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Single particle cryo-EM analysis of 6S-Eσ 70 particle orientation distributions in KGlu + NP40S and KCl + CHAPSO. A-B (Top, middle, bottom panels) Refer to Fig. 1. A. 2D classes and angular distribution of 6S-Eσ 70 particles in KGlu + NP40S (see Supplemental Table 1). B. 2D classes and angular distribution of 6S-Eσ 70 particles in KCl + CHAPSO (see Supplemental Table 1). C. Particles for each dataset were grouped into Euler angle bins (20° rotation angle × 20° tilt angle bind) and then the bins were ranked according to the number of particles populating that bin (bin #1 has the most particles, so on). Plotted on a semi-log scale is the percent of total particles in each dataset by bin #. The horizontal dashed line represents a totally random particle orientation distribution (equal number of particles in each bin). (Inset) Plotted is the cumulative percent particles by bin #. The random distribution is denoted by the dashed line. D. Cross-sections through the middle of the expected PSFs (calculated using cryoEF (Naydenova and Russo, 2017) are superimposed, illustrating the anisotropy for the KGlu (red), KCl (blue), and KGlu + NP40S (orange) samples, while the KCl + CHAPSO sample yields an isotropic PSF (green). Parameters further characterizing the orientation distributions (the orientation efficiency, E od , and the fraction of unsampled Fourier space, f empty (Naydenova and Russo, 2017) are also tabulated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Preferred particle orientation presents a major challenge for many single particle cryo-electron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed...
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... of the particles in KGlu + NP40S ( Fig. 2A) also yielded two mirror image peaks [(rot −130°, tilt −47°) and (rot 50°, tilt 47°)]. The distribution was broadened with respect to the KGlu and KCl distributions, with standard deviation ± 20°. Thus, the addition of the detergent still gave only one effective orientation, but the bias was slightly ameliorated. In contrast to KGlu, ...
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... deviation ± 20°. Thus, the addition of the detergent still gave only one effective orientation, but the bias was slightly ameliorated. In contrast to KGlu, KCl, and KGlu + NP40S, particles prepared in KCl + CHAPSO did not exhibit peaks of preferred orientation; instead the particle orientations were spread over a large fraction of Euler angles (Fig. 2B), resulting in isotropically uniform 3D reconstructions (Supplemental Fig. 1B). Scheres, 2012) based on particle population. Absolute number and percentage of particles for each class are designated in white text. (Middle Panel) 3D distribution plot of particle orientations. Particles were 3D classified into one class using Eco core ...
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... angles (corresponding to 20° increments in rotation and tilt angles) and ranked according to the percentage of particles in each bin (bin #1, highest %; bin #2, next highest %; so on). The orientation distribution of the particles prepared in each condition was compared by plotting the histograms of the % particles in each bin according to bin # (Fig. 2C). A completely random distribution of particle orientations would yield a flat distribution, with 0.61% particles in each bin (dashed horizontal line in Fig. 2C). Visualizing the orientation distributions this way highlights the bias of the KGlu, KCl, and KGlu + NP40S samples. The KCl + CHAPSO sample, while not completely randomized, ...
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... next highest %; so on). The orientation distribution of the particles prepared in each condition was compared by plotting the histograms of the % particles in each bin according to bin # (Fig. 2C). A completely random distribution of particle orientations would yield a flat distribution, with 0.61% particles in each bin (dashed horizontal line in Fig. 2C). Visualizing the orientation distributions this way highlights the bias of the KGlu, KCl, and KGlu + NP40S samples. The KCl + CHAPSO sample, while not completely randomized, approaches the random orientation distribution more closely. The inset of Fig. 2C plots the cumulative % of particles across the bins. This plot reveals that 50% ...
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... yield a flat distribution, with 0.61% particles in each bin (dashed horizontal line in Fig. 2C). Visualizing the orientation distributions this way highlights the bias of the KGlu, KCl, and KGlu + NP40S samples. The KCl + CHAPSO sample, while not completely randomized, approaches the random orientation distribution more closely. The inset of Fig. 2C plots the cumulative % of particles across the bins. This plot reveals that 50% of the particles are binned into only 5, 9 and 12 bins for the KGlu, KCl, and KGlu + NP40S conditions, respectively, while 50% of the KCl + CHAPSO particles are spread out over 36 bins (Fig. ...
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... approaches the random orientation distribution more closely. The inset of Fig. 2C plots the cumulative % of particles across the bins. This plot reveals that 50% of the particles are binned into only 5, 9 and 12 bins for the KGlu, KCl, and KGlu + NP40S conditions, respectively, while 50% of the KCl + CHAPSO particles are spread out over 36 bins (Fig. ...
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... reconstructions were also analysed using cryoEF (Naydenova and Russo, 2017). A cross-section through the middle of the expected point spread functions (PSFs) calculated from the particle orientation distributions reveals the severe anisotropy of the KGlu, KCl, and KGlu + NP40S PSFs while the KCl + CHAPSO PSF appeared as roughly a spherical ball (Fig. 2D). Also tabulated in Fig. 2D is the orientation Supplemental Table 1). B. 2D classes and angular distribution of 6S-Eσ 70 particles in KCl + CHAPSO (see Supplemental Table 1). C. Particles for each dataset were grouped into Euler angle bins (20° rotation angle × 20° tilt angle bind) and then the bins were ranked according to the number ...
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... using cryoEF (Naydenova and Russo, 2017). A cross-section through the middle of the expected point spread functions (PSFs) calculated from the particle orientation distributions reveals the severe anisotropy of the KGlu, KCl, and KGlu + NP40S PSFs while the KCl + CHAPSO PSF appeared as roughly a spherical ball (Fig. 2D). Also tabulated in Fig. 2D is the orientation Supplemental Table 1). B. 2D classes and angular distribution of 6S-Eσ 70 particles in KCl + CHAPSO (see Supplemental Table 1). C. Particles for each dataset were grouped into Euler angle bins (20° rotation angle × 20° tilt angle bind) and then the bins were ranked according to the number of particles populating that ...
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... that adsorption of the 6S-E σ 70 particles at air/water interfaces gives rise to the severe orientation bias seen in the KGlu, KCl, and KGlu + NP40S samples (Figs. 1, 2). The addition of CHAPSO at the critical micelle concentration (CMC, 8 mM) completely eliminates surface interactions and significantly randomizes the particle orientations ( Fig. 2B-D, 3D). To investigate if CHAPSO at CMC was required for the full effect, we compared particle orientations for datasets collected with 0, 4 mM (0.5XCMC), and 8 mM (1XCMC) CHAPSO 4. Effect of CHAPSO on particle orientations is concentration dependent. A -C. (Top Panel) 3D distribution plot of particle orientations. Particles were 3D ...
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... The addition of CHAPSO at the CMC (8 mM) completely prevents adsorption of the complexes to air/water interfaces ( Fig. 3D) and dramatically broadens the distribution of particle orientations (Figs. 2B-D, 3D, 4C-E), allowing for the determination of isotropically uniform maps (Figs. 2D, 4E; Supplemental Fig. 1B) (Naydenova and Russo, ...
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... The addition of CHAPSO at the CMC (8 mM) completely prevents adsorption of the complexes to air/water interfaces ( Fig. 3D) and dramatically broadens the distribution of particle orientations (Figs. 2B-D, 3D, 4C-E), allowing for the determination of isotropically uniform maps (Figs. 2D, 4E; Supplemental Fig. 1B) (Naydenova and Russo, ...
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... binding to the surface of RNAP (Fig. 5), altering the RNAP surface properties to prevent adsorption at the air/ water interface. Our result that 8 mM CHAPSO is required for the full effect (Fig. 4) strongly supports hypothesis (1) as the primary factor, since at 4 mM CHAPSO we still observe CHAPSO molecules bound to the RNAP (Supplemental Fig. ...
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... grid preparation of samples comprising Eco RNAP transcription complexes is relatively functionally inert, completely eliminates interaction and orientation of the particles at air/water interfaces (Fig. 3), and significantly broadens the particle orientation distributions to allow determination of high-resolution cryo-EM maps with isotropic PSFs (Figs. 2D, 4E). These properties greatly facilitate high-resolution structure determination of these complexes using modern cryo-EM approaches Kang et al., 2017;Kang et al., 2018a,b;Boyaci et al., ...
Citations
... Speculating that the aggregation might be associated with the air-water interface issues 27,28 , we introduced a non-ionic detergent into the Rituximab-diluting buffer since this approach had effectively mitigated the air-water interface issues 29 . We chose n-Dodecyl-beta-Maltoside (DDM) as it had been proven compatible with Rituximab, given its previous use in cryo-EM studies of Rituximab in complex with CD20 8,9 . ...
While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.
... By contrast, stainless steel is more prone to causing electrochemical reactions on proteins, therefore damaging protein structures severely. denaturation and/or preferred orientation of target molecules 17,18 , compromising successful structure determination 19,20 . Previous attempts to address the issue involved reducing the spot-to-plunge time 21 . ...
Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air–water or graphene–water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.
... G. Han et al. 2022;Weissenberger, Henderikx, and Peters 2021). These problems including aggregation, denaturation, disassembly, preferred orientation, etc. are mostly caused by the air-water interface binding of the sample during freezing (Chen et al. 2019) (Fig.S1). ...
... Other sample preservation approaches have been used such as glutaraldehyde fixation or fixation in agar which may help to stabilize heterogenous samples (Adamus et al. 2019). Many samples have seen success by using a binding surface like graphene or graphene oxide to trap the sample and keep it away from the air-water interface (Chen et al. 2019;D'Imprima et al. 2019). Alternatively some approaches have used fast automated plunging with self-wicking grids to have a much faster plunge/blot time and involve less human error during the freezing (Levitz et al. 2022). ...
In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.
... Here, we asked whether the improved strategies for processing cryo-EM data can help understand how this polymerase transcribes unnatural base pairs not found in the standard DNA or RNA "alphabets"in particular, how Us bind, how mispairing is recognized, and how next generation UBPs might be designed. This is especially challenging for RNAP, which is known to exhibit strong preferential orientation on cryo-EM grids 39 ; conventional data collection strategies have typically yielded maps characterized by severe resolution anisotropy characterized by elongated features. The inclusion of detergent can produce additional views 39 , leading to resolvable features within cryo-EM maps, however, the resulting orientation distribution remains non-uniform. ...
... This is especially challenging for RNAP, which is known to exhibit strong preferential orientation on cryo-EM grids 39 ; conventional data collection strategies have typically yielded maps characterized by severe resolution anisotropy characterized by elongated features. The inclusion of detergent can produce additional views 39 , leading to resolvable features within cryo-EM maps, however, the resulting orientation distribution remains non-uniform. Further, it is well-known that the addition of detergent is Apoferritin cryo-EM data was collected at increasing stage tilt angles (0°black bars, 10°light orange bars, 20°light blue bars, 30°green bars, 40°yellow bars, 50°dark blue bars, 60°dark orange bars) and equalized for defocus, with 17,000 particles used per tilt angle. ...
... In stark contrast, the reconstruction from 60°-tilted data produced a map with features fully consistent with the model (Fig. 9a bottom). This latter map was comparable in quality to typical reconstructions derived using cryo-EM 39,40 and readily lent itself to biological interpretations. Merging the untilted and tilted datasets did not yield an improvement in the quality of reconstructions compared to the tilted dataset alone (see Supplementary Note 2). ...
Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.
... Several methods have been developed to improve particle behavior in vitreous ice. In some cases, adding detergent, like CHAPSO, can eliminate particle adsorption to AWI 10 . Coating of cryo-EM grids with a thin layer of graphene materials as supporting film also improves sample quality in vitreous ice [11][12][13] . ...
... Using a larger dataset for HA with MSBP, we finally obtained a high quality cryo-EM density map for structural analysis (Fig. 4a, Supplementary Fig. 13a, b). In some cases, addition of detergent can also improve particle distribution in vitreous ice 10 . Systematical study shows nonionic and zwitterionic but not ionic detergent could separate protein particles from AWI 31 . ...
Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air–water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air–water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.
... The freshly prepared complexes were incubated with 3-([3-cholamidopropyl] dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO, 8 mM; Hampton Research) before grid preparation 52 . Then, a drop of 3 µl was placed on a glow-discharged holey gold grid (UltrAuFoil, R 1.2/1.3 ...
Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA–NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a ‘DNA looping’ mechanism of cooperative transcription activation in bacteria.
... Speculating that the aggregation might be associated with the air-water interface issues [27][28], we introduced a non-ionic detergent into the Rituximab-diluting buffer since this approach had effectively mitigated the air-water interface issues [29]. We chose n-Dodecyl-beta-Maltoside (DDM) as it had been proven compatible with Rituximab, given its previous use in . ...
While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating cancer cell toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.
... Due to certain limitations, in some cases it is not possible to obtain a high resolution structure of a membrane protein:VHH complex using CryoEM, even with the assistance of a marker such as Fab-V1. These limitations include lack of ready access to an electron microscope or computational resources to process the data, insufficient particle adsorption to the grid (Cianfrocco & Kellogg, 2020;Xu & Dang, 2022), lack of preferred particle orientation at the air-water interface (Chen et al., 2019;Xu & Dang, 2022), and deviations from perfect symmetry reducing the attainable resolution (Tsumoto et al., 1996). Therefore, we decided to investigate whether the crystallizability of the Fab-V1:VHH complex is enhanced when the S1, Crystal Kappa, and elbow substitutions are incorporated into the framework, thus improving the capacity of Fab-V1 to act as a chaperone in the crystallization of membrane protein:VHH complexes (which cannot be solved by CryoEM for instance) (Figure 5a(ii)). ...
The atomic‐resolution structural information that X‐ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage‐display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.
... Final 4 mM of MgCl 2 and BTP or STP or UTP in 1X EB were added to elongation complex and incubated in ice for 30 min. Before sample preparation, final 4 mM CHAPSO dissolved in 1X EB were added to complex to reduce particle orientation bias 47 . The final E. coli RNAP concentration was 15−18 mg/ml. ...
Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.
... The most widely used approach in decreasing the preferred orientation of macromolecules is to add surfactants to the sample buffer prior to grid preparation (Chen et al., 2019;B. Li et al., 2021). ...
... It is now known that the tendency of macromolecules to adopt preferential orientation when frozen on ice using cryo-EM grids is a consequence of protein-AWI interaction (J. Chen et al., 2019;, but the nature of this interaction remains unclear. On the other hand, amphipathic molecules have been used in cryo-EM grid preparation since the 1980s (Frederik et al., 1989). ...
... Their use in solving the preferred orientation of proteins by modulating the air-water interface has recently been appreciated in cryo-EM and is now being used more routinely for high-resolution structure determination (J. Chen et al., 2019;S. Chen et al., 2022;Li et al., 2021). ...
The formation of vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge freeze method described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the airwater interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, through an analysis of selected wellstudied macromolecules, the effect of physical and chemical factors on the orientations of macromolecules was investigated, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and provide a framework to design small-molecule additives to enhance sample stability and behaviour.
