cryo-EM Imaging Research Articles

Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy.

Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.

Electrified Operando-Freezing of Electrocatalytic CO2 Reduction Cells for Cryogenic Electron Microscopy.

The ability to freeze and stabilize reaction intermediates in their metastable states and obtain their structural and chemical information with high spatial resolution is critical to advance materials technologies such as catalysis and batteries. Here, we develop an electrified operando-freezing methodology to preserve these metastable states under electrochemical reaction conditions for cryogenic electron microscopy (cryo-EM) imaging and spectroscopy. Using Cu catalysts for CO2 reduction as a model system, we observe restructuring of the Cu catalyst in a CO2 atmosphere while the same catalyst remains intact in air at the nanometer scale. Furthermore, we discover the existence of a single valence Cu (1+) state and C-O bonding at the electrified liquid-solid interface of the operando-frozen samples, which are key reaction intermediates that traditional ex situ measurements fail to detect. This work highlights our novel technique to study the local structure and chemistry of electrified liquid-solid interfaces, with broad impact beyond catalysis.

Structure and function of the human apoptotic scramblase Xkr4.

Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create an electronegative surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.

P-097 Studying epididymal sperm using cryo-electron microscopy

Abstract Study question What are the structural changes in epididymal sperm leading to gain of motility? Summary answer We resolved the contents of the cytoplasmic droplet in immature sperm. We further found that the outer dynein arms are in a pre-power stroke conformation. What is known already Cells exiting the testis are morphologically complete but incapable of fertilization. They first need to mature in the epididymis, where sperm gain progressive motility. Intriguingly, during this epididymal maturation the cytoplasmic droplet, a cytoplasmic remnant from spermiogenesis, migrates distally from the connecting piece to the principal piece. At the heart of the sperm’s flagellum is the axoneme, the molecular motor of the cell. It is a microtubule-based structure that houses protein complexes with different functions. For instance, it has radial spokes for signaling and rigidity. It also houses dyneins, motor proteins attached to neighboring doublets that ultimately generate flagellar beating. Study design, size, duration Bovine testes were acquired from a slaughterhouse, and sperm were isolated from different regions of the epididymis. After a washing step, these cells were directly used for imaging. Participants/materials, setting, methods Cells were prepared for imaging by rapid freezing on cryo-Electron Microscopy (cryo-EM) grids. Frozen cells were either imaged directly or first thinned by Focused Ion Beam (FIB) – milling. We applied the image analysis approaches subtomogram averaging and single particle analysis. Main results and the role of chance We resolved the structure of the 96-nm repeat of the axonemal doublet from immature sperm and showed a complete axoneme, containing radial spokes and different sets of dynein arms. Focusing our refinement on a specific set of dyneins, the outer dynein arms, revealed that they are in the pre-power stroke state. In this primed state the dyneins have ATP bound and are active. Interestingly, these cells display no movement. This could indicate that a different blocking mechanism is present in the axoneme. Furthermore, we used 3D cryo-EM to elucidate the contents of the cytoplasmic droplet. We showed that it contains many vesicles, some display a shape that is reminiscent of an autophagosome. We also find vesicles displaying a double membrane with a highly repetitive density connecting in between. This suggests that sperm cells bring along auxiliary proteins and molecules in the cytoplasmic droplet that may be required for proper maturation. Altogether we show that cryo-EM can be used to study immature sperm cells in detail, with a focus on structures important in the gain of movement. Limitations, reasons for caution Cryo-EM imaging requires frozen cells and uses electrons as imaging source, therefore we lack information on cell state (e.g. viability/motility) or protein identity. By complementing our visual data with other techniques such as CASA and mass spectrometry, we aim to get a full picture. Wider implications of the findings Elucidating protein conformation or the structural mechanism behind sperm activation could open doors into developing new therapeutics for diseases on one hand. On the other hand, understanding how sperm motility arises (and how to potentially put it to a halt) could also lay ground for developing male contraception. Trial registration number not applicable

Mucin-Inspired Polymeric Fibers for Herpes Simplex Virus Type 1 Inhibition.

Mucus lines the epithelial cells at the biological interface and is the first line of defense against multiple viral infections. Mucins, the gel-forming components of mucus, are high molecular weight glycoproteins and crucial for preventing infections by binding pathogens. Consequently, mimicking mucins is a promising strategy for new synthetic virus inhibitors. In this work, synthetic mucin-inspired polymers (MIPs) as potential inhibitors of herpes simplex virus 1 (HSV-1) are investigated. By using a telechelic reversible addition-fragmentation chain-transfer (RAFT) polymerization technique, a new dendronized polysulfatep(G1AAm-OSO3)PDSwith an amide-backbone similar to the native mucin glycoproteins is synthesized.p(G1AAm-OSO3)PDSshows mucin-like elongated fiber structure, as revealed in cryo-electron microscopy (cryo-EM) imaging, and its HSV-1 inhibition activity together with its previously reported methacrylate analoguep(G1MA-OSO3)PDS is tested. Both of the sulfated MIPs show strong HSV-1 inhibition in plaque reduction assays with IC50 values in lower nanomolar range (<3× 10-9 m) and demonstrate a high cell compatibility (CC50>1.0mgmL-1) with lower anticoagulant activity than heparin. In addition, the prophylactic and therapeutic activity of both MIPs is assessed in pre- and post-infection inhibition assays and clearly visualize their high potential for application using fluorescent microscopy imaging of infected cells.

Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call “honeycomb gold discs”, replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

Subtraction of liposome signals in cryo-EM structural determination of protein–liposome complexes

Abstract Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy (cryo-EM). However, the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins. Here, we propose a liposome signal subtraction method based on single-particle two-dimensional (2D) classification average images, aimed at enhancing the reconstruction resolution of membrane proteins. We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain. Based on this analysis, we designed a method to subtract the liposome signals from the original particle images. After the subtraction, the accuracy of single-particle three-dimensional (3D) alignment was improved, enhancing the resolution of the final 3D reconstruction. We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

Abstract 116: Apolipoprotein E Registry In Discoidal Lipoproteins Influences Uptake

Introduction: Apolipoprotein E (apoE) is of great interest due to its role as a risk factor for Alzheimer's disease and atherosclerosis. ApoE is secreted by astrocytes in the central nervous system as HDL-like lipoparticles. Currently there is no high-resolution structural model of lipidated apoE, resulting in a limited mechanistic understanding of how apoE functions in health and disease. Research Questions: The goal of this research was to utilize cryoEM to characterize the structure of apoE in discoidal lipoproteins. Methods: Astrocyte secreted apoE was immunopurified from culture media and characterized by TEM and cryoEM. Recombinant apoE (rapoE) was lipidated with DMPC to model astrocyte secreted lipoproteins. Results: TEM and cryoEM imaging of astrocyte apoE and rapoE found that apoE forms a dimer on discoidal lipoproteins. These results provide support for the “double belt” model of two apoE in an anti-parallel conformation. This conformation was independent of apoE isoform, suggesting it is a general feature of apoE in discoidal lipoproteins. ApoE isoforms differ by the addition or loss of a Cys residue. ApoE2 has 2 Cys residues (Cys112, Cys158), apoE3 has 1 Cys residue (Cys112), and apoE4 has no Cys residues. Accordingly, apoE4 cannot form disulfide bonds, apoE3 can form intermolecular disulfide bonds, and apoE2 can form both intramolecular and intermolecular disulfide bonds. Due to the presence of two apoE on discoidal lipoproteins, apoE3 can form a disulfide bridge between the two apoE protein. Disulfide bridge formation and experimental reduction of these disulfide bridges revealed that disulfide-linked apoE3 was uptaken significantly more by Neuro2a cells (P ≤ 0.0001) than nondisulfide-linked apoE3. Conclusion: All isoforms of apoE adopt an antiparallel dimer in discoidal lipoprotein. Different registries, regulated through the formation of disulfide bonds between the anti-parallel apoE polypeptides, leads to differential uptake of apoE by Neuro2a cells. Since the different isoforms of apoE have inherently different capacity to generate disulfide bridges, we conclude that this property affecting receptor uptake may strongly impact differential biological properties of the different isoforms.

Structural insights into the transporting and catalyzing mechanism of DltB in LTA D-alanylation

DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of gram-positive bacteria. This process stabilizes the cell wall structure, influences bacterial virulence, and modulates the host immune response. Despite its significance, the role of DltB is not well understood. Through biochemical analysis and cryo-EM imaging, we discover that Streptococcus thermophilus DltB forms a homo-tetramer on the cell membrane. We further visualize DltB in an apo form, in complex with DltC, and in complex with its inhibitor amsacrine (m-AMSA). Each tetramer features a central hole. The C-tunnel of each protomer faces the intratetramer interface and provides access to the periphery membrane. Each protomer binds a DltC without changing the tetrameric organization. A phosphatidylglycerol (PG) molecule in the substrate-binding site may serve as an LTA carrier. The inhibitor m-AMSA bound to the L-tunnel of each protomer blocks the active site. The tetrameric organization of DltB provides a scaffold for catalyzing D-alanyl transfer and regulating the channel opening and closing. Our findings unveil DltB’s dual function in the D-alanylation pathway, and provide insight for targeting DltB as a anti-virulence antibiotic.

Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.

Drug-induced transitions from micelles to vesicles in ionic surfactant solutions

Surfactant solutions play key roles in scientific research and technological applications. Here we investigated solutions that combined an ionic surfactant, Cetylpyridinium Chloride (CPyCl), with a non-steroidal, anti-inflammatory drug, Sodium Diclofenac (Diclo). The study covered a wide range of compositions and utilized both rheology and cryogenic-Electron Microscopy (cryo-EM). The solutions were prepared at three different concentrations of CPyCl: 5.0 mM, 16.7 mM and 33.0 mM, and we maintained the ratio of surfactant to salt, R=CPyCl/Diclo, within the range of 0.24<R<1.2. Linear viscoelasticity was measured at 25°C. The use of dilute solutions allowed for detailed cryo-EM imaging. By tracking the evolution of the zero-frequency viscosity as a function of Diclo concentration, we identified distinct regions of different rheological responses. The correlation between frequency sweep data and cryo-EM images enabled us to link these regions to specific morphological transitions ranging from spherical micelles to long and entangled wormlike micelles, to branched networks and, eventually, to perforated vesicles. For some systems, we were able to measure the complete spectrum of relaxation times, including the shortest characteristic Rouse time at high frequencies, previously only achieved by Diffusive Wave Spectroscopy. The use of existing models to evaluate the relevant important microstructural parameters such as entanglement, contour, and persistence lengths allowed us to establish a clear correlation between the microstructural parameters derived through rheology and the insights from the cryo-EM analysis.

Structural basis for the regulatory mechanism of mammalian mitochondrial respiratory chain megacomplex-I2III2IV2

Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria. Although this system has been studied for more than a century, its composition and molecular mechanism are still largely unknown. Here we report the high-resolution cryo-electron microscopy (Cryo-EM) structures of porcine respiratory chain megacomplex-I2III2IV2 (MCI2III2IV2) in five different conformations, including State 1, State 2, Mid 1, Mid 2, and Mid 3. High-resolution Cryo-EM imaging, combined with super-resolution gated stimulated emission depletion microscopy (gSTED), strongly supports the formation of MCI2III2IV2 in live cells. Each MCI2III2IV2 structure contains 141 subunits (70 different kinds of peptides, 2.9 MDa) in total with 240 transmembrane helices. The mutual influence among CI, CIII, and CIV shown in the MCI2III2IV2 structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping. The conformational changes from different states suggest a plausible regulatory mechanism for the MCI2III2IV2 activation/deactivation process.

Accurate global and local 3D alignment of cryo-EM density maps using local spatial structural features.

Advances in cryo-electron microscopy (cryo-EM) imaging technologies have led to a rapidly increasing number of cryo-EM density maps. Alignment and comparison of density maps play a crucial role in interpreting structural information, such as conformational heterogeneity analysis using global alignment and atomic model assembly through local alignment. Here, we present a fast and accurate global and local cryo-EM density map alignment method called CryoAlign, that leverages local density feature descriptors to capture spatial structure similarities. CryoAlign is a feature-based cryo-EM map alignment tool, in which the employment of feature-based architecture enables the rapid establishment of point pair correspondences and robust estimation of alignment parameters. Extensive experimental evaluations demonstrate the superiority of CryoAlign over the existing methods in terms of both alignment accuracy and speed.

Using the YOLO DeepLearning algorithm to quantify the rAAV empty/filled ratio from Cryo-EM imaging

Using the YOLO DeepLearning algorithm to quantify the rAAV empty/filled ratio from Cryo-EM imaging

Correlative cryo-microscopy pipelines for in situ cellular studies.

Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning. The first approach is a complete cryogenic pipeline in which both fluorescence and electronic imaging are performed on frozen-hydrated samples, the second is a hybrid cryoCLEM approach in which fluorescence imaging is performed at room temperature, followed by rapid freezing and subsequent cryoEM imaging. We provide a detailed description of the two methods we have employed for imaging fluorescently labeled cellular structures with thickness below 350-500nm, such as cell protrusions and organelles located in the peripheral areas of the cells.

Multilayer DNA Origami with Terminal Interfaces That Are Flat and Wide-Area.

DNA origami is a popular nanofabrication strategy that employs self-assembly of a long single scaffold strand, typically less than 10 kilobases in length, with hundreds of shorter staple strands into a desired shape. In particular, origami arranged as a single-layer rectangle has proven popular as flat pegboards that can display functionalities at staple-strand breakpoints, off the sides of the constituent double helices, with a ∼5.3 nm rhombic-lattice spacing. For applications that demand tighter spacing, functionalities can be displayed instead on the termini of helices of multilayer DNA origami. However, pegboards with the greatest addressable surface area are often found to be the most versatile. Given the practical limitations of the length of the scaffold that can be easily realized, designs that minimize the length of each helix would have advantages for maximizing the number of helices and therefore the number of addressable pixels on each terminal surface. Here we present an architecture for multilayer DNA origami displaying flush terminal interfaces from over 200 helices that each are only 5.3 turns in length. We characterize an example using cryo-EM imaging paired with single-particle analysis for further analysis of the global structure.

Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions.

mRNA LNPs can experience a decline in activity over short periods (ranging from weeks to months). As a result, they require frozen storage and transportation conditions to maintain their full functionality when utilized. Currently approved commercially available mRNA LNP vaccines also necessitate frozen storage and supply chain management. Overcoming this significant inconvenience in the future is crucial to reducing unnecessary costs and challenges associated with storage and transport. In this study, our objective was to illuminate the potential time frame for nonfrozen storage and transportation conditions of mRNA LNPs without compromising their activity. To achieve this goal, we conducted a stability assessment and an in vitro cell culture delivery study involving five mRNA LNPs. These LNPs were constructed by using a standard formulation similar to that employed in the three commercially available LNP formulations. Among these formulations, we selected five structurally diverse ionizable lipids─C12-200, CKK-E12, MC3, SM-102, and lipid 23─from the existing literature. We incorporated these lipids into a standard LNP formulation, keeping all other components identical. The LNPs, carrying mRNA payloads, were synthesized by using microfluidic mixing technology. We evaluated the shelf life stability of these LNPs over a span of 9 weeks at temperatures of 2-8, 25, and 40 °C, utilizing an array of analytical techniques. Our findings indicated minimal impact on the hydrodynamic diameter, zeta potential, encapsulation efficiency, and polydispersity of all LNPs across the various temperatures over the studied period. The RiboGreen assay analysis of LNPs showed consistent mRNA contents over several weeks at various nonfrozen storage temperatures, leading to the incorrect assumption of intact and functional LNPs. This misunderstanding was rectified by the significant differences observed in EGFP protein expression in an in vitro cell culture (using HEK293 cells) across the five LNPs. Specifically, only LNP 1 (C12-200) and LNP 4 (SM-102) exhibited high levels of EGFP expression at the start (T0), with over 90% of HEK293 cells transfected and mean fluorescence intensity (MFI) levels exceeding 1. Interestingly, LNP 1 (C12-200) maintained largely unchanged levels of in vitro activity over 11 weeks when stored at both 2-8 and 25 °C. In contrast, LNP 4 (SM-102) retained its functionality when stored at 2-8 °C over 11 weeks but experienced a gradual decline of in vitro activity when stored at room temperature over the same period. Importantly, we observed distinct LNP architectures for the five formulations through cryo-EM imaging. This highlights the necessity for a deeper comprehension of structure-activity relationships within these complex nanoparticle structures. Enhancing our understanding in this regard is vital for overcoming storage and stability limitations, ultimately facilitating the broader application of this technology beyond vaccines.

Gentle ions for cryo-FIB milling

Cryo-EM imaging of vitreous samples is limited to a few hundred nanometers in thickness. Focused ion beams can mill windows into cells and tissues for imaging, but they damage biological samples. In this issue of Structure, Yang etal. (2023) quantitatively describe this damage and suggest ways to minimize it.

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy.

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Copper Oxide Spike Grids for Enhanced Solution Transfer in Cryogenic Electron Microscopy.

The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.