the scattering potential of the molecule (6). If the resulting map approaches a resolution of 3 Å, it allows building an atomic model of the molecules, from which useful information about their function may be derived.A major hurdle in single-particle analysis is the need to recover the relative viewing angles...

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Section: Discussionmentioning

confidence: 99%

Design of a molecular support for cryo-EM structure determination

Martin

Bharat

Joerger

et al. 2016

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

100

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“…One interesting category of grid modification includes variations on the theme of “affinity grids”, where, for example, antibodies attached to a grid can capture untagged proteins or complexes out of lysate, without requiring prior purification [61,62]. This has successfully been used for the high resolution 3D reconstruction of an icosahedral virus [63].…”

Section: Grid Modification Methods For Specimen Preparationmentioning

confidence: 99%

Cryo-EM: beyond the microscope

Earl

Falconieri

Milne

et al. 2017

Current Opinion in Structural Biology

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The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

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“…For example, the particle to PFU ratio for human herpes simplex virus 1 is ~10:1 [68] but ~40,000:1 for Varicella-zoster virus, another human herpes virus [69]. Affinity cryo-EM methods [70,71,72] capture target particles directly from cell lysate without pre-purification. The virus particles are more likely to resemble the native states.…”

Section: Structural Virology Beyond Purified Virus Particlesmentioning

confidence: 99%

Atomic cryo-EM structures of viruses

Jiang

Tang

2017

Current Opinion in Structural Biology

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During the development of single particle cryo-EM in past five decades, icosahedral viruses have led the resolution progress owing to their large mass and high symmetry. Many technical advances in cryo-EM were first established with viruses. Since reaching ~4 Å resolution in 2008, it has become a relatively routine task to solve the atomic structure of isolated viruses. The future of structural virology will be increasingly focused on remaining challenges including solving structures of jumbo viruses, intermediate functional states during assembly, maturation, and infection, and in situ structures. Recent demonstrations of near-atomic resolution structure with electron tomography and sub-tomogram averaging opens a new direction for high resolution studies of pleomorphic viruses and the pleomorphic states of icosahedral viruses that have defied past efforts using the single particle cryo-EM approach.

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Antibody-based affinity cryo-EM grid

Cited by 70 publications

References 43 publications

Design of a molecular support for cryo-EM structure determination

Design of a molecular support for cryo-EM structure determination

Cryo-EM: beyond the microscope

Atomic cryo-EM structures of viruses

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