Need for Speed: Examining Protein Behaviour During CryoEM Grid Preparation at Different Timescales

Cryo-EM structure and functional analyses show that human iron exporter ferroportin (Fpn) can bind and transport calcium ions and the transport activity is reduced when the concentration of ferrous ions is high.

Protein carboxyl methyltransferase domain-containing protein 1 (PCMTD1) may be the substrate adaptor protein of a potential E3 ubiquitin ligase that ubiquitylates proteins harboring isoaspartyl damage for proteasomal degradation. PCMTD1 is able to multimerize with cullin-RING ligase (CRL) components to form the putative CRL: CRL5-PCMTD1. We set out to structurally characterize this novel E3 ubiquitin ligase to elucidate its potential role in preventing the accumulation of age-damaged proteins. Because CRLs are known to function dynamically to facilitate ubiquitylation of substrates, we pursued single particle cryoEM of recombinantly reconstituted heterogenous assemblies, in hopes of mapping the conformational states adopted by the multimeric complex. Since studies have noted preferred orientation and complex dissociation of CRLs at the air-water interface during sample preparation for cryoEM, we pursued new methods in our cryoEM sample preparation. Specifically, we chose to prepare frozen hydrated grids of the CRL5-PCMTD1 complex via fast-plunge sample vitrification using self-wicking grids compatible with the automated Chameleon system. This sample preparation approach enabled the interrogation of minute amounts of freshly gel filtrated preparations that alleviated freeze-thaw induced denaturation and dissociation of the protein complexes. Our approach was also compatible with and benefitted from native top-down mass spectrometry of the same sample to determine the stoichiometric make-up of CRL5-PCMTD1 complex, which validated the solution architecture observed during cryoEM structure determination.

Single-particle cryogenic electron microscopy (cryo-EM) enables reconstruction of atomic-resolution 3D maps of proteins by visualizing thousands to millions of purified protein particles embedded in vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of a very sensitive analytical method for delivering high-resolution protein structure as a readout. In practice, millions of times more starting biological material is required to prepare cryo-EM grids. Here we show that using a micro isolation (MISO) method, which combines microfluidics-based protein purification with cryo-EM grid preparation, cryo-EM structures of soluble bacterial and eukaryotic membrane proteins can be solved starting from less than 1 µg of a target protein and progressing from cells to cryo-EM grids within a few hours. This scales down the amount of starting biological material hundreds to thousands of times, opening possibilities for the structural characterization of hitherto inaccessible proteins.

A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Mitigating risk and enhancing reproducibility in cryo-EM sample preparation

Nanocrates: Inside-Out Scaffolds for Sample Protection from the Air-Water Interface During Cryo-EM Sample Preparation

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

Ribosomes and cryo-EM: a duet

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Specimen preparation remains the principal bottleneck in cryo-electron microscopy (cryo-EM). Achieving uniformly thin ice without denaturation, preferred particle orientation, or air- water-interface damage is challenging. The demands of time-resolved experiments and sub-nanoliter sample volumes raise the bar even further. Commercial plungers are closed, high-cost platforms that limit experimental flexibility, constraining users to a few vendor-defined "custom" routines. By contrast, an open, modular and automated system would allow researchers to tailor experimental conditions by tweaking mixing, dispensing, and freezing parameters while lowering barriers to adoption. We present VitriFlex, an open-source cryo-EM grid preparation platform built around an industrial-grade SCARA robot and 3D-printed components. Designed for modularity and ease of assembly, the system enables programmable control of grid handling, sample deposition, and blotting, supporting both standard and time-resolved workflows. VitriFlex's modularity allows flexible implementation of sample delivery strategies, including pre-mixing and on-grid mixing modes, combined with acoustic-assisted spray application. The system allows for spray-to-plunge delays as short as ∼130 ms, enabling initiation of biochemical reactions within sub-second timescales prior to vitrification. Protocols are easily modifiable, and new components can be integrated with minimal redesign. We validated the platform using standard single-particle samples including apoferritin and dGTPase. Additionally, we demonstrate effective mixing capabilities by generating spike-ACE2 and α7-bungarotoxin complexes. In all cases, VitriFlex reproducibly yielded high-quality, collectable grids, allowing us to achieve high-resolution reconstructions. These results highlight VitriFlex as a flexible, accessible solution for both routine and time-resolved cryo-EM applications.

Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis. For complete details on the use and execution of this protocol, please refer to Michael et al.1.

Mass spectrometers as cryoEM grid preparation instruments

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.