Challenges and opportunities in cryo-EM single-particle analysis

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Abstract

<p class="first" id="d5817462e119">Cryogenic electron microscopy (cryo-EM) enables structure determination of macromolecular objects and their assemblies. Although the techniques have been developing for nearly four decades, they have gained widespread attention in recent years due to technical advances on numerous fronts, enabling traditional microscopists to break into the world of molecular structural biology. Many samples can now be routinely analyzed at near-atomic resolution using standard imaging and image analysis techniques. However, numerous challenges to conventional workflows remain, and continued technical advances open entirely novel opportunities for discovery and exploration. Here, I will review some of the main methods surrounding cryo-EM with an emphasis specifically on single-particle analysis, and I will highlight challenges, open questions, and opportunities for methodology development. </p>

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Most cited references 139

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Structure of the TRPV1 ion channel determined by electron cryo-microscopy

Maofu Liao, Erhu Cao, David Julius … (2013)

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here, we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane helices S5–S6 and the intervening pore loop, which is flanked by S1–S4 voltage sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including N-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

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TRPV1 structures in distinct conformations reveal mechanisms of activation

Erhu Cao, Maofu Liao, Yifan Cheng … (2013)

TRP channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (S1-S4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiologic modulation exhibited by TRPV1 and other TRP channels.

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The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

R. Henderson (1995)

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

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Author and article information

Journal

Title: Journal of Biological Chemistry

Abbreviated Title: J. Biol. Chem.

Publisher: American Society for Biochemistry & Molecular Biology (ASBMB)

ISSN (Print): 0021-9258

ISSN (Electronic): 1083-351X

Publication date Created: March 29 2019

Publication date (Print): March 29 2019

Publication date (Electronic): February 25 2019

Volume: 294

Issue: 13

Pages: 5181-5197

Article

DOI: 10.1074/jbc.REV118.005602

PMC ID: 6442032

PubMed ID: 30804214

SO-VID: 98b57a1e-8cd8-48bb-b3ac-3b773124bfa0

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Challenges and opportunities in cryo-EM single-particle analysis

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Core Readings in Statistical Mediation Analysis

Most cited references 139

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

TRPV1 structures in distinct conformations reveal mechanisms of activation

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

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