N-halosuccinimides as the halogen source under neat grinding or liquid-assisted grinding conditions in a ball mill has been described. Depending on the azobenzene substrate used, halogenation of the C–H bonds occurs in the absence or only in the presence of PdII catalysts. Insight into the reaction dynamics and characterization of the products was achieved by in situ Raman and ex situ NMR spectroscopy and PXRD analysis. A strong influence of the different 4,4’-substituents of azobenzene on the halogenation time and mechanism was found.The direct and selective mechanochemical halogenation of C–H bonds in unsymmetrically substituted azobenzenes using  Electrophilic aromatic substitution –3 and liThe need for green and sustainable chemistry  has led 2]2 in combination with AgSbF6 as catalyst and N-halosuccinimide  as halogen source [N-bromosuccinimide (NBS) and MCM-41-SO3H catalyst and no liquid additives [ortho-halogenation of acetanilide with NXS  using Pd(OAc)2 as precatalyst in the presence of p-toluenesulfonic acid (TsOH) as an additive under solvent-free conditions [In 2015, Bolm and Hernandez reported the halogenation of 2-phenylpyridine in a ball mill using [Cp*RhCln source . Two yeadditives . In 2018nditions . Recentlnditions .II-catalyzed bromination of unsubstituted azobenzene (L1) by N-bromosuccinimide (NBS) under neat grinding (NG) and liquid-assisted grinding (LAG) conditions in a ball mill [II species, cyclopalladated intermediates, and products 2 as precatalyst in the presence of TsOH and MeCN as solid and liquid additives, respectively, led to the ortho-halogenated products relative to the azo group of the azobenzenes. In situ Raman monitoring of these reactions confirmed that the most favorable reaction pathway is via the monomeric cyclopalladated intermediate, as in the halogenation reaction of unsubstituted L1 [L1 and its para-halogenated derivatives were unsuccessful without the PdII catalyst and TsOH, the halogenation of azobenzenes with the strong electron-donating substituents in the para position occurred in the absence of the added PdII catalyst and additives, in the ortho position to the substituent, which is typical for the products of electrophilic aromatic substitution.Here we present the mechanochemical selective halogenation of unsymmetrically substituted azobenzenes by NXS . The liquid-assisted grinding of tuted L1 . While tII catalyst was developed for the selective imidation of azobenzenes containing a dimethylamino group as substituent in the para position to the azo group.In addition, an additive- and solvent-free protocol without the added PdL1 [Inspired by our findings on the mechanochemical halogenation of L1  and the L1 , we inveL1 [L2), 4-aminoazobenzene (L3), 4-dimethylaminoazobenzene (L4), and 4-(dimethylamino)-4'-nitroazobenzene (L5)) with NXS (X = Cl and Br) occurred in the absence of the added PdII catalyst and additives. These reactions in most cases produced electrophilic substitution products that were halogenated in the ortho position(s) relative to the electron-donating substituent  relative to the electron-donating substituent, which may be attributed to competition between PdII-catalyzed reactions and uncatalyzed electrophilic substitution.In contrast to the reactions of L1 , the halstituent , as confstituent  and NMR stituent . All expII catalyst [Our results are consistent with those reported by Sanford's group for the halogenation of various substrates by NXS with and without Pdcatalyst . Most ofcatalyst , resulteL3 and L4 with NCS produced the monochlorinated products L3Cl-I and L4Cl-I as single isomers within one hour (L5 with NCS resulted in a mixture of mono- and dichlorinated regioisomers (L5Cl-I and L5Cl-II) , the chlorination of L5 proceeded much more slowly (Neat grinding of L5Cl-II) , entry 4L2 (L2 with NBS gave the monobrominated product L2Br-I in 96% yield after 15 hours of milling (L2 confirmed its conversion to the product L2Br-I (L2Br-I was accompanied by the intensity decrease of L2ν(N=N) and ν(C–N) bands in the range 1400–1450 and 1080–1200 cm−1, respectively. Compounds L2 and L2Br-I were identified in the reaction mixture by the Raman spectra of isolated L2 and L2Br-I  at the para position of the second phenyl ring in L5 significantly slowed the halogenation reaction and impaired the reactivity of the azobenzene. This result suggests that NO2 has a long-range effect that spreads through the azobenzene skeleton. Compared to the bromination of substrates L3–5 containing amino substituents, the analogous reaction of L2 is slower because the methoxy substituent has weaker donor strength than amino substituents [The reactivity trend of electron-rich azobenzenes tituents .II catalysts for the halogenation of Csp2–H and imidation of Csp3–H bonds of azobenzenes with electron-donating substituents by electrophilic activation with NXS.The protocols described above provide a solvent- and additive-free approach without added PdL1 [para position relative to the azo group: 4-chloroazobenzene (L6), 4-bromoazobenzene (L7), and 4-iodoazobenzene (L8)  .Ln/NXS/TsOH 1:1.2:0.5 equiv, 5 mol % Pd(OAc)2 precatalyst, and 15 µL MeCN as liquid additive. Under these conditions, the reactions of L6–8 with NBS resulted in moderate to good yields of monobrominated products LnBr-IV 2(MeCN)2 was identified as the active catalyst, formed in situ, in the bromination reaction of L1 [L6–8 were carried out using Pd(OTs)2(MeCN)2 as the catalyst instead of Pd(OAc)2. As expected, the yields of LnBr-IV products were comparable to those obtained in the reactions with the Pd(OAc)2 precatalyst.The bromination of on of L1 , the anaII-catalyzed iodination of L6–8 was conducted with N-iodosuccinimide (NIS) as the iodine source. The reaction time for the iodination of L6 was the same as for the analogous bromination reaction  gave no chlorinated product, which was also observed in the reaction of L1 with NCS  data  data , are simL6–8 in the presence of 30 mol % PdII catalyst revealed a new band around 1380 cm−1 (ν(N=N) bands of the In-I intermediates, confirming that the bromination of L6–8 proceeds via monopalladated In-I intermediates as in the reactions of L1 2 with L6–8 and TsOH were performed using 25 µL of MeCN as a liquid additive in a molar ratio of 1:1:1 (Ln/Pd(OAc)2/TsOH). In situ Raman monitoring of C–H bond activation was possible for L6 and L7, while in the case of L8, fluorescence prevented a more detailed insight into this reaction. The monitoring results confirmed the complete conversion of azobenzenes L6 and L7 to their monopalladated products after about one hour of milling, as well as a reaction course similar to that of the analogous reaction of L1 [To gain insight into the dynamics of the formation of cyclopalladated intermediates on of L1  2 as precatalyst, as confirmed by NMR spectroscopy.The isolated cyclopalladated intermediates L1 [L6–8 begins with the formation of the catalytically active PdII species, Pd(OTs)2(MeCN)2, from Pd(OAc)2, TsOH, and MeCN. It is followed by the formation of monomeric cyclopalladated intermediates In-I, at which halogenation occurs. Based on the similarities between the halogenation of L6–8 and the bromination of L1 [I1-I are also possible for the halogenation of I6-I, I7-I, and I8-I [The results of in situ and ex situ spectroscopic monitoring along with the structural characterization of the intermediates have shown that the mechanism of the halogenation of azobenzenes with electron-accepting substituents is consistent with the proposed mechanistic schemes for the bromination of the unsubstituted azobenzene L1 . Thus, ton of L1 , we assuand I8-I .L1 and its para-halogenated derivatives L6–8 toward NBS or NIS. The competition experiments showed that the azobenzenes with electron-withdrawing substituents are much less reactive to halogenation than L1. The reactivity of azobenzenes in the case of NBS decreases in this order: L1 >> L6 > L7 ≈ L8  as the halogen source.II catalyst. These transformations, which take place via electrophilic aromatic substitution, resulted in products halogenated in the ortho position to the electron-donating groups. The reactions of azobenzenes containing a dimethylamino group as substituent with NIS led to imidation products. A different reactivity of the dimethylamino group compared to the other substituents was also observed in these reactions.Halogenation of azobenzenes with strong electron-donating groups was carried out without an added Pdpara-halogenated azobenzenes required the presence of the PdII catalyst and TsOH as additive. In situ spectroscopic monitoring of these reactions revealed that the PdII-catalyzed halogenation proceeds via monomeric cyclopalladated intermediates formed by activation of the C–H bond in azobenzenes with the in situ generated Pd(OTs)2(MeCN)2 catalyst. The described results indicate a strong dependence of the halogenation outcome of C–H bonds in 4,4’-functionalized azobenzenes on the nature of their substituents.On the other hand, the halogenation of File 1Detailed experimental procedures, complete characterization data for new compounds, X-ray structures of compounds, and the results of in situ Raman monitoring.File 2X-ray crystallographic data.


Unique features of the Boudouard reaction coupled with CO‐driven catalyst engineering ensure not only suppression of the second layer growth but also provide a simple and reliable technique for surface cleaning. Aside from being a novel carbon source, carbon monoxide ensures peculiar modification of catalyst and in general opens avenues for breakthrough graphene‐catalyst composite production.Following the game‐changing high‐pressure CO (HiPco) process that established the first facile route toward large‐scale production of single‐walled carbon nanotubes, CO synthesis of cm‐sized graphene crystals of ultra‐high purity grown during tens of minutes is proposed. The Boudouard reaction serves for the first time to produce individual monolayer structures on the surface of a metal catalyst, thereby providing a chemical vapor deposition technique free from molecular and atomic hydrogen as well as vacuum conditions. This approach facilitates inhibition of the graphene nucleation from the CO/CO Unique features of Boudouard reaction facilitate high‐purity, wafer‐scale single‐crystal graphene on metal catalysts. Nevertheless, the method for the state‐of‐the‐art graphene, i.e., “natural” exfoliation, is strictly limited in terms of the sample size and geometry. Therefore, a large‐scale graphene synthesis is a challenge to bring lab‐scale prototypes to a wide range of applications. One of the most advanced approaches to graphene preparation is chemical vapor deposition (CVD). This method comprises catalytic decomposition of gaseous carbon sources  on metallic substrates. The CVD synthesis of single‐crystal monolayer graphene is an unprecedented challenge for catalysis: the cycle of carbon precipitation should be terminated after the first graphitic layer, and carbon‐, hydrogen‐, and oxygen‐based intermediates should control the nucleation of new seeds.Graphene has become a milestone discovery in contemporary solid‐state physics and materials science.1 Prop4 and C2H4 on the surface of metallic  and semiconducting (Ge) substrates. One of the strategies to obtain single‐crystal graphene is the alignment of the graphene nuclei that prevents defect formations caused by grain boundaries. This method demands a single‐crystalline metallic foil, which requires additional technological steps. Another strategy relies on the inhibition of graphene nucleus density so that each seed grows up into a large graphene crystal. Such an approach requires a balance between nucleation control and the growth rate of a single grain. In this case, the pressure within the reactor plays a dominant role to control the nucleation process. Remarkable progress in obtaining large graphene samples with outstanding electronic properties and superior crystallinity was achieved using molten and resolidified substrates. However, according to the recent review, rapid synthesis technique facilitating single grain growth is still a challenge.During the past decade, wafer‐scale single‐crystal graphene synthesis was achieved using hydrocarbons such as CH and “graphene‐like structures” were recently observed, CO has never been used for the CVD synthesis of monolayer graphene on a metallic substrate. Carbon monoxide is truly a unique carbon source. Unlike the conventional methods based on pyrolysis of hydrocarbons requiring hydrogen to control carbon intermediates, the unique thermodynamics of the Boudouard reaction, 2CO ⇆ C + CO2, under high temperatures favors CO, not the carbon precipitation. The high stability of CO molecule restricts noncatalytic pyrolysis, and, thereby, formation of amorphous deposits or second graphitic layer. Moreover, carbon monoxide appears to have a peculiar interaction with the surface of transition metals, especially with copper.A carbon source with specific stability to prevent nucleation of multiple grains by both pyrolysis and surface decomposition may help to overcome the abovementioned problems. Surprisingly, one of the simplest carbon sources, carbon monoxide, has acquired little attention. Though the first multilayer graphitic structures were reported as early as in the 1950s17 and and single‐walled carbon nanotubes (SWCNTs) both on lab‐ and industrial scales. Indeed, HiPco (high‐pressure CO) was one of the very first technologies to deliver a high yield SWCNT synthesis, while the combination of the aerosol CVD method with CO provides state‐of‐the‐art transparent conducting films. It should be mentioned that SWCNT growth based on the Boudouard reaction allows nucleation control by the addition of CO2. At the same time, CO2 is also a superior pre‐treatment “cleaning” tool during the graphene synthesis. However, the utilization of CO2 with hydrocarbons to control nucleation and growth processes during graphene synthesis is limited. In contrast, the Boudouard reaction allows the implementation of both nucleation control and perfect cleaning by CO2. Finally, the CO‐based synthesis route is completely hydrogen‐free, favorable in atmospheric or high‐pressure CVD implementations.Taking into account all benefits of CO utilization, the absence of the method for monolayer graphene synthesis is even more interesting, since the well‐studied Boudouard reaction has been actively used for the synthesis of graphite26 and2Figure 2 rich atmosphere at 1135 °C . We have constructed a horizontal tubular hot‐wall reactor equipped with a fast load‐in∖out ceramic manipulator Figure 1a–d. AftC Figure , a CO/COC Figure . After aC Figure . The sysFigureI2D/IG greater than 2 over the entire area of the sample. Dendritic boundaries are likely formed due to high growth rates, while the produced domains still preserve hexagonal shape that suggests a single‐crystal nature of the layer. X‐ray photoelectron spectroscopy (XPS) data collected from the graphene‐coated copper surface reveal the outstanding purity of both the graphene and the copper substrate  in Figure LMM lines  measurements performed on the same sample. These data reveal classical electronic landscape and graphene Dirac cone with a common electron doping shift. It is worth noting that a Shockley surface state can be clearly observed near the Fermi level (0.285 eV). This electronic state is another evidence of pure reduced copper, which surface is protected by a graphene layer. Such an observation is rather unusual for a CVD graphene on a copper catalyst. Note that this state, intrinsic to Cu (111) surface, has never been observed for as‐grown samples when non‐single‐crystal substrates were used.Catalytic disproportionation of carbon monoxide leads to the formation of graphene grains of up to several mm in size Figure . These ae Figure . Obtainel Figure , indicatFigure  Such an unprecedented alignment facilitates the formation of single‐crystal grains of graphene, which is also confirmed by LEED obtained from individual graphene crystals of ≈4 mm in size  plane Figure  alone spFigure 24 Suce Figure . The twoÅ Figure  and reve] Figure . For mor copper pellet on top of a molybdenum foil (acting as a wetting layer by heating to 1135 °C and cooling to 1085 °C) and added a procedure of CO2 annealing during the melting phase for additional copper cleaning. It should be stressed that we observed no traces of carbon species on Cu after CO2 annealing with XPS and Raman spectroscopy. Thereby, the rate of graphene nucleation was reduced by recrystallization of copper by several orders of magnitude reaching 102–103 cm−2s−1 while no impurities leading to multilayer graphene were observed as well.A single crystal graphene synthesis is a result of a thorough kinetic study for the mechanisms of grain nucleation and growth. When working with copper foils, we have observed the graphene nucleation to be extremely sensitive to the surface roughness or impurities; we even observed the formation of the multilayer graphene  and nuclei surface density (N)  graphene synthesis terminates. The dependence of the growth rate on the average grain perimeter , diffusion (Ed = 1 eV), catalytic decomposition of CO molecule (Edec = 2–4 eV), and attachment of the carbon atom to the growing front of the nucleus (Eatt = 2 eV). The last process is more likely to contribute the most to the limiting process.The temperature dependences of the growth rate WG and nuc2 removal shows the highest barrier in the sequence  surface using density functional theory (DFT) methods  allows to significantly reduce the time (down to 10 min) required for millimeter size crystals , a smooth balance between CO2 concentration and total pressure value is the key for facile synthesis of single‐crystal graphene.Small amounts of COreaction.29 As e Figure  while ths Figure . Thus, sFigure dθx/dt):θx, θCO, θCO2, θ—surface coverages for carbon nuclei, CO, CO2, and free surface, respectively; W2+—the rate for decomposition of an adsorbed CO, kdiff, kdes—kinetic constants for diffusion and desorption of carbon intermediates; β—arbitrary constant for the diffusion of carbon intermediates and carbon dimers and nuclei, which can be estimated experimentally; k7 + , k7 − —kinetic constants for CO2 surface assembly from CO and O and decomposition, respectively, k8—kinetic constants for CO2 etching of carbon intermediates.To reveal the graphene nucleation mechanism for the process based on the Boudouard reaction, we developed a model following Donohoe and Robins formalism.55FigAs for the experiments on nucleation temperature dependence, the carbon dioxide concentration was zero, we can omit corresponding terms to describe two cases denoted as “high” and “low” temperature by Donohoe and Robins.55k we did not observe in experiment two distinct regimes for nucleation, only “high temperature one.” We attribute this to the high stability of CO molecule that prevents decomposition at conditions where diffusion controls the nucleation. Moreover, depending on CO2 concentration, we observe two distinct regimes meeting the experimental findings: CO2‐independent  does not lead to the complete termination of the growth, but rather to a self‐etching of individual crystals. Even more, the critical concentration of CO2 . Above 500 cm−1 the spectra practically coincide, while the difference below ≈500 cm−1 is due to free charge carriers in the graphene layer  = σDC × [1 − iωτ]−1, where σDC = neμ = ne2/ɣme* is the direct current conductivity, n is charge carrier concentration, ɣ = 1/(2πτ) is charge carriers scattering frequency (inverse scattering time τ), me* is the electron effective mass, and μ is the electron mobility. The calculated parameters of the Drude‐model fit for the samples grown with different CO2 concentrations are shown in Figure 2 concentration (the red curve), as one would expect, since adding CO2 promotes the growth of greater graphene crystals. At the same time, the conductivity value decreases (the black curve), which suggests a decreasing charge carrier concentration. The obtained UV‐vis‐NIR spectra of the samples are consistent with the single‐layer nature of graphene: indeed, the π plasmon is located at ≈270 nm, and the transmittance is close to 97.7%  × 104 cm2V−1s−1, likely indicating that the graphene transfer on PF affected its intrinsic properties. Subsequent transfer to Si/SiO2 substrate most likely caused more cracks and further decrease in the mobility. Clearly observable Dirac cone with vf = 106 m s−1 also indicates high quality of as‐grown graphene electronic properties.Next, we investigated the charge carrier transport in the transferred graphene by means of contactless Fourier‐transform far‐infrared (FIR) and time‐domain terahertz (THz) spectroscopy and measurements of the field‐effect transistors (FETs) based on graphene with Au/Cr side‐contacts in a Hall‐bar geometry. In the latter devices, the graphene single crystal is encapsulated in parylene‐N, and the heavily doped Si substrate is used as a back gate. To obtain electrodynamic properties of synthesized graphene, we have measured transmission coefficient spectra of graphene supported by a parafilm (PF) substrate and that of a substrate alone  is reduced. Furthermore, the proposed procedure shows significant reduction in duration of heating and cooling procedures to a few tens of minutes. The catalyst preparation step, which might take 7–18 h, is reduced to 15 min. At the same time, high and competitive quality of the produced graphene can be easily achieved.From the engineering point of view, the utilization of CO simplifies the synthesis procedure: there is no need in vacuum12 or 2 allows synthesis of patterned graphene full of holes with perfect boundaries. The presented approach also allows further investigation of transition metal's electronic surface states protected by the graphene layer from degradation. Specific chemistry of various elements with carbon monoxide may allow formation of transfer‐free graphene, e.g. Ti + 2CO ⇆ TiO2 + 2C, TiCl4 + 2CO ⇆ TiO2 + 2C + 2Cl2 or Al2S3 + 3CO ⇆ Al2O3 + 3C + 3S which has been demonstrated for aluminum sulfide powder and can be extended to an atomic layer deposition or CVD films of aluminum sulfide.Future development of the proposed technique might include the design of one‐time‐fill operation under elevated pressure in the autoclave‐bomb of a cold‐wall reactor in order to reach extreme growth rates, which potentially can be upscaled to the wafer size graphene grown in a matter of several minutes. Aside from the well‐known applications of graphene in optoelectronic devices (assuming optimized transfer protocol), the reported novel synthesis opens several new avenues for the scientific community to explore. To name a few, the self‐etching mechanism of Boudouard reaction equilibrium shifting by COundaries.58 The32 concentration) altogether make the system safe and easy‐to‐build when compared to the hydrocarbon‐derived routes.To sum up, we show carbon monoxide to be a promising carbon source for the naturally self‐limiting synthesis of graphene on metallic substrates with low carbon solubility. High temperature annealing of the Cu surface in CO facilitates both a high degree of crystallinity of the formed graphene and modifies the catalyst resulting in the single‐crystal formation. The experimental design and thorough engineering of the growth governed by the Boudouard reaction allowed us to dramatically inhibit the nucleation rate reaching the regime of single‐crystal graphene growth highlighting the route toward the high pressure CO process—HiPco‐G. Besides, it also significantly streamlines the reactor design—operation at ambient or elevated pressures, absence of hydrogen, tuning a smaller set of parameters  to anneal, melt, and clean the surface. Subsequently, the furnace was cooled down to 1085 °C within 4 min and the atmosphere was changed to a combination of CO and CO2, depending on the desired sample type. Next, the sample was rapidly pulled out to a water‐cooled flange zone and kept for 10 min under the synthesis atmosphere . Finally, the chamber was purged with the inert gas and the sample was taken out for further analysis.Chemical vapor deposition hot‐wall reactor was implemented in the form of a horizontal tube furnace with corundum tubular chamber. One of the tube flanges is connected to the set of mass flow controllers to introduce high purity Ar, CO, N2, during 3 h.Ambient pressure: 1085 °C, 3000 sccm CO, 8 sccm CO2, during 12 min at 3500 mbar reactor pressure.Elevated pressure: 1085 °C, 3000 sccm CO, 18 sccm COAmbient pressure: 1085 °C, 3000 sccm CO for 12 min.Elevated pressure: 1085 °C, 1000 sccm CO, for less than 1 min at 3500 mbar reactor pressure.2 during 3 h. The Raman spectra and mapping in Figures 2 substrate. XPS spectra in Figure 2 within 1 h. NEXAFS and ARPES data in Figure 2 within 1 h was used, while for synchrotron studies in Figure 2 for 2 h was used. For LEED measurements  per point were captured by SEM or optical microscope. For high pressure experiments the CO2 concentration was fixed at 0.5% vol and at least 20 grains were collected. These measurements were complicated for lower CO2 concentration by the fact of extremely high growth rates and inability to capture the time required for the synthesis regime when individual grains are observable.For the kinetics investigation, samples synthesized on the copper foil were used. For each temperature point on the graph Figure  foil wasα radiation with wavelength of 0.7136 A was used. Further X‐ray diffraction experiments were executed at beamlines No. 2 and No. 4 of VEPP‐3 storage ring at Siberian Synchrotron and Terahertz Radiation Centre, Budker Institute of Nuclear Physics of Siberian Branch of RAS, Novosibirsk, Russian Federation. The beamline No. 2 is dedicated for powder diffraction experiments with high instrumental resolution and is equipped with Si(111) channel‐cut monochromator, sample holder moving along theta angle, and Ge(111) crystal‐analyzer mounted in front of scintillation detector on the 2θ arm of the diffractometer. The working wavelength is 0.154 nm. The beamline No. 4 operates with a wavelength of 0.03685 nm and the diffracted radiation is registered by 2D image plate detector MAR345. The sample is set in transmission mode and can be rotated around the axis perpendicular to the incident beam.Diffraction experiments in reflection geometry have been performed at Institute of Solid State Physics of Russian Academy of Sciences (RAS) using monocrystal diffractometer Oxford Diffraction Gemini equipped by charge coupled device detector with resolution of 0.35 A. MoKand No. 460 of θ–2θ scanning, 2θ scanning at fixed theta angle of 4°, θ scanning at fixed 2θ positions corresponding to Cu(111) and Cu(200) reflections, scanning with sample spinned around normal to the surface. The diffraction patterns of the sample were obtained at beamline No. 4 both in conventional transmission mode with no sample movement and with the sample rotated around the horizontal axis within the range of 30°.The experiment at beamline No. 2 was carried out in different modes including conventional −10–3 × 10−9 Torr using the irradiation of AlKα (mono) 1486.69 eV . Binding energy of Cu 2p3/2 at 932.7 eV is typical of metallic copper, while spin‐orbit splitting with Δ = 19.7 eV, minor satellite features at ≈943 eV, and finally the position of Auger line CuLMM at kinetic energy of 918.7 eV limit the possibility of any chemical modification of the substrate during synthesis.XPS spectra were collected by XPS spectrometer Kratos Axis Ultra DLD with spherical sector analyser, ion gun, ultraviolet and x‐ray sources. Experiments were conducted under ultra‐high vacuum 5 × 10Raman spectroscopy was performed with the DXRxi Raman Imaging Microscope equipped with ×50 objective and a 532 nm excitation laser operating at 1 mW output power. Raman spectra were measured at several points along with the sample with the exposure timeLEED embodied in Kratos Ultra DLD was used for tracking the crystallinity of the samples. The distortion of the image, clearly seen from the imperfection of the hexagons formed in the diffraction pattern of the graphene, is caused by the form of the lens, non‐flat samples’ surface and edge effects of the signal capturing camera.−10 mbar. The angular and energy resolutions of the photoemission experiments were 0.1° and 10 meV, respectively. The sample was introduced to the ultra‐high vacuum UHV chamber from the atmosphere, and before measurements, it was annealed at 300 °C for 20 min. During the measurements, the sample was kept at 20 K.ARPES measurements were carried out using linearly polarized undulator radiation at the U112‐2‐PGM beamline of BESSY‐II in Berlin. Photoelectrons were detected with a Scienta R8000 analyzer at the “One square” ARPES instrument, and the base pressure of the experimental setup was better than 1 × 10 NEXAFS spectra were recorded in total electron yield mode. The measurements were performed for the as‐loaded as well as for the annealed (≈300 °C) samples of graphene on copper.Room temperature NEXAFS measurements were performed at Russian‐German dipole beamline of BESSY‐II electron storage ring operated by Helmholtz–Zentrum Berlin für Materialien und Energie.61 NEX−6 mbar pressure. Spectroscopic data were collected with Opus and TPS Spectra software and processed using least‐square fitting procedure with the home‐made WASF software. Deep minima at higher frequencies correspond to absorption lines of PF . The spectrum of the PF was processed using a well‐known expression for transmissivity of the plane‐parallel layer with the absorption minima modeled with Lorentzians. The obtained electrodynamic parameters of PF were employed for least‐square fitting of the transmission coefficient of graphene on PF using corresponding expression for two‐layered systems  under the conditions of secondary electrons detection mode (ETD detector) and 10 kV electron beam accelerating voltage. For larger grains and various catalyst substrates, Jeol JSM‐7001F at acceleration voltages from 10 to 30 kV, as well as optical microscope Leica with copper oxidation contrasting  were used. Dual beam scanning electron microscope Helios G4  was used to capture EBSD maps. Low‐temperature JT‐SPM SPECS was used for atomic reoslution imaging of graphene samples.Bruker Multimode V8 atomic force microscope was used for graphene grains visualization on early stages employing the difference in adhesion force, young modulus, and sample deformation maps obtained in Quantitative NanoMechanical Mapping PeakForce tapping mode. Bruker RTESPA300 cantilevers with FIJI software with a built‐in function was used for particle size distribution measurements. All images were converted to 16‐bit and the contrast was thresholded so that the background stays white, and the particles are black. After processing the obtained perimeters and areas of the particles were averaged. For processing of Raman, UV‐vis, IR, and XPS were processed using python lmfit, rampy, pandas, matplotlib, and numpy packages.CVD parylene deposition was performed in a home‐built set‐up. A quartz tube was heated by two individual horizontal tube furnaces. One zone was kept at 175 °C, where the parylene‐N dimer was kept in a quartz boat. The second zone was heated to 700 °C. Passing through the tube, sublimed parylene dimer undergoes the pyrolysis in the hot zone and polymerization (at a rate of ≈20 nm/min) in the room‐temperature zone holding the samples. The room‐temperature zone can be isolated by a valve, allowing precise control of the final parylene thickness. The system is pumped by an Edwards scroll pump via a liquid nitrogen cold trap. Silicon wafers coated with the parylene were used as a substrate for graphene FETs and subsequent additional deposition of parylene on top of graphene for encapsulation.3PO4, 18 mL HNO3, 10 mL CH3COOH, 65 mL H2O). After the etching was complete, the sample was transferred to pure water to remove the etchant residuals and scooped it with Si/SiO2 wafer in case of cellulose acetate or PMMA or the PF was taken out by tweezers with graphene for air drying. Following removal of the sacrificial layer was performed in cold acetone for PMMA and CA within 12 h. For the case of PF, the polymer film with graphene was applied to the surface of substrate under mild pressure by a rubber roller and heated to 120 °C on a hotplate to ensure uniform coverage. Subsequently, the substrate was placed in o‐xylene and kept overnight at room temperature, transferred to cyclohexane at 70 °C for 2 more hours and again to o‐xylene at 120° for 2 h. Afterwards, the sample was rinsed by isopropanol, deionized water, and vacuum dried.Poly(methyl methacrylate) (PMMA), cellulose acetate (CA), and parafilm M were employed as sacrificial layers during the transfer. Due to large adhesion between graphene and copper substrate, most probably caused by van der Waals interactions, this work failed to tune the electrochemical delamination and SpEED peeling of the graphene. However, able to use a combination of electrochemical etching of the Molybdenum‐Copper substrate in “Hell”‐etchant (30 mL H4He cryostat (4.2 K) where a sample was in the vapor of 3He (exchange gas). Before cooling down an insert with the sample was pumped during >5 h with residual pressure <5 × 10−4 mbar. Either standard four‐probe low‐frequency lock‐in or DC techniques were used for electrical transport measurements.Under ambient conditions transfer curves have been captured with the help of a probe station and Keysight B1500A Semiconductor Analyser. Low temperature measurements were carried out in a  with the Perdew‐Burke‐Ernzerhof (PBE) approximation for the Exchange and Correlation energy. For most calculations only one k‐point was used; nevertheless, some results were tested with (2 × 2 × 1) k‐points and the relative energies changed only around 0.1 eV verifying the proposed procedure. Climbing Image Nudged Elastic Band (CI‐NEB) methods were implemented in atomic simulation environment module in GPAW. All NEB calculations need the initial and final geometries while an estimation of the transition state (TS) is also beneficial. Linear initial trajectory interpolation usually with initial, TS, and final geometries was used. In some cases, the reacting molecules moved significantly and the reaction path could have two barriers, the NEB calculations were restarted so that it would contain only one transition state. As the process occurs at the temperatures slightly above the melting point for copper, the surface is likely liquefied. Since the accuracy of DFT models for the liquid metal surface is substantially lower than that for the solid structure, for the DFT studies Cu(110) surface was employed. Even though the exact values for the activation energies might slightly differ for the different surfaces, the main trend would remain the same.DFT calculations were performed to assess the reaction pathway for the graphene synthesis based on Boudouard reaction on a copper surface. A periodic 3 × 3 surface unit cell for Cu surface was used and the Cu slab was 7 layers thick. All the calculations were done with the grid‐based projector‐augmented wave (GPAW) code64 witThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Take-Home Naloxone (THN) is a core intervention aimed at addressing the toxic illicit opioid drug supply crisis. Although THN programs are available in all provinces and territories throughout Canada, there are currently no standardized guidelines for THN programs. The Delphi method is a tool for consensus building often used in policy development that allows for engagement of stakeholders.We used an adapted anonymous online Delphi method to elicit priorities for a Canadian guideline on THN as a means of facilitating meaningful stakeholder engagement. A guideline development group generated a series of key questions that were then brought to a 15-member voting panel. The voting panel was comprised of people with lived and living experience of substance use, academics specializing in harm reduction, and clinicians and public health professionals from across Canada. Two rounds of voting were undertaken to score questions on importance for inclusion in the guideline.Nine questions that were identified as most important include what equipment should be in THN kits, whether there are important differences between intramuscular and intranasal naloxone administration, how stigma impacts access to distribution programs, how effective THN programs are at saving lives, what distribution models are most effective and equitable, storage considerations for naloxone in a community setting, the role of CPR and rescue breathing in overdose response, client preference of naloxone distribution program type, and what aftercare should be provided for people who respond to overdoses.The Delphi method is an equitable consensus building process that generated priorities to guide guideline development.The online version contains supplementary material available at 10.1186/s12954-022-00650-4. A total of 26,690 people died from opioid toxicity between January 2016 and September 2021 in Canada due to the toxic drug supply [Naloxone is a µ-opioid receptor antagonist that reverses opioid overdose and aids in preventing death with minimal adverse effects , 3. In CThe Canadian THN Guideline development project was funded through the Canadian Research Initiative in Substance Misuse Emerging Health Threat Implementation Science program in response to escalating mortality associated with opioid toxicity. THN programs are widely recognized as an important response to the to toxic drug supply yet there are discrepancies within THN programs in Canada, such as whether intranasal THN kits are available and the number of ampoules/vials of naloxone included in THN kits .Guidelines are based on current evidence and used to optimize consistent service provision and administration . There aWhile past THN guidelines offer valuable information, we identified a gap in guidelines developed specifically for community THN distribution programs, which have different informational needs and challenges compared to professional groups. The Canadian harm reduction context may differ from other countries as there is regional variation regarding the scope and quantity of harm reduction policies , availabThe Delphi method is a consensus building approach used to inform policy decisions , 18. ThrGuideline development groups should be multi-disciplinary and represent diverse views, involving a variety of methodological experts and clinicians, as well as any population affected by the guidelines . The DelThe aim of the adapted online Delphi method was to investigate which research questions stakeholders believed should be prioritized in a Canadian THN guideline.All meetings and data collection were conducted online in order to get perspectives of stakeholders from across Canada and to respect physical distancing measures during the COVID-19 pandemic. Author MF facilitated all subcommittee meetings with support from author JN. Authors AM, CS, KR, TDR sat on the Guideline Steering Committee.Guideline Development Group recruitment was an iterative process aiming for pan-Canadian membership and diverse perspectives. Four Guideline Development Group subcommittees participated in the Delphi method.Recruitment occurred between November 2020 and January 2021. The study principal investigators and the affiliated Canadian Research Initiative in Substance Misuse (CRISM) working group members nominated members for the Guideline Development Group. These initial members nominated other potential stakeholders to expand and diversify recruitment using a snowball sampling approach.PWLLE were explicitly invited via the snowball sampling process. PWLLE who were connected or unconnected to advocacy, research, or frontline harm reduction organizations were eligible for participation. We recruited from existing communities of PWLLE such as the Canadian Association of People who Use Drugs, the People with Lived and Living Expertise of Drug Use National Working Group, and Professionals for Ethical Engagement of Peers, from our professional networks, and sought feedback from identified PWLLE to ensure diversity of experiences and cross-country representation.We invited participants from all subcommittees to introduce themselves in whatever manner made them feel comfortable, including lived and living experience, but did not explicitly ask all group members whether they identify as people who use drugs. We recruited sufficient self-identified PWLLE to ensure representation in all subcommittees. PWLLE in the Guideline Development Group who were not being compensated for participation through their workplace were provided $25/hr honoraria .Effort was made to find representation across Canadian provinces and territories through the local and national networks of existing members. Indigenous, Black, and People of Color and 2S/LGBTQ group members were explicitly invited in the snowball sampling process.The Guideline Steering Committee (composed of eight members) steered discussions, encouraged constructive debate, summarized main points and key decisions, and gave oversight to the guideline development process.The Affected Community Committee (composed of seven members) provided guidance and recommendations based on the values and preferences of people with lived experience related to naloxone distribution and use in opioid overdose.The Clinical Expert Committee (composed of 13 members) provided guidance and recommendations on the use of naloxone in opioid overdose from a clinical perspective.The Guideline Development Panel (composed of 15 members) voted on the key questions to be addressed by the guideline  as part of a Delphi method. Members of the Guideline Steering Committee, Affected Community Committee, and Clinical Expert Committee were invited to participate in voting as part of the Guideline Development Panel.Subcommittees of the Guideline Development Group included:Author AM, a member of the Guideline Steering Committee, introduced meeting facilitation strategies drawing on experiences facilitating with community groups and varied stakeholders on harm reduction topics in British Columbia . MeetingWe identified initial key questions based on a recent scoping review of the literature on the use of naloxone for reversal of opioid overdose in a community setting . Themes Three subcommittees  reviewed these initial questions and suggested new questions before the Guideline Development Panel voted on the importance of each question. The Guideline Development Group discussed whether to include questions on cost-effectiveness due to concern about the potentially stigmatizing effect of comparing lives lost to public health programming cost. However, some Guideline Development Group members described challenges getting funding for services in the absence of evidence on cost-effectiveness. Questions on cost remained in the voting process but were flagged for careful consideration for presentation of evidence.The Guideline Steering Committee, Affected Community Committee, and Clinical Expert Committee recommended that plain language questions rather than academic research questions or statements be developed to guide the process . The GuiA total of 15 Guideline Development Panel members participated in voting on which key questions should be addressed in the guideline. The Guideline Development Group was composed of people that had overlapping lived or living/clinical/academic experience. A table with descriptions of the panel members’ professional and advocacy roles is included as a Additional file Methodology for the Delphi method was drawn from McMillan et al. . Data coThe method was adapted from early literature on the Delphi by moving data collection online to accommodate for the national scope of the project and to avoid limitations related to the ability to meet in-person during the COVID-19 pandemic .All Guideline Development Panel members participated in the first round of voting (15/15) while 80% of members (12/15) participated in the second round of voting.The research team used the CREDES tool to inform reporting of the Delphi method in this manuscript .A total of 17 of the 35 original research questions scored below 2.0 in the first round of voting and so were retained for the second round. Nine of the 17 key questions were rated below 2.0 in the second round of voting and qualified for inclusion in the THN guideline. The mean scores for both rounds are presented in Table The Delphi method was conducted to identify what questions community stakeholders thought important to prioritize in a Canadian guideline on THN. The following key questions were identified as important to address: What equipment should be in THN kits, whether there are important differences between intramuscular and intranasal naloxone administration, how stigma impacts access to distribution programs, how effective are THN programs at saving lives, what distribution models are most effective and equitable, storage considerations for naloxone in a community setting, the role of CPR and rescue breathing in overdose response, client preference of naloxone distribution program type, and what aftercare should be provided for people who assist with overdoses.Group heterogeneity among those making decisions can lead to more thorough consideration of all relevant aspects of a topic including applicability; by including a diversity of stakeholders, we were able to thoroughly weigh the pros and cons of key questions from different perspectives . The DelPrior Delphi studies in harm reduction report including people from advocacy groups  or advocWe found the online Delphi method to be especially beneficial in the context of a pan-Canadian project occurring during dual public health emergencies of drug toxicity and the COVID-19 pandemic. Public health and community health organizations reported feeling increasingly burdened trying to respond to the pandemic while concurrently seeing increases in overdose-related mortality , 35. TheThe Delphi method is the first step in community engagement for a Canadian THN guideline and will inform the next stage of the guideline development project. Three of the highly ranked key questions will be translated to research questions for systematic reviews and the evidence will be evaluated using the GRADE approach . EvidencDespite targeted recruitment, Newfoundland and Labrador, the Northwest Territories, Nunavut, and Prince Edward Island were not represented in the Guideline Development Group. Some of the nominees from these provinces and territories reported a higher than usual workload due to the COVID-19 pandemic as a barrier to participation. While two of the three territories were not represented, perspectives from the Yukon were included. Conclusions drawn through this Delphi method may not be generalizable to the provinces and territories not represented.A key limitation is reliance on online meetings. While use of online meetings and data collection allowed us to engage people from across Canada, it could present barriers to engagement especially for those with limited access to technology or limited technological knowledge including PWLLE. Additionally, our project relied on participants’ comfort with sharing ideas. While data collection was conducted using survey software, other meetings occurred over ZOOM video conferencing software. Prior qualitative research shows that while participants may feel able to express ideas during online focus groups, they also felt less engaged with other participants and less able to have a conversation that flowed compared to face-to-face discussions . We obseThe research team drafted the guideline questions using Population/Problem, Intervention/Exposure, Comparison, Outcome/Findings (PICO) language, then reworded them into accessible language for the Delphi method. However, this created uncertainty with the research team around the interpretation of some of the prioritized questions. The team was unable to ascertain how individuals interpreted the common language questions, which led to challenges in creating the final PICO questions used in the literature searches in the absence of more extensive pilot testing. Capturing comments on the questions or asking the voters to explain their understanding of the questions may have ensured that voters all had similar interpretations. Additionally, key questions included multiple outcomes, but the research team needed to narrow down questions for feasibility of executing the systematic reviews that will generate evidence for the THN guidelines.The Delphi method allowed Guideline Development Group members from across disciplines, life experiences, and geographical space to share insights and help establish priorities for a Canadian guideline. A guideline on Take-Home Naloxone informed by key stakeholders will provide evidence to support consistent, effective, and equitable service delivery across Canada. The evidence on the nine questions identified through this Delphi method study will be explored through systematic review.Additional file 1. Table 1. Guideline Development Panel Breakdown.


Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml−1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of β-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO Automated vitrification devices have been developed5, and various efforts have been made to produce good grids with well-dispersed particles and without much sample loss by denaturation8. In general, proteins often localize to the air–water interface and denature, resulting in poor particle density and preferred orientation and thereby making high-resolution 3D reconstruction inefficient9. Due to these problems, optimizing the conditions for cryoEM grid preparation requires much time and effort11. To overcome these problems, thin support films, such as graphene, have been used to change the physical properties of grid surface14, and various types of functionalization have been achieved: Nα, Nα-dicarboxymethyllysine (NTA)16; SpyTag/SpyCatcher17; amino-polyethylene glycol (PEG) linkers18; as well as various organic compounds19. Although synthesis of graphene oxide is the first important step for graphene functionalization, a limited number of methods have been available: modified Hummers’ methods21 ; conventional glow discharge; and chemical modification by plasma treatment22 . However, these methods suffer from problems such as undesirable breakage of the graphene framework (C–C bonds) and low selectivity of reaction points between the basal plane and the edges of graphene due to the harsh reaction conditions. It would be more useful if we can oxidize graphene in a milder condition and introduce appropriate functional groups into the basal plane of graphene selectively.Despite the recent improvements in hardware and software for electron cryomicroscopy (cryoEM) single particle image analysis for high-throughput and high-resolution structure determination of biological macromolecules, specimen grid preparation remains a bottleneck due to various factors. In single particle analysis (SPA), protein particles must be well dispersed in aqueous solution to be flash-frozen and embedded in thin vitreous ice film formed within micron-sized holes on a few 10s nm thick carbon film laid on a metal grid substrate2· gas as a oxidant; it can convert a gaseous fuels of methane into liquid fuels of methanol and formic acid without producing CO223 and can also make surface oxidation modification of polymers, such as polypropylene24. We applied this oxidation reaction to introduce hydroxy groups into the basal plane of graphene surface on the EM grid and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N2 atmosphere. This is the first effective method as a chemical dry process for oxidation of graphene on substrate. We then successfully introduced epoxy groups by epichlorohydrin (ECH) treatment, which has been used for functionalization of graphene oxide26. The resulting epoxidized graphene grid (EG-grid™) effectively adsorbed proteins so that they stayed on the surface even after washing with buffer. This was confirmed using electron cryotomography (cryo-ET), which showed that the protein particles were located close to the graphene surface and away from the air–water interface to avoid their denaturation.We have recently discovered a useful chemical reaction using photoactivated ClOThe EG-grid allows protein particles to be immobilized on graphene in a variety of orientation, not only making the adjustment of sample protein concentration less needed but also solving the problem of preferred orientation caused by adsorption to the air–water interface. In its application tests using standard proteins, such as GroEL and β-galactosidase, as well as difficult proteins with preferred orientation, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and SARS-CoV-2 spike protein, the numbers of cryoEM images needed for high-resolution reconstruction were much fewer than the cases where standard holey carbon grids were used. The resolution of apoferritin easily reached 1.29 Å resolution, also indicating its atomic-resolution imaging capability. We believe this EG-grid will become a powerful tool for accelerating high-resolution cryoEM structure determination.13 with some modification. Then, the surface of graphene is oxidized by photoactivated ClO2· treatment for 10 min to introduce hydroxy groups (see “Methods” for detail and Supplementary Movie 1). The following reaction with ECH in the third step introduces epoxy groups on the surface of graphene. The hydrophilicity of graphene grids was evaluated by the water contact angle (WCA) measurement. The WCA of graphene on the Au grid decreased from 76° ± 0.4° to 60° ± 0.3° by the ClO2· oxidation treatment  showed several characteristic peaks such as those at 3400 cm−1, v(C–OH); 1620 cm−1, v(C=C); and 1300 cm−1, 850 cm−1, v(C–O–C), suggesting that oxygen-containing functional groups were successfully introduced to the graphene surface. Contrary to this, the plasma-treated graphene showed a peak at 1740 cm−1 corresponds to the C=O bond and almost no C–O bond derived peaks. The Raman spectra of GP and GO are shown in Fig. −1) and 2D (2690 cm−1), and their intensity ratio (I2D/IG) was about 1.8, proving the high quality of single layer graphene. After oxidation, the D and D’ peaks appeared whereas the 2D peak nearly diminished. These features of the Raman spectra indicated the formation of sp3-carbon by the introduction of functional groups to graphene basal plane.To confirm the oxidation state of the graphene surface, we performed infrared (IR) and Raman spectral measurements and analyses. While pristine graphene (GP) was IR inactive as shown in Fig. 2· treatment. After plasma treatment, the composition ratios of the C–O groups (corresponding to the peak at 286.0 eV) and the C=O groups (corresponding to the peak at 287.8 eV) were 22.5% and 29.2%, respectively . On the other hand, in the ClO2·-treated graphene, they were 28.4% (C–O) and 8.5% (C=O), respectively . Generally, the increase of C=O ratio means the cleavage of the C=C bond of graphene. The increase of C–O ratio indicated that the hydroxy groups were introduced dominantly to the basal plane in the ClO2·-treated graphene rather than the cleavage of the C=C bond in the plasma-treated graphene, in agreement with the IR measurement.To investigate the effect of oxidation on the chemical state of the graphene surface more comprehensively, the chemical compositions of the functional groups were determined by X-ray photoelectron spectroscopy (XPS) measurement. Graphene was transferred onto a silicon wafer and then oxidized either by plasma or photoactivated ClOTo confirm the introduction of epoxy groups, we used 1H,1H-undecafluorohexylamine (UFHA) because the amino groups in this compound should react with the epoxy groups on the graphene surface and fluorine peaks can be clearly observed in XPS measurement . Only carbon and oxygen were detected with non-oxidized graphene (black line). No fluorine was detected with the UFHA-treated oxidized graphene and ECH-/UFHA-treated graphene (red and green lines), while a clear fluorine peak appeared with the UFHA-treated EG-grid (purple line). These results indicated that UFHA was successfully introduced on the graphene surface and that the epoxy groups enabled further chemical modification of the EG-grid.2· oxidized graphene grid (Ox-Gra); and EG-grid, and compared the peak intensities of the diffraction spots. We counted the maximum values of six equivalent diffraction peaks from the {2· oxidation, epoxidation, and vitrification.We observed the EG-grid using an electron cryomicroscope (cryoTEM) equipped with a cold field emission gun and an Ω-type in-column energy filter (JEM-Z300FSC (CRYO ARM™ 300), JEOL) operated at 300 kV and recorded images on a K3 direct electron detector . Most part (> 90–95%) of the EG-grid was covered with graphene  while we sometimes found graphene-detached area by recognizing rolled-up edges of graphene . The surface of graphene within the holes was quite clean , and the existence of suspended graphene was confirmed by electron diffraction patterns . To estimate the graphene damage caused by the oxidation and epoxidation, we recorded diffraction images of suspended graphene in 5 holes of three different types of grids: non-oxidized graphene grid (Gra); ClOEscherichia coli (Molecular weight (Mw) ~ 800 kDa) as a benchmark protein because the resolution of the cryoEM 3D reconstruction of this protein has been limited to 3.26 Å possibly by limited number of particles due to elimination of damaged particle images and by preferred orientation towards its end-on views27. A 3 μl solution of GroEL at a protein concentration of 0.3 mg ml−1 was applied on an EG-grid, and the protein solution was blotted after 5 min incubation. The grid was washed three times with 5 μl of buffer each, and then the grid was negatively stained with uranyl acetate. Many GroEL particles remained on the EG-grid and were packed densely . We also prepared a grid in the same way except skipping the epoxidation process by ECH and observed no or very small number of GroEL particles , which indicates that the EG-grid strongly adsorbed the protein particles.To investigate the protein binding activity of the EG-grid, we used GroEL from −1 GroEL solution was loaded onto the EG-grid and allowed to stand for 5 min at room temperature, followed by blotting and plunge freezing using Vitrobot™ (Thermo Fisher Scientific). A short blotting time around 1.0–1.5 s usually worked well for getting appropriate ice thickness on the EG-grid. We observed the grid and collected a dataset using CRYO ARM™ 300 operated at 300 kV (referred to as GroEL-EG). For comparison with a conventional grid preparation method, we also prepared a cryoEM grid using a Quantifoil grid and collected a dataset under the same EM imaging condition (referred to as GroEL-Q). We collected around 500 micrographs in both cases. The micrographs of GroEL-EG showed much more densely packed GroEL particles with more side view orientations than GroEL-Q  showed a much lower particle density than GroEL-EG , while those obtained from some areas of the other grid (dataset referred to as GroEL-Gh) showed densely packed and side-view oriented particles similarly to GroEL-EG . GroEL-Gh showed more side-view preference compared to GroEL-Gl , indicating that the orientation distribution of GroEL is somehow strongly affected by the particle density. To collect about 200,000 particle images of GroEL for these two datasets, we collected 5614 micrographs for GroEL-Gl and 649 for GroEL-Gh. The final reconstructions were obtained from 215,011 and 198,677 particle images, with resolutions of 2.08 and 2.09 Å for the GroEL-Gl and GroEL-Gh datasets, respectively  was smaller (better) than those of GroEL-Q (98.5 Å2), GroEL-Gl (83.3 Å2), and GroEL-Gh (85.4 Å2) , demonstrating a higher homogeneity of the particle structures on the EG-grid to reach such higher resolution. We also calculated the FSC curves and sphericities of these datasets using the 3DFSC server30 and found that the sphericity of GroEL-EG (0.991) was slightly higher than those of GroEL-Q (0.964), GroEL-Gl (0.985) and GroEL-Gh (0.985) . The efficiency , which represents the uniformity of orientation distribution used in the reconstruction (the perfect orientation distribution is presented as Eod = 1.0), of GroEL-EG (0.71) was slightly lower than those of GroEL-Gl (0.75) and GroEL-Gh (0.74) , which would reflect the efficiency of data collection. For the GroEL-EG and GroEL-Gh datasets, 315 and 306 particles/image were used for final reconstruction, respectively, while only 36 and 38 particles/image were used for the GroEL-Q and GroEL-Gl datasets, respectively (Supplementary Table 1). Although both the GroEL-EG and GroEL-Gh datasets showed high particle density and side-preferred orientation, the resolution of the final map of GroEL-EG (1.99 Å) was slightly higher than that of GroEL-Gh 2.09 Å) . It was clear from highly tilted images that these ice contaminants were located on the surface of vitreous ice . Reconstruction of tomographic images from this dataset revealed three unique layers from the top to bottom. In the top layer, small ice contaminants were observed and no GroEL particles were found . The middle layer 240 Å below the top layer contained densely packed GroEL particles . Unexpectedly, at 225 Å below the middle layer, there were a few GroEL particles that seemed to be bound on the back side of the graphene layer . The estimated distance from the air–water interface to the bottom of the major GroEL layer was ~ 160 Å, which is reasonable to accommodate a single GroEL layer . These results indicate that GroEL particles were located close to the graphene surface and away from the air–water interface . We also collected a cryo-ET dataset from the GroEL-Gh grid and selected a hole containing both areas with and without graphene support for comparison. The graphene area showed a high density of GroEL particles in a single layer like the EG-grid, demonstrating the compatibility with the EG-grid, while fewer particles were observed in the non-graphene area .2· oxidized graphene grids are storable or not. After ClO2· treatment for 10 min, the grids were washed with water, air-dried, and stored under N2 atmosphere at room temperature for 3 months. Then, the grids were chemically modified by 1% (v/v) ECH solution, and 3 μl of 1.5 mg ml−1 GroEL solution was loaded in the same way as GroEL-EG. We found many GroEL particles adsorbed on the EG-grid , and the calculated sphericity (0.976) and efficiency factor (0.70) were comparable to those of GroEL-EG  and NPPI-final (104.1) compared to the GroEL-EG dataset (Supplementary Table 1) may reflect the way of the data collection, because, unlike the GroEL dataset described above, we collected three micrographs from one hole in the GroEL-EG3m, leading to carbon areas included in some micrographs to cause many particles on the carbon areas picked automatically.We also tested whether the ClOMw ~ 420 kDa), for which the present demand for high-resolution structures is quite high. We prepared three specimen grids: EG-grid (referred to as Spike-EG) and glow discharged graphene grid (referred to as Spike-G), both at a protein concentration of 0.1 mg ml−1, and conventional Quantifoil grid (referred to as Spike-Q) at a protein concentration of 0.5 mg ml−1. None of the grids were washed with the buffer after protein loading. The particle density was much higher in Spike-EG than that in Spike-Q, although the protein concentration was five times lower for Spike-EG  were all similar in Spike-EG and Spike-Q  . The value of NPPI-final for Spike-EG (129.2) was about twice as much as that for Spike-Q (56.5), while the FI ratio values were quite similar (24.9% and 23.8%) (Supplementary Table 1).Next, we examined the performance of the EG-grid on the SARS-CoV-2 spike protein trimer Mw ~ 20 kDa, f1-ATPase) composed of the A3B3 hexametric ring from Thermus thermophilus and the DF subunits from Homo sapiens (Mw ~ 400 kDa), the tetramer of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Mw ~ 144 kDa), β-galactosidase  (Mw ~ 460 kDa), and apoferritin (Mw ~ 500 kDa). The GAPDH structure has not been solved by cryoEM except for the part of the large ternary complex32. We loaded each sample solution onto an EG-grid and made a cryoEM grid by plunge freezing in the same way as we did for GroEL. In all samples, we saw many protein particles in the micrographs . Datasets were collected and processed . The 2D class averages showed wide varieties in particle orientations . During 3D classification of V1-ATPase-EG, we found two classes, one with and the other without the human DF subunits corresponding to the central rotary shaft. We only selected the class without the shaft, because the particle number of this class was much larger than that of the other. The 3D map and model with the shaft will be presented elsewhere. The orientation distributions of the particles used for the final maps were quite uniform as expected from the 2D class averages . The overall map resolutions of V1-ATPase, GAPDH, β-galactosidase, and apoferritin reached 3.03 Å, 2.16 Å, 1.81 Å, and 1.29 Å. respectively  and apoferritin-EG (36.4 Å2) are also as small as those of the previous reconstructions35 . So the EG-grid allowed to reach the highest resolution of β-galactosidase with 40% less number of micrographs. The FI ratios and the NPPI-finals of V1-ATPase, GAPDH, β-galactosidase, and apoferritin were 20.0%, 29.4%, 47.0%, and 66.5%, and 116.4, 368.2, 71.4, and 70.3, respectively (Supplementary Table 1).We applied the EG-grid to four samples: the chimeric complex of the soluble domain of V/A-type ATPase  and 2D class averages with an improved orientation distribution , although the resolution of the 3D map was limited to 6–7 Å.We also applied the EG-grid to hemagglutinin (HA), which suffered from preferred orientation and needed grid tilting data collection for 3D reconstruction19. It has also been reported that UV/O3 or KMnO4 treatment leads to the introduction of carboxy groups, implying the cleavage of C=C bonds in graphene, and that was confirmed by the formation of nanopores13. As can be seen in these reports, it is difficult to oxidize chemically stable graphene to introduce oxygen functional groups on the surface without disruption of its atomic layer sheet structure. The distinctly different results of GroEL-Gl and GroEL-Gh obtained by plasma-treated graphene grids  also indicate the difficulty in controlling the property of graphene surface by plasma treatment to reproduce good results in the cryoEM grid preparation.Oxidation of graphene films on EM grids without damaging is not an easy task. In fact, the plasma method (glow discharge) has been reported to cause graphene to collapse in a short time2·. This method can introduce reactive functional groups such as hydroxy (OH) group, while maintaining the graphene framework intact. In our method, C–O single bond groups (hydroxy groups) are more preferentially introduced compared to C=O type functional groups such as carboxy groups , and therefore the intrinsic sheet structure of graphene is better maintained. We also confirmed that these hydroxyl groups react with ECH to produce epoxy groups and that they further react with amine compounds . CryoEM observation confirmed that little damage was introduced to the graphene after oxidation, epoxidation, and vitrification . This suggests not only a variety of applications of the EG-grid but also a much wider potential application of the method we used for graphene oxidation by combination with further chemical modifications.We have successfully developed a novel oxidative modification method of graphene on substrates using photoactivated ClO2· oxidized graphene grids can be stored for at least 3 months under N2 atmosphere at room temperature  compared to those of Spike-Q and Spike-G  and also proved that near 2 Å resolution is reachable with ~ 144 kDa protein only from ~ 240 images , or PMMA-coated single-layer CVD graphene on a Cu foil from any suppliers  can be used. If necessary, the backside (the opposite side of PMMA) of graphene was removed by mechanical polishing using a waterproof abrasive paper . The Cu foil was etched by floating the PMMA-coated CVD graphene on a 0.5 M ammonium persulfate (APS) solution for more than 30 min. Then, the PMMA-coated graphene was scooped up on a filter paper, transferred to water and left for 10 min. This washing process was repeated twice. Or, a much easier and quicker method is to use Trivial Transfer Graphene™ , which can be transferred to water directly without polishing, etching, and washing. The graphene was transferred to water in a stainless container in which a number of Quantifoil grids (R1.2/1.3 Au 200 mesh) were aligned at the bottom with their carbon side facing up. The water was slowly drained, and the graphene layer was deposited on the grids. After air-dried for more than 1 h, each grid was separated and baked at 130 °C for 15 min. The grids were immersed in acetic acid twice for PMMA removal: for 2–3 h in the first step and then overnight. Then the grids were soaked in isopropanol for 10 min, air-dried on a filter paper, and baked at 100 °C for 10 min. The grids were stored in a grid box in a desiccator.We prepared graphene grids using Quantifoil grids based on the protocol described previously with some modifications2· gas generated from an aqueous solution (20 ml) of NaClO2 (200 mg) and 37% HCl (aq) (100 μl) by irradiation with an UV LED lamp  for 10 min. A square-shaped dish-like glassware with an inner and outer compartment that we developed before  was used to perform this oxidation process. The aqueous solution of NaClO2 was filled into the outer compartment, the graphene grids were laid in the inner compartment, and a glass plate was put on the glassware to seal the entire chamber. To avoid any damage on the graphene layer by direct photoirradiation, an aluminum foil was laid on the grids to shield them from the UV light . The total reaction volume can be reduced to 5 ml. For the storage experiment (GroEL-EG3m), the ClO2· oxidized graphene grids were washed with 5 μl of ultrapure water three times, air-dried, and stored under N2 gas at room temperature for 3 months. The graphene on a silicon wafer were also oxidized in the same way. Because the ClO2· gas is toxic and potentially explosive especially at high concentration, all the oxidation process should be performed under controlled concentration in a fume hood. Plasma treated hydrophilized graphene on a silicon wafer were also prepared as a control for XPS measurement by glow discharge  using a JEC-3000FC sputter coater (JEOL) with an aluminum foil mask to shield them from plasma irradiation.The graphene grids prepared as above were oxidized by photoactivated ClOThe preparation of EG-grid  was performed as described in manuscript for negative staining. A 3 μl aqueous solution of 1% 1H,1H-undecafluorohexylamine (UFHA) was loaded on the EG-grid. After 5 min, the solution was blotted with a filter paper and the grid was washed with 5 μl water for 3 times. After washing process, the fluorinated graphene grid was dried in vacuo overnight.−1 over 16 scans in a scan range of 600–4000 cm−1. The surface chemical composition of the graphene was determined using a JPS-9010MC XPS instrument (JEOL). The XPS insert parameters included the power of analysis  and monochromatic Mg Kα radiation. The survey and high-resolution XPS profiles were obtained at the constant analyzer pass energies of 160 and 10 eV, respectively. The CasaXPS Version 2.3.15 software was used for the peak-differentiation-imitating analysis of the C1s narrow XPS profile. Two independent experiments were conducted to obtain the standard deviation of the data. The static water contact angles of the graphene surfaces were determined using a Drop Master DM300 (Kyowa Interface Science) contact angle meter. A 1.0 μl of water droplet was placed on the surface of the graphene on the grid, and the contact angle was determined 5 s (scanning time) after the attachment of the droplet. Data are obtained as the mean of three independent experiments with standard deviation. All the cryoEM images and diffraction patterns of the grids were acquired using SerialEM39 and JEM-Z300FSC  operated at 300 kV with a K3 direct electron detector . The peak values of the diffraction intensities were identified using Fiji40.Raman spectra were obtained with an NRS-3100 T laser Raman spectrometer (JASCO) with a 532 nm laser at room temperature. Fourier-transform infrared (FTIR) spectroscopic measurements were performed using a Spectrum Two spectrometer (Perkin-Elmer) equipped with an UATR two attachment. All the spectra were acquired at a resolution of 4 cmEscherichia coli was purchased from TaKaRa Bio Inc. (Product code 7330). A 5 mg powder sample of GroEL was dissolved with 500 μl of GroEL buffer , and it was purified using a Superdex 200 Increase 10/300 GL gel filtration column with the GroEL buffer. The main peak was collected and concentrated to 3.2 mg ml−1, flash-frozen with liquid nitrogen and stored at − 80 °C.GroEL from 2+ affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva), followed by size exclusion chromatography using Superdex 200 Increase 10/300 GL (Cytiva) equilibrated with a buffer containing 50 mM HEPES (pH 7.0) and 200 mM NaCl.The codon-optimized gene of SARS-CoV-2 Spike (S) protein (GenBank: QHD43416) was designed for expression in mammalian cells and synthesized from GeneArt DNA Synthesis (Thermo). For expression of recombinant S protein, the sequence encoding the S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at furin cleavage site (residues 682–685), and C-terminal foldon trimerization motif followed by an octa-histidine tag was cloned into a pcDNA3.1 expression vector (Invitrogen). Further, S protein D614G mutation was introduced by inverse PCR method. Recombinant S proteins were transiently expressed in Expi293f. cells (Thermo) maintained in HE400AZ medium . The expression vector was transfected by using Gxpress 293 Transfection Kit  as following the manufacturer’s protocol. After 5 days post-transfection, culture supernatants were harvested, and His-tagged S proteins were purified by NiEscherichia coli strain BL21-CodonPlus-RP (Stratagene) was used for expression of the human DF-V1-ATPase chimeric complex. The recombinant complex was isolated as described previously42. The expression plasmids for V1-ATPase containing DF from Homo sapiens were constructed by the same method as described previously41. The genes encoding the D and F subunits were amplified from human cDNA. The amplified fragment was then digested with appropriate restriction enzymes and inserted into the corresponding region of the Thermus thermophilus V1-ATPase expression plasmid42. The mutant V1-ATPase  was used for preparation of the complex.Escherichia coli strain, BL21 Star™ (DE3) (Thermo Fisher Scientific). The transformed cells were cultured in Luria–Bertani (LB) medium with 50 μg ml−1 Kanamycin at 37 °C until the OD600 reached ~ 0.6. After adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and culturing for 16 h at 37 °C, cells were harvested by centrifugation, flash-frozen with liquid nitrogen, and stored at − 80 °C. Frozen bacteria pellets were resuspended in a buffer containing 20 mM Tris–HCl pH 8.0, 30 mM NaCl, 1 mM 1,4-dithiothreitol (DTT) supplemented with 10 μl Benzonase, 1 mg ml−1 lysozyme, and EDTA-free cOmplete™ protease inhibitor cocktail (Roche) and lysed by sonication. The suspension was centrifuged for 30 min at 200,000×g and 4 °C. The supernatant was collected and purified using a HisTrap HP 5 ml column  equilibrated with a buffer containing 20 mM Na4P2O7 pH 7.4, 500 mM NaCl, 30 mM imidazole, and subsequently eluted with the same buffer containing 500 mM imidazole. The desired fractions were collected and dialyzed overnight at 4 °C against a buffer composed of 20 mM Na4P2O7 pH 7.4, 200 mM NaCl supplemented with TEV protease. The protein was further purified using a HiLoad 16/600 Superdex 200 column  equilibrated with 20 mM Tris–HCl pH 8.0, 30 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP). The desired fractions were collected, concentrated to ~ 10 mg ml−1, flash-frozen with liquid nitrogen, and stored at − 80 °C.The gene of human GAPDH was cloned into pET-28a(+) expression vector (Novagen) with an N-terminal His-tag followed by the Tobacco Etch Virus (TEV) protease cleavage site. The recombinant protein was expressed using an 2, 1 mM TCEP) to a final concentration of 4.0 mg ml−1. IPTG was supplemented to a final concentration of 5 mM, and the solution was incubated on ice for 30 min. β-gal was purified using Superdex 200 Increase 10/300 GL column  with the same buffer. The main peak was collected and concentrated to 2.0 mg ml−1, flash-frozen with liquid nitrogen and stored at − 80 °C.β-galactosidase  was purchased from SIGMA-Aldrich (Product code G5635) and dissolved with β-gal buffer  (Thermo Fisher Scientific). After the plasmid was transformed, the cells were cultured in 4 ml of LB medium with 50 μg ml−1 Kanamycin and 0.5% glucose overnight. The culture was transferred into 500 ml of fresh culture medium and cultured at 37 °C until the optical density at 600 nm reached 0.5. Then, 1 mM IPTG was added, and the cultivation was continued for 4 h at 37 °C. Cells were harvested and resuspended in buffer A  supplemented with 1 mg ml−1 lysozyme and lysed by sonication. The suspension was centrifuged, and the supernatant was incubated at 70 °C for 10 min. After centrifugation and collecting supernatant again, the protein was precipitated by adding ammonium sulfate to a final concentration of ~ 53% and stirring on ice for 30 min. After centrifugation and collecting pellet, the pellet was washed with buffer A supplemented with ~ 53% ammonium sulfate. This washing step was repeated twice. The washed pellet was resuspended in 2 ml of phosphate buffered saline (PBS) and dialyzed against 500 ml of PBS at 4 °C overnight. Precipitation was removed by ultracentrifugation, and the solution was concentrated to ~ 0.9 ml, which was further purified using a Superose 6 10/300 column  equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl. The desired fractions were collected separately, flash-frozen with liquid nitrogen and stored at − 80 °C.Mouse apoferritin was expressed using mFth1-pET24a plasmid and an −1.Influenza hemagglutinin trimer (HA)  was purchased from MyBioSource  and dissolved in 1× PBS buffer to a concentration of 0.5 mg ml−1) was loaded on the graphene side of the grid. After 5 min, the protein solution was blotted, and the grid was washed with 5 μl of the same buffer three times . Then the grid was immediately stained with 3 μl of 2% uranyl acetate solution. Images were taken using a JEM-1011 electron microscope (JEOL) at 100 kV.A 3 μl solution of 1% (v/v) epichlorohydrin (ECH) in water was loaded on the graphene side of an oxidized graphene grid held with a tweezer. After 5 min, the ECH solution was blotted with a filter paper, and the grid was washed with 5 μl of ultrapure water (or protein buffer not containing a compound with amino group such as Tris) three times. Then, 3 μl of GroEL solution  and 150 mM NaCl. In the washing step after protein loading, the final (third) washing buffer on the grid was not blotted, and the tweezer holding the grid was placed in a chamber of Vitrobot™ Mark IV (Thermo Fisher Scientific) equilibrated at 4 °C and 100% humidity. For Spike-EG, GAPDH-EG, and HA, the grid was not washed after the protein loading. After a liquid ethane container was placed, the grid was blotted for 1.5 s  or 2.0 s (for Spike-EG) and immediately plunged into liquid ethane. Excessive ethane was manually blotted with a filter paper, and the grid was stored in liquid nitrogen. For comparison, non-oxidized graphene grid  or Quantifoil grids  was hydrophilized by glow discharge using a JEC-3000FC sputter coater (JEOL). To avoid graphene breakage, a loose mask made of a thin aluminum foil was placed above the grids during the glow discharge for the graphene grid. A tweezer holding the grid was placed in a chamber of Vitrobot™ and after the liquid ethane container was placed, 3 μl of 1.5 mg ml−1 (for GroEL-Q and GroEL-Gl) or 1.0 mg ml−1 (for GroEL-Gh) GroEL, or 0.1 mg ml−1 (for Spike-G) or 0.5 mg ml−1 (for Spike-Q) spike protein, or 0.5 mg ml−1 (for GAPDH-Q) GAPDH solution was loaded on the graphene or carbon side. Then the grid was blotted for 1.5 s (for GroEL-Gl and GroEL-Gh) or 2.0 s  or 3.0 s (for GAPDH-Q) and immediately plunged into liquid ethane. Excessive ethane was manually blotted with a filter paper, and the grid was stored in liquid nitrogen.The surface functionalization of oxidized graphene by 1% ECH and the protein loading step were performed as described above for negative staining. Protein concentration was 1.5 mg ml39, yoneoLocr43, and JEM-Z300FSC  operated at 300 kV with a K3 direct electron detector  in the CDS mode. The Ω-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. For the GroEL-EG, GroEL-Gl, GroEL-Gh, GroEL-EG3m, and GAPDH-EG dataset, nominal magnification was 60,000×. Defocus varied between − 0.5 and − 2.0 μm. Each movie was fractionated to 40 frames  with a total dose of 40 e−/Å2 for the GroEL-EG, GroEL-Gl, GroEL-Gh, and GAPDH-EG dataset. For the GroEL-Q, GroEL-EG3m, and GAPDH-Q dataset, three movies were collected from one hole, and each movie was fractionated to 40 frames  with a total dose of 40 e−/Å2. For the Spike-EG, Spike-Q, and HA dataset, nominal magnification was 60,000×. Defocus varied between − 0.5 and − 2.0 μm. Each movie was fractionated to 60 frames  with a total dose of 60 e−/Å2. For the V1-ATPase-EG dataset, nominal magnification was 50,000×. Defocus varied between − 1.0 and − 2.5 μm. Each movie was fractionated to 48 frames  with a total dose of 62 e−/Å2. For the β-gal-EG dataset, nominal magnification was 100,000×. Defocus varied between − 0.5 and − 2.0 μm. Each movie was fractionated to 40 frames  with a total dose of 40 e−/Å2. For the apoferritin-EG dataset, nominal magnification was 120,000×. Defocus varied between − 0.3 and − 1.3 μm. Each movie was fractionated to 64 frames  with a total dose of 40 e−/Å2. More parameters are summarized in Supplementary Table 1.All the cryoEM image datasets were acquired using SerialEM44, and CTFs were estimated using CTFFIND 4.145. All the following steps were performed using RELION 3.146 except GroEL-Q, GroEL-EG3m, and GAPDH-Q. RELION 4.047 was used for processing for GroEL-Q, GroEL-EG3m, and GAPDH-Q. Only the images whose CTF max resolutions were beyond 5 Å were selected, and the resulting number of images are referred to as Initial images. For GroEL-EG3m, 1209 movies were processed, and 500 micrographs were randomly selected after the CTF max resolutions beyond 5 Å selection and processed in the following steps. Laplacian of Gaussian (LoG)-based auto-picking was performed for small number of images to generate the 2D class averages, which were then used as a template for second auto-picking for all images. Picked particles were extracted with 1–4× binning, and the resulting number of particles was referred to as the initial particle number. Several rounds of 2D classification were carried out to remove bad particles. For GroEL-EG3m, one round of 2D classification was performed. Initial 3D model was generated with C1 symmetry, and then D7 (for GroEL) or D2  symmetry was applied to the resulting maps. No symmetry was applied for SARS-CoV-2 spike protein and V1-ATPase. 3D classification (into 4 classes) with C1 symmetry was conducted to exclude bad particles, and the number of remaining particles was referred to as the final particle number except the GAPDH-EG and β-gal-EG datasets. The remaining particles were re-extracted with a larger box size, and 3D auto-refine applying D7 (for GroEL) or D2  symmetry was performed. After a soft mask around the map was generated, the resulting map was post-processed and sharpened. After several rounds of CTF refinement and Bayesian polishing with divided optics groups of 6 (for GroEL-Q), 8 (for GAPDH-EG), 10 , 11 (for GroEL-Gl and V1-ATPase-EG), 12 (for Spike-EG), 13 (for GroEL-Gh), and 16 , 3D auto-refine and post-processing were conducted again. For the GAPDH-EG and β-gal-EG datasets, particles were re-selected with rlnMaxValueProbDistribution = 0.05 criteria , and 3D auto-refine and post-processing were conducted to generate the final map.The movies were motion corrected using MotionCor2For apoferritin, in total 793,398 particles picked by using 2D class averages template were extracted from 7500 images (Supplementary Table 1) with a box of 512 × 512 pixels and divided into 25 groups as optical groups according to the time of data collection. After the particle selection by 2D classification, an initial model was generated, and the selected particles were subjected to several rounds of 3D auto-refine with O symmetry and post-processing with CTF refinement and Bayesian polishing. Then the particles were regrouped into 12 by the range of beam tilt X, Y (0.1 milli-radian increments) and further regrouped into 300 (= 12 × 25:5 × 5 multi-hole shots) by image shift. Regrouped particles were subjected to another round of CTF refinement, 3D auto-refine, and post-processing. Then the particles were further selected with rlnMaxValueProbDistribution = 0.04 criteria, and the selected 527,261 particles were subjected to another rounds of Bayesian polishing with a larger box size (600 × 600), 3D auto-refine, and post-processing. Finally, the Ewald sphere correction and another round of post-processing was applied to generate the final map.30 . The efficiencies in particle orientational distributions were calculated using cryoEF31. The Rosenthal-Henderson B-factors29 were calculated and plotted using bfactor_plot.py script in RELION34. Structural analysis and figure preparation were performed using UCSF Chimera48 and ChimeraX49.The map resolutions were estimated by the gold-standard Fourier shell correlation (FSC = 0.143) criterion. The FSC curves and sphericities were calculated using 3DFSC server39, and the order of tilt series was as the following tilt angle in degree:  ->  ->  ->  ->  ->  ->  ->  ->  ->  -> . Each movie was fractionated to 5 frames  with a total dose of 98 e−/Å2. For GroEL-Gh, the nominal magnification was 20,000×, corresponding to 2.37 Å/pixel. Defocus was set to − 3.0 μm. The tilt angle varied from − 60º to 60º with 3º increment to acquire 41 movies using SerialEM39, and the order of tilt series was as the following tilt angle in degree:  ->  ->  ->  ->  ->  ->  ->  ->  ->  ->  ->  ->  ->  ->  -> . Each movie was fractionated to 5 frames  with a total dose of 40 e−/Å2. Tomograms were reconstructed using IMOD50. Images were aligned without using gold nanoparticles by patch tracking (for GroEL-EG) or by tracking ice contaminations as seeds (for GroEL-Gh).The datasets for tomography were acquired for the same grid as the GroEL-EG or GroEL-Gh dataset was collected using JEM-Z300FSC  operated at 300 kV with a K3 direct electron detector  in CDS mode. The Ω-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. For GroEL-EG, the nominal magnification was 40,000×, corresponding to 1.25 Å/pixel. Defocus was set to − 3.0 μm. The tilt angle varied from − 60º to 60º with 3º increment to acquire 41 movies using SerialEMSupplementary Figures.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.