Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication.

We report new advancements in the determination and high-resolution structural analysis of beam-sensitive metal organic frameworks (MOFs) using microcrystal electron diffraction (MicroED) coupled with focused ion beam milling at cryogenic temperatures (cryo-FIB). A microcrystal of the beam-sensitive MOF, ZIF-8, was ion-beam milled in a thin lamella approximately 150 nm thick. MicroED data were collected from this thin lamella using an energy filter and a direct electron detector operating in counting mode. Using this approach, we achieved a greatly improved resolution of 0.59 Å with a minimal total exposure of only 0.64 e-/A2. These innovations not only improve model statistics but also further demonstrate that ion-beam milling is compatible with beam-sensitive materials, augmenting the capabilities of electron diffraction in MOF research.

Microcrystal electron diffraction (MicroED) is a powerful tool for determining high-resolution structures of microcrystals from a diverse array of biomolecular, chemical, and material samples. In this study, we apply MicroED to DNA crystals, which have not been previously analyzed using this technique. We utilized the d(CGCGCG)₂ DNA duplex as a model sample and employed cryo-FIB milling to create thin lamella for diffraction data collection. The MicroED data collection and subsequent processing resulted in a 1.10 Å resolution structure of the d(CGCGCG)₂ DNA, demonstrating the successful application of cryo-FIB milling and MicroED to the investigation of nucleic acid crystals.

In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.

Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.