Author response: Routine single particle CryoEM sample and grid characterization by tomography (2018)

Article Figures and data Abstract Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment. https://doi.org/10.7554/eLife.34257.001 Introduction For decades, single particle cryo-electron microscopy (cryoEM) grids have commonly been imaged and processed under the assumption that most particles imaged were not adsorbed to the air-water interfaces and were in a single layer as they were plunge-frozen. An ideal grid and sample for single particle collection would have the majority of areas in holes maximally occupied by non-adsorbed, non-interacting particles 10 nm or farther from the air-water interfaces, particles oriented randomly, vitreous ice thin enough to contain the particles plus about 20 nm of additional space, where none of the particles overlap in the beam direction, and where the beam direction is normal to the areas of interest (Figure 1). Collection in such ideal areas of a grid would then be the most efficient use of resources and would result in the highest resolution structure possible for a given number of particles, collection hardware, and collection parameters. Figure 1 Download asset Open asset Schematic diagrams of grid hole cross-sections containing regions of ideal particle and ice behavior for single particle cryoEM collection. (A) A grid hole where all regions of particles and ice exhibit ideal behavior. (B) Grid holes where there are areas that exhibit ideal particle and ice behavior. Green arrows indicate areas with ideal particle and ice behavior. The generic particle shown is a low-pass filtered holoenzyme, EMDB-6803 (Yin et al., 2017). The particles were rendered with UCSF Chimera (Pettersen et al., 2004). https://doi.org/10.7554/eLife.34257.002 In practice, during single particle grid preparation and data collection there are many issues that contribute to preventing a sample from following this ideal behavior. As depicted in Figure 2, numerous combinations of air-water interface, particle, and ice behavior are possible for each hole and for regions within each hole, without taking into account surface ice contamination. Each air-water interface might be: (i) free from sample solution constituents (Figure 2, A1), (ii) covered with a layer of primary, secondary, and/or tertiary protein structures (either isolated or forming protein networks) from denatured particles (A2), or (iii) covered with one or more layers of surfactants if present during preparation (A3). It is difficult to distinguish between air-water interfaces that are clean, covered in primary protein structures, or covered in surfactants as they are likely indistinguishable by cryoEM or cryo-electron tomography (cryoET) analysis without a sample-free control for comparison (cryoET may be able to resolve lipid layers at the air-water interface if high tilt angles are collected [Vos et al., 2008]). Bulk particle behavior in regions of holes might include any combination of: (i) non-adsorbed particles without preferred orientation (B1), (ii) particles at an air-water interface without preferred orientation (B2), (iii) particles at an air-water interface with N-preferred orientations (B3), (iv) partially denatured particles at an air-water interface with M-preferred orientations (B4), and/or (v) significantly denatured particles at an air-water interface (B5). Protein degradation in A2 might be considered to be a continuation of the denaturation in B4 and B5. Interactions between neighboring particles at the air-water interface might induce different preferred orientations in B3 and B4, particularly at high concentrations. Ice behavior at the air-water interfaces of each hole might be characterized by any two combinations of: convex ice (C1), flat ice (C2), concave ice where the center is thicker than the particle's minor axis (C3), and/or concave ice where the center is thinner than the particle's minor axis (C4). In the case of a convex air-water interface, the particle's minor axis might be larger than the ice thickness at the edge of the hole. Figure 2 Download asset Open asset Depictions of potential ice and particle behavior in cryoEM grid holes, based on Figure 6 from (Taylor and Glaeser, 2008). A region of a hole may be described by a combination of one option from (A) for each air-water interface and one or more options from (B). An entire hole may be described by a set of regions and one or more options from (C). (A) Each air-water interface might be described by either (1), (2), or (3). Note that cryoET might only be able to resolve tertiary and secondary protein structures/network elements at the air-water interface. (B) Particle behavior between air-water interfaces and at each interface might be composed of any combination of (1) through (5), with or without aggregation. B3 is different from B4 if, for example, a particle prone to denaturation is frozen before or after denaturation has begun, thus potentially changing the set of preferred orientations. At high enough concentrations additional preferred orientations might become available in B3 and B4 due to neighboring protein-protein interactions. (C) Ice thickness variations through a central cross-section of hole may be described by one option for one air-water interface and one option for the apposed interface. Note that in C1 the particle's minor axis may be larger than the ice thickness. In both C1 and C4, the particle may still reside in areas thinner than its minor axis if the particle is compressible. Phenomenon such as bulging or doming (Brilot et al., 2012) may be represented as a combination of C1-4. https://doi.org/10.7554/eLife.34257.003 The most common technique for preparing cryoEM grids, pioneered in the labs of Robert Glaeser (Jaffe and Glaeser, 1984; Taylor and Glaeser, 1974; Taylor and Glaeser, 1976) and Jacques Dubochet (Adrian et al., 1984; Dubochet et al., 1985; Dubochet et al., 1982), involves applying about 3 microliters of purified protein in solution onto a metal grid covered by a holey substrate that has been glow- discharged to make hydrophilic, blotting the grid with filter paper, and plunge-freezing the grid with remaining sample into a cryogen to form vitreous ice. Incubation times before and after blotting are on the order of seconds, allowing for the possibility of protein adsorption to the air-water interface due to Brownian motion. Concerns regarding deleterious air-water interface interactions with proteins have been often discussed in the literature. For instance, Jacques Dubochet et al., 1988 observed issues with regards to air-water interface and particle orientation for a small number of samples. In a recent review by Robert Glaeser (Glaeser and Han, 2017), evidence (Trurnit, 1960) using Langmuir-Blodgett (LB) troughs (Langmuir, 1917) was used to propose that upon contact with a clean air-water interface, proteins in solution will denature, forming an insoluble, denatured protein film. This film reduces the surface tension at the air-water interface and might act as a barrier between the remaining particles in solution and the air. Particles in solution might then adsorb to the denatured layer of protein depending on the local particle affinity with the interface, thus creating an ensemble of preferred orientations. Estimates for the amount of time a particle with a mass of 100 kDa to 1 MDa in solution might take to first reach the air-water interface (bulk diffusion) range from 1 ms to 0.1 s (Naydenova and Russo, 2017; Taylor and Glaeser, 2008). More recent literature, using LB troughs, substantiates that 10–1000 mL volumes of various proteins (commonly 10–1000 kDa and at ≲1 mg/mL) in buffer commonly adsorb to the air-water interface and form <10 nm thick (Gunning et al., 1996; Vliet et al., 2002) denatured viscoelastic protein network films (Birdi, 1972; Damodaran and Song, 1988; de Jongh et al., 2004; Dickinson et al., 1988; Graham and Phillips, 1979; Yano, 2012). The time it takes for adsorption to begin due to bulk diffusion may be on the order of 0.1 to 1 ms, depending on the protein (Kudryashova et al., 2005). For a protein that denatures at the air-water interface (surface diffusion), the surface diffusion time might be on the order of tens of milliseconds (Kudryashova et al., 2005), depending on factors including protein and concentration, surface hydrophobicity, amount of disordered structure, secondary structure, concentration of intramolecular disulfide bonds, buffer, and temperature. Higher bulk protein concentrations have been shown to increase the protein network thickness (Meinders et al., 2001). When several proteins and/or surfactants in solution are exposed to a clean air-water interface, competitive and/or sequential adsorption may occur (Ganzevles et al., 2008; Le Floch-Fouéré et al., 2010; Stanimirova et al., 2014). It has been shown using atomic force microscopy (AFM) imaging of LB protein films that these protein network films may not completely denature down to individual amino acids: adding surfactants to protein solutions in which a protein network film has already formed at the air-water interface will displace the protein layer (desorption [MacRitchie, 1998]) (Gunning and Morris, 2018; Mackie et al., 1999; Wilde et al., 2004) and the resulting protein network segments might partially re-fold in solution (Gunning and Morris, 2018; Mackie et al., 1999; Morris and Gunning, 2008). Time-resolved AFM surfactant-protein displacement experiments for a specific protein, β-lactoglobulin, and different surfactants, Tween 20 and Tween 60, show that displacement of the protein network film by the surfactants occurs at equivalent surface pressures and results in non-uniform surfactant domain growth, implying that the protein network is not uniform (Gunning et al., 2004). Different surfactant displacement behavior and patterns are observed while varying only the proteins, where the degree of protein network displacement isotropy by surfactant decreases for more ordered, globular proteins (Mackie et al., 1999). Non-uniformity of the protein network has also been seen by 3D AFM imaging of β-lactoglobulin LB-protein network films placed on mica (Gunning et al., 1996; Morris and Gunning, 2008). Similar experiments using LB troughs have also shown that proteins with β-sheets partially unfold, with the hydrophobic β-sheets remaining in-tact at the air-water interface and with potentially one or more layers of unstructured, but connected, hydrophilic amino acid strands just below the air-water interface (Yano et al., 2009). This potential for β-sheets to survive bulk protein denaturation is likely due to β-sheets commonly consisting of alternating hydrophobic and hydrophilic (polar or charged) sidechains (Zhang et al., 1993), with the hydrophobic sidechains orienting towards the air. Intermolecular β-sheets may also bind together, strengthening the protein network (Martin et al., 2005; Renault et al., 2002). Moreover, the number of random coils, α-helices, and β-sheets for a protein in bulk solution might each increase or decrease when introduced to a hydrophobic environment (Reddy and Nagara, 1989; Zangi et al., 2002), including the air-water interface (Martin et al., 2003; Yano, 2012), implying that protein conformation when adsorbed to the air-water interface could be different than when in solution (Lad et al., 2006; Vance et al., 2013; Yano, 2012). Measurements of shear stress and compressibility of protein network films versus the internal cohesion of the constituent protein show a correlation: the more stable a protein in bulk solution, the more robust the resulting protein network film at the air-water interface (Martin et al., 2005). At high enough surface concentrations and depending on surface charge distribution, neighboring globular proteins might interact to induce additional preferred orientations as has been shown in surface-protein studies (Billsten et al., 1995; Rabe et al., 2011; Tie et al., 2003). Such nearest neighbor protein-protein interactions may in turn decrease protein affinity to the interface and increase desorption. Similar effects might occur at protein-air-water interfaces. Given the length of incubation time commonly permitted before plunging a grid for cryoEM analysis, the cross-disciplinary research discussed above suggests that some particles in a thin film on a cryoEM grid will form a viscoelastic protein network film at the air-water interface. The composition and surface profile of the resulting protein network film will vary depending on the structural integrity of the bulk protein and the bulk protein concentration. Bulk protein affinity to the protein network film will then vary depending on the local affinity between the film and the proteins. To better understand the range of particle behaviors with respect to the air-water interfaces in cryoEM grid holes, a representative ensemble of grid and sample preparations needs to be studied in three dimensions. One method of studying single particle cryoEM grids is using cryoET. CryoET is typically practiced by adding gold fiducials to the sample preparation for tilt-series alignment, which requires additional optimization steps and might not be representative of the same sample prepared without gold fiducials. To avoid the issues imposed by gold fiducials, we have employed the fiducial-less tilt-series alignment method of Appion-Protomo (Noble and Stagg, 2015), allowing for cryoET analysis of all single particle cryoEM grids we have attempted. We used this fiducial-less cryoET method to investigate over 50 single particle cryoEM samples sourced from dozens of users and using grids prepared using either conventional grid preparation techniques or the new Spotiton (Jain et al., 2012) method. Our aim was to determine the locations of particles within the vitreous ice and the overall geometry of the ice in grid holes (related to the possible combinations in Figure 2). We have also found that the usefulness of performing cryoET on a single particle cryoEM grid extends beyond the goal of understanding the arrangements of particles in the ice. CryoET allows for the determination of optimal collection locations and strategies, single particle post-processing recommendations, understanding particle structural heterogeneity, understanding pathological particles, and de novo model building. We contend that cryoET should be routinely performed on single particle cryoEM grids in order to fully understand the nature of the sample on the grid and to assist with the entire single particle collection and processing workflow. We have made available a standalone Docker version of the Appion-Protomo fiducial-less tilt-series alignment suite used in these investigations at https://github.com/nysbc/appion-protomo. Results and discussion The fiducial-less tomography pipeline at the New York Structural Biology Center (NYSBC) consisting of Leginon (Suloway et al., 2005; Suloway et al., 2009) or SerialEM (Mastronarde, 2003) for tilt-series collection and Appion-Protomo (Noble and Stagg, 2015; Winkler and Taylor, 2006) for tilt-series alignment allows for the routine study of grids and samples prepared for single particle cryoEM in three dimensions. The resulting analysis sheds light on long standing questions regarding how single particle samples prepared using traditional methods (manual, Vitrobot, and CP3 plunging), or with new automated plunging with Spotiton (Jain et al., 2012), behave with respect to the air-water interfaces. In the following sections, we report and discuss how tomography collection areas were determined and analyzed, the observation that the vast majority of particles are local to the air-water interfaces and the implications with regards to potential denaturation, the prevalence of overlapping particles in the direction orthogonal to the grid, the observation that most cryoEM imaging areas and particles are tilted several degrees with respect to the electron beam, the value of cryoET to determine optimal collection locations and strategies, the benefits of using cryoET to understand pathological particle behavior, and the use of fiducial-less cryoET for isotropic de novo model generation. Determination of tomography collection locations The single particle samples studied here were sourced from a diverse set of grids, samples, and preparation techniques. Grid substrates include carbon and gold holey films, either lacey or with a variety of regularly spaced holes, and various nanowire grids (Razinkov et al., 2016) prepared using Spotiton. Grid types also include carbon Quantifoil (Ermantraut et al., 1998), gold Quantifoil (Russo and Passmore, 2014), and C-flat carbon on metal (Quispe et al., 2007). Plunging methods include plunging manually, with a Vitrobot (FEI Company, Hillsboro, OR) or CP3 (Gatan, Inc., Pleasanton, CA), and with Spotiton (Jain et al., 2012). With such diversity in samples and preparation techniques, we determined that the most feasible and representative collection strategy for analyzing particle and ice behaviors over dozens of preparations would be to collect in areas typical of where the sample owner intended to collect or had already collected single particle micrographs. For a typical grid, a low-magnification grid atlas or montage is collected, promising squares are imaged at increasing magnifications, and potential exposure locations are examined at high magnification until sufficient particle contrast and concentration is found as determined by the sample owner. Then before or after a single particle collection, typically three or more tilt-series are collected as described in the Materials and methods. Tilt-series were typically collected from −45° to 45° with a tilt increment of 3°, defocus of ~5 microns, total dose of ~100 e-/Å2, and a pixelsize between 1 and 2 Å. For most grids, one or two tilt-series are collected at the center of a typical hole and one or two tilt-series are collected at the edge of a typical hole, often including the edge of the hole if the grid substrate is carbon. Tilt-series are then aligned with Appion-Protomo (Noble and Stagg, 2015; Winkler and Taylor, 2006) for analysis as described in the Materials and methods. Analysis of single particle tomograms Single particle tomograms of samples described in Table 1 have each been analyzed visually using 3dmod from the IMOD package (Kremer et al., 1996). After orienting a tomogram such that one of the air-water interfaces is approximately parallel to the visual plane, traversing through the slices of the tomogram allows for the determination of relative particle locations, orientations, ice thickness variations in holes, and measurement of the minimum particle distance from the air-water interfaces. For many of the samples shown here and made available in the data depositions, particle orientations can be explicitly determined by direct visualization. Contamination on the surface of the air-water interface is used to determine the approximate location of the interface and to measure the ice thicknesses. After analyzing hundreds of single particle tomograms, we have concluded that sequestered layers of proteins in holes always correspond to an air-water interface, thus providing a second method for determining the location of the interface. Table 1 Ice thickness measurements, number of particle layers, preferred orientation estimation, and distance of particle layers from the air-water interface as determined by cryoET of single particle cryoEM grids for 46 grid preparations of different samples. The table is ordered in approximate order of increasing particle mass. Several particles are un-named as they are yet to be published. Sample concentration in solution is included with the sample name if known. Distance measurements are measured with an accuracy of a few nanometers due to binning of the tomograms by a factor of 4 and estimation of air-water interface locations using either contamination or particle layers. Grid types include carbon and gold holey grids and lacey and holey nanowire grids, plunged using conventional methods or with Spotiton. Edge measurements are made ~100 nm away from hole edges. '--' indicates that these values were not measurable. Samples highlighted with blue contain regions of ice with near-ideal conditions (<100 nm ice, no overlapping particles, little or no preferred orientation). Samples highlighted with green contain regions of ice with ideal conditions (non-ideal plus no particle-air-water interface interactions). Incubation time for the samples on the grid before plunging is on the order of 1 s or longer. https://doi.org/10.7554/eLife.34257.004 Sample #Sample nameGrid typeIce thickness (center, edge, substrate) in nm ± a few nm# of Layers (center, edge, substrate)Apparent preferred orientation in layer?Min. particle/layer distance from air- water interface (nm ± a few nm)1*32 kDa KinaseCarbon Spotiton6545--000Unknown<5232 kDa KinaseGold Spotiton30----0----Unknown<53Insulin ReceptorGold Spotiton55----1–2----No54*†HemagglutininCarbon Spotiton25–95100–210--0 or 22--Some55*HIV-1 Trimer Complex 1Carbon Spotiton75–210----2----Yes5–106*HIV-1 Trimer Complex 1Gold Spotiton20----1----Some57*HIV-1 Trimer Complex 2Carbon Spotiton190265--222Yes58147 kDa KinaseGold Spotiton15----1----Unknown<59150 kDa ProteinHoley Carbon Spotiton3570--222Some<510*Stick-like Protein 1‡Carbon Spotiton80----1----No<511Stick-like Protein 2 (150 kDa)‡Carbon CFlat100100--11--Unknown512*Stick-like Protein 2‡Gold Spotiton135–190----1----Some513*Neural Receptor‡Carbon Spotiton60–90----1----Yes514*Neural Receptor‡Carbon Spotiton80–90100–140135111Yes515200 kDa ProteinCFlat Carbon + Gold mesh40–6095110112No516Small, Popular ProteinCarbon with with in ProteinHoley in kDa ProteinCarbon Carbon Carbon Gold with with Carbon and with Carbon on is included for this is for this thick ice. Table 1 is with the single particle sample mass in 1000 single particle tomograms of over 50 different sample preparations have been collected over a of these samples are on samples include studied such as and and with various such as a and particles on affinity grids and Samples that are not have yet to be published. of the samples were prepared on gold or carbon nanowire grids, while the remaining were prepared on a variety of carbon and gold holey grids using common and techniques. Samples regions of ice in grid holes with near-ideal conditions than 100 nm ice thickness, no overlapping particles, and little or no preferred orientation are highlighted in blue of 46 in 1 and Samples regions of ice in grid holes with ideal conditions near-ideal conditions plus no particle-air-water interface are highlighted in green of 46 of the samples only areas that are not ideal for collection due to ice thickness than 100 overlapping particles, and/or preferred orientation. Table 2 air-water interface, particle, and ice behavior of the same samples in Table 1 using the in Figure Tilt-series were aligned and using the same and thus are oriented in the same the direction relative to the sample is not known. The air-water interface to and the to values as rendered in 3dmod from the IMOD package (Kremer et al., 1996). that the air-water interface is clean and be visually between A2 or in are particle layer angles are the angles between the particle normal and the electron beam direction, measured using in It is often difficult to distinguish between flat and ice at the air-water interfaces Figure 2, or or or most of not entire holes. indicates that the layer of is the same layer as the '--' indicates that these values were not measurable. Sample #Sample interface, particle behavior, and interface, particle behavior, and behavior interface, particle behavior, and interface, particle behavior, and behavior kDa or or B3 or or B3 or or or or into kDa or or B3 or or or or are or or B3 or or or or are particles, B3 or B3 or particles, or B3 B3 or thin ice in the center of holes particles, protein Trimer Complex B3 or B3 or and/or are adsorbed to air-water Trimer Complex B3 and/or are adsorbed to air-water Trimer Complex or B3 or B3 or or B3 or B3 or kDa or B3 or or or are kDa or B3 or B3 or or or B3 or Protein and B4 and B4 and Protein 2 (150 B3 and B4 and B3 and B4 and B3 and B4 and B3 and B4 and are not due to over and tilt Protein B3 or B3 or Note B3 or particles, or Note particles, or B3 B3 or particles, Note tomograms