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Relevant publications

Alvarez, F. J., He, S., Perilla, J. R., Jang, S., Schulten, K., Engelman, A. N., ... & Zhang, P. (2017). CryoEM structure of MxB reveals a novel oligomerization interface critical for HIV restriction. Science Advances, 3(9), e1701264.

Human dynamin-like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein-fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction.

Cheng, Y. (2018). Single-particle cryo-EM—How did it get here and where will it go. Science, 361(6405), 876-880.

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

D'Imprima, E., D. Floris, M. Joppe, R. Sánchez, M. Grininger and W. Kühlbrandt (2018). "The deadly touch: protein denaturation at the water-air interface and how to prevent it." bioRxiv: 400432.

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Drulyte, I., Johnson, R. M., Hesketh, E. L., Hurdiss, D. L., Scarff, C. A., Porav, S. A., ... & Thompson, R. F. (2018). Approaches to altering particle distributions in cryo-electron microscopy sample preparation. Acta Crystallographica Section D: Structural Biology, 74(6).

Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.

von der Ecken, J., Heissler, S. M., Pathan-Chhatbar, S., Manstein, D. J., & Raunser, S. (2016). Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution. Nature, 534(7609), 724.

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated during a complex mechanochemical reaction cycle. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins, a high-resolution structure of the F-actin–myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release) states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

Frederik, P. M. and D. H. W. Hubert (2005). "Methods in Enzymology." 391: 431-448.

A thin aqueous film of suspended lipid vesicles⧸micelles is the object of choice for vitrification and subsequent study by cryoelectron microscopy. Just prior to vitrification, a thin film (compare with a soap film) is vulnerable to heat and mass exchange. Preparation of thin films in a temperature- and humidity-controlled environment is essential to prevent osmotic and temperature-induced alterations of the lipid structure, as will be explained in this chapter. Further automation of the preparative procedure by automatic blotting and PC control over the timing of critical steps (including vitrification) may further assist in the reproducible throughput of high-quality specimens. By cryotomography, taking a tilt series under low-dose conditions, a three-dimensional reconstruction of the specimen can be analyzed.

Kuhlbrandt, W. (2014). "Biochemistry. The resolution revolution." Science 343(6178): 1443-1444.
Liu, Z., Wang, J., Cheng, H., Ke, X., Sun, L., Zhang, Q. C., & Wang, H. W. (2018). Cryo-EM Structure of Human Dicer and Its Complexes with a Pre-miRNA Substrate. Cell, 173(5), 1191-1203.

Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate ∼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer’s multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA’s stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer’s N-terminal helicase domain.

Passmore, L. A. and C. J. Russo (2016). "Specimen preparation for high-resolution cryo-EM." Methods in enzymology 579: 51-86.

Imaging a material with electrons at near-atomic resolution requires a thin specimen that is stable in the vacuum of the transmission electron microscope. For biological samples, this comprises a thin layer of frozen aqueous solution containing the biomolecular complex of interest. The process of preparing a high-quality specimen is often the limiting step in the determination of structures by single-particle electron cryomicroscopy (cryo-EM). Here, we describe a systematic approach for going from a purified biomolecular complex in aqueous solution to high-resolution electron micrographs that are suitable for 3D structure determination. This includes a series of protocols for the preparation of vitrified specimens on various supports, including all-gold and graphene. We also describe techniques for troubleshooting when a preparation fails to yield suitable specimens, and common mistakes to avoid during each part of the process. Finally, we include recommendations for obtaining the highest quality micrographs from prepared specimens with current microscope, detector, and support technology.

Ravelli, R. B., Nijpels, F. J., Henderikx, R. J., Weissenberger, G., Thewessem, S., Gijsbers, A., ... & Peters, P. J. (2020). Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification. Nature communications, 11(1), 1-9.

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device’s performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Sun, F. (2018). Orienting the future of bio-macromolecular electron microscopy. Chinese Physics B, 27(6), 063601.

With 40 years of development, bio-macromolecule cryo-electron microscopy (cryo-EM) has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques: single-particle analysis (SPA), electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air–water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.

Tyler Engstrom,a Jonathan A. Clinger,b Katherine A. Spoth,cOliver B. Clarke,d,e David S. Closs,aRichard Jayne,a Benjamin A. Apkera and Robert E. Thornea,b* High-resolution single-particle cryo-EM of samples vitrified in boiling nitro­gen

aMiTeGen, LLC, PO Box 3867, Ithaca, NY 14850-3867, USA, bPhysics Department, Cornell University, Ithaca, NY 14853, USA, cCornell Center for Materials Research, Cornell University, Ithaca, NY 14853, USA, dDepartment of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA, and eDepartment of Anesthesiology, Columbia University, New York, NY 10032, USA
*Correspondence e-mail: robert.thorne@mitegen.com

Edited by E. Bullitt, Boston University School of Medicine, USA (Received 2 July 2021; accepted 5 August 2021; online 7 September 2021)

Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitro­gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro­gen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only 250 000 K s−1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitro­gen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitro­gen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitro­gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.