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Proteins, Interfaces, and Cryo-Em Grids Lawrence Berkeley National Laboratory Recent Work Title PROTEINS, INTERFACES, AND CRYO-EM GRIDS. Permalink https://escholarship.org/uc/item/4b23r25h Author Glaeser, Robert M Publication Date 2018-03-01 DOI 10.1016/j.cocis.2017.12.009 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Available online at www.sciencedirect.com ScienceDirect Proteins, interfaces, and cryo-EM grids Robert M. Glaeser⁎ It has become clear that the standard cartoon, in which macromolecular particles prepared for electron cryo-microscopy are shown to be https://doi.org/10.1016/j.cocis.2017.12.009 surrounded completely by vitreous ice, often is not accurate. In 1359-0294/© 2017 Elsevier Ltd. All rights reserved. particular, the standard picture does notincludethefactthatdiffusion to the air-water interface, followed by adsorption and possibly denaturation, can occur on the time scale that normally is required to make thin specimens. The extensive literature on interaction of proteins 1. Introduction with the air-water interface suggests that many proteins can bind to the interface, either directly or indirectly via a sacrificial layer of already- The requirements are quite demanding for preparing thin denatured protein. In the process, the particles of interest can, in some specimens of randomly disbursed biological macromole- cases, become preferentially oriented, and in other cases they can be cules that can be used for high-resolution electron damaged and/or aggregated at the surface. Thus, although a number of microscopy [1•,2•]. Ideally, these vitrified, aqueous methods and recipes have evolved for dealing with protein complexes specimens should be no N100 nm in thickness, and possibly thatprovetobedifficult,makinggood cryo-grids can still be a major as thin as 20 nm or 30 nm. Such thin specimens are challenge for each new type of specimen. Recognition that the air-water required because, among other reasons, the mean free interface is a very dangerous place to be has inspired work on some novel path for inelastic scattering [3] is estimated to be about approaches for preparing cryo-grids. At the moment, two of the most promising ones appear to be: (1) thin and vitrify the specimen much 350 nm or less for high-energy (300 keV) electrons. In faster than is done currently or (2) immobilize the particles onto a addition, the macromolecular particles must remain fully structure-friendly support film so that they cannot diffuse to the air- hydrated after being inserted into the vacuum of the water interface. electron microscope. The most practical way to maintain a well-hydrated state has proven to be to put a few μLof Address sample onto a thin, holey film, supported on a fine-mesh, Lawrence Berkeley National Laboratory, University of California, 3 mm diameter metal grid, and then blot off excess sample Berkeley, CA 94705, United States with filter paper. This is usually done in an environment of controlled temperature and humidity, in order to mini- 363B Donner Laboratory, Lawrence Berkeley National Laboratory, mize evaporation of the remaining water. The resulting, University of California, Berkeley, CA 94705, United States. thin sample then is rapidly quenched to low temperature; • ([email protected]) for further detail see [4 ] and for historical background see [5•]. Keywords: A very simple picture has been used for decades to Interfacial adsorption explain why blotting and subsequent quenching results in Preferential orientation nearly ideal specimens, at least some of the time. As is Protein denaturation illustrated in Fig. 1, macromolecular particles are imagined Affinity support films to be embedded within a vitrified layer of buffer. According to this picture, the spatial distribution, orientation and – Current Opinion in Colloid & Interface Science (2018) 34,1 11 structure of the macromolecules are expected to be For a complete overview see the Issue and the Editorial identical to what they previously were in bulk solution, Article History: unperturbed by the process of making and freezing the thin Received 20 October 2017 film. If every grid were as shown in this picture, regardless of Received in revised form 14 December 2017 what protein complex was used, then all of them would give Accepted 14 December 2017 superb images. Many samples do, in fact, give superb results Available online 22 December 2017 in electron microscopy, thus leading to the widely-held Current Opinion in Colloid & Interface Science (2018) 34,1–11 www.sciencedirect.com RM Glaeser 2 2. Many proteins are known to adsorb to air- water interfaces 2.1. Adsorption progresses in stages Adsorption of proteins to the air-water interface has historically been pictured to progress through at least three distinct steps. Fig. 1 Cartoon showing the standard picture that is A review published in 1950, for example, spoke of an initial “ ” envisioned in order to explain why embedding macromolecular adsorption of proteins at the interface in the globular form , “ ” complexes within a thin film of vitrified buffer should preserve followed by unrolling of the peptide chains at the interface , “ the structure in a near-native state. Macromolecular particles and subsequent aggregation of the unrolled chains into a ” •• arerandomlydistributedinthesamplewhenonaholeysupport coagulum [8 ]. Later, it seemed perhaps self-evident to film, just as they were in the test tube. When everything above include a step in which additional proteins bind to the layer of denatured-protein at the air-water interface, after which the dotted line is blotted away, a thin film remains in the hole. •• This thin film is then vitrified by plunging into cryogen, leaving further denaturation and aggregation might follow [9 ].A the particles embedded in amorphous ice. contrary view has long been presented in the literature, however, at least for some proteins [10•]. For example, [11] concluded that preferential orientation and some structural deformation of bovine serum albumin may occur, but never- belief that the standard picture shown in Fig. 1 is, indeed, theless there is no denaturation. correct. In either event, the first step involves structurally intact Many macromolecules, however, prove to be difficult to particles colliding with and sticking to the interface. Initial prepare in the form of single-particle cryo-EM specimens adhesion to a clean air-water interface presumably involves (referred to here as cryo-grids), leading one to doubt dewetting of individual hydrophobic side chains or even whether the standard picture is always correct. As a result, small hydrophobic patches, both of which normally exist on an effort has begun to develop more sophisticated models, the surfaces of native proteins. This initial-adsorption step whichtakeintoaccountthefactthattherequiredthin, can be diffusion limited, i.e. the activation energy for aqueous films have a very high surface-to-volume ratio in binding to the (hydrophobic) interface may be very small, the brief moment before vitrification. Such models take and thus the sticking coefficient (the number of times that into account the fact that macromolecular particles can – particles stick, relative to the number of times that they indeed must – diffuse and collide with the air-water impinge upon an interface) can be close to 1.0. If that is the interface, where they may adsorb and even possibly case, the initial rate of adsorption is expected to be denature before vitrification occurs. Other consequences proportional to the bulk concentration of the particles. include preferential orientation of particles, or – in some The second step, at least when it does occur, involves cases – the number of particles seen does not correspond partial or complete unfolding of the native-protein structure to their concentration in bulk. Although numerous cau- at the interface. In some cases the thickness of the resulting tionary remarks about these hazards were, in fact, made in protein monolayer is estimated to be b2nm [12,13•]. Section 6.6 of the review by Dubochet et al. [6••],and Unfolding of the native structure at an air-water interface more recently in a retrospective by Taylor and Glaeser [7•], is imagined to involve a rapid, step-by-step movement of little has yet been done to address the issue in a systematic hydrophobic residues from the interior of a protein to air, way. while still leaving the hydrophilic residues on the aqueous The primary goal in this Opinion is to critically examine, side of the interface. As is discussed in Section 3, the energy on the basis of the known behavior of proteins at air-water landscape of protein unfolding at interfaces is thus expected interfaces, what might be wrong with what has been the to trend monotonically downhill, interrupted only by small standard picture of single-particle cryo-EM specimens, which activation barriers as the reaction progresses, which is very is shown in Fig. 1. The heart of this critique is presented in different from what it is in bulk. Section 4, preceded first by a limited review (Section 2)of The third step envisioned in this review, as mentioned some of the literature showing that many proteins do adsorb above, involves the adsorption of additional, structurally to air-water interfaces, followed by a similarly short review intact proteins, possibly via a mixture of hydrophilic and (Section 3) of work showing that many proteins do, in fact, hydrophobic interactions with the pre-existing layer of denature shortly after they have first been adsorbed. denatured proteins. Since the binding of proteins to Finally, the critique of what is wrong with the standard hydrophilic surfaces is often much weaker than it is to picture is followed, in Section 5, by a discussion of hydrophobic surfaces [14–16], the sticking coefficient may alternative approaches that are being taken for preparing be much lower for a denatured-protein monolayer than it is cryo-EM specimens, all of which can be seen as ways to for a pristine air-water interface.
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