Imagine seeing the natural shape of biological molecules so closely that you can start to identify individual atoms. This dream is a reality for many researchers using a technique called cryoelectron microscopy (cryoEM). Although cryoEM can be a powerful tool, it is often associated with a high-cost barrier and requires specialized training in sample preparation, data collection, and data processing. The Common Fund’s Transformative High-Resolution Cryoelectron Microscopy program is broadening access for biomedical researchers to cryoEM, and a related technique called cryoelectron tomography (cryoET), through the creation of national service centers and training materials. The Centers focus on outreach to institutions and research groups in Institutional Development Award (IDeA)-eligible states as a major component of their mission. IDeA-eligible states are geographical regions of the United States that have historically received low levels of NIH support and often lack infrastructure supporting cryoEM research.

Successful engagement with investigators from IDeA-eligible states requires access to technology for data collection and might also require more hands-on training experiences to introduce researchers from these regions to the entire cryoEM workflow, including the sample preparation and screening steps that precede data collection. The Pacific Northwest Center for Cryo-EM (PNCC) supports a variety of projects from IDeA-eligible states, covering topics such as cancer biology, pharmacology, and therapeutics to combat COVID-19. At PNCC, these often resource-limited researchers can become engaged with cryoEM in a variety of ways. Dr. Lejla Zubcevic is an Assistant Professor at University of Kansas Medical School who studies how molecules are transported across cell membranes to result in temperature and pain sensation. Dr. Zubcevic was previously trained in cryoEM at another institution but her lab lacks the resources to use cryoEM at her current institution. Dr. Zubcevic’s lab is now an independent user at PNCC and uses screening and data-collection resources through a remote-access platform. To further support Dr. Zubcevic’s lab members in preparing samples at their home institution, PNCC also loaned them a cryo-grid clipping station, equipment that enables their complete independence in the cryoEM workflow. PNCC is looking forward to growing engagement between the National Centers for CryoEM and researchers from IDeA-eligible states, like Dr. Zubcevic. Interested researchers should reach out to any of the National Centers for CryoEM for more information about screening and data collection access.

References:
Information about the Common Fund’s National Centers for CryoEM and the National Network for CryoET
Information about the NIGMS Institutional Development Award (IDeA) program

In neurodegenerative diseases including Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS), proteins form abnormal clumps in the brain that can contribute to neuron loss. Among these proteins, Tau is an important protein for normal physiologic function, but is also well known to form rope-like aggregates, or clumps, known as tau fibrils in neurodegenerative diseases. Evidence suggests that RNA binds to tau proteins and contributes to the development of these tau fibril structures, but little is known about the structure of these complexes. 

With the help of the Stanford-SLAC CryoEM Center (S2C2), one of three Common Fund-supported National Centers for Cryoelectron Microscopy (CryoEM), a team of researchers led by David S. Eisenberg, Ph.D., at the University of California, Los Angeles, determined the structure of full-length tau fibrils bound to RNA. According to the authors, this structure could represent an early step in formation of tau fibrils and may offer useful information about how development of tau-RNA fibrils might be reversed.

To study the relationship between tau fibrils and RNA, researchers formed tau fibrils with RNA from mice and viewed their structure using cryoEM, a method that provides detailed images of biological molecules without the use of structure-altering dyes or fixatives necessary in other methods. The structure revealed strands of RNA molecules running up and down along the sides of the long tau fibrils. When they added enzymes to break up the RNA, the tau fibrils dissolved, revealing that the RNA was necessary for protein integrity.

According to the researchers, the cryoEM structure, which is nearly detailed enough to see the individual atoms of tau RNA fibrils, provides a structural explanation of how tau-RNA fibrils could be reversed. If this structure found using cryoEM samples also forms within the brains of human patients, it could be a step toward designing structure-based therapeutics to break up tau fibrils that lead to neurodegeneration.

The project used resources from the NIH Common Fund’s Transformative High-Resolution CryoEM program, which is broadening access to CryoEM for biomedical researchers by creating national service centers and cultivating a skilled workforce through CryoEM training materials, providing detailed images of molecules that could not be obtained using previous methods such as X-ray crystallography. 

Reference:
Abskharon R, Sawaya MR, Boyer DR, Cao Q, Nguyen BA, Cascio D, Eisenberg DS. Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation. Proc Natl Acad Sci U S A. 2022 Apr 12; 119 (15):e2119952119. doi: 10.1073/pnas.2119952119. PMID: 35377792

Neuropeptides are important molecules in the body that act as signals and enable nerve cells, or neurons, in the brain and nervous system to communicate with each other. Substance P was one of the first neuropeptides to be identified and is known to play an important role in things like pain, mood, respiration, and nausea. To deliver its message to a neuron, Substance P interacts with a specialized protein located on the neuron’s surface, called neurokinin 1 receptor (NK1R). How Substance P interacts with NK1R remains poorly understood, making it difficult to design drugs that target NK1R and treat challenging neuropsychiatric issues such as pain, inflammation, and mood disorders.

One way to study interactions between molecules and proteins like Substance P and NK1R is by collecting detailed images of their architecture. Cryoelectron microscopy (cryoEM) is a method used to image biological molecules that have been frozen, without the use of structure-altering dyes or fixatives necessary in other methods. Due to recent advances in cryoEM, researchers are obtaining detailed images of molecules that could not be obtained using previous methods such as X-ray crystallography. The NIH Common Fund’s Transformative High-Resolution Cryoelectron Microscopy (CryoEM) program is broadening access to cryoEM for biomedical researchers by creating national service centers and cultivating a skilled workforce through cryoEM training materials.

The Stanford-SLAC Cryo-EM Center (S2C2), one of three Common Fund-supported National Centers for Cryoelectron Microscopy, was accessed by a team of researchers led by Dr. Aashish Manglik to study how Substance P interacts with NK1R. The state-of-the-art cryoEM equipment and technical support made available by S2C2 allowed Dr. Manglik’s team to collect images of Substance P delivering its message inside the cell through interactions with NK1R. From these images, the researchers found that interaction with Substance P causes NK1R to shift into an unexpected arrangement. They also gained insight on how different molecular parts of Substance P come into contact with NK1R to influence this arrangement and the signal ultimately delivered to the inside of the cell. This knowledge, enabled by the CryoEM program, may one day lead to the design of better therapies for neurological and psychiatric diseases.

Reference:
The specific research projects listed here were not financially supported by the Common Fund's CryoEM program but did use resources funded by the CryoEM program.
Harris JA, Faust B, Gondin AB, Dämgen MA, Suomivuori CM, Veldhuis NA, Cheng Y, Dror RO, Thal DM, Manglik A. Selective G protein signaling driven by substance P-neurokinin receptor dynamics. Nat Chem Biol. 2022 Jan;18(1):109-115. doi: 10.1038/s41589-021-00890-8. Epub 2021 Oct 28. PMID: 34711980
 

NCCAT staff provide remote training to Dr. Jogl, as seen here, by performing each step leading to cryoEM data collection using samples from Dr. Jogl’s lab.

Understanding the form and function of biological molecules expands our knowledge of how living things work. Researchers can apply that knowledge to improve health, like in the process of developing new antibiotics. To answer questions about the form and function of biological molecules, many structural biologists are moving from traditional X-ray crystallography methods to cryoelectron microscopy (cryoEM). Images obtained through cryoEM methods provide similar amounts of detail as those of X-ray crystallography, allow for more protein variability than X-ray crystallography methods, and eliminate the need to find crystallization conditions. The Common Fund’s Transformative High-Resolution Cryoelectron Microscopy (CryoEM) program is broadening access to cryoEM for biomedical researchers. One way to reach this goal is by training researchers so that they may independently conduct cryoEM research projects. Dr. Gerald Jogl, a laboratory head at Brown University, studies antibiotic resistance in bacterial ribosomes using structural biology techniques such as X-ray crystallography. Ribosomes are particles in the cell that read the genetic blueprints encoded in RNA to make proteins. Many antibiotics work by targeting bacterial ribosomes so that they can no longer make proteins critical for bacterial cell survival. Antibiotic resistance can occur when the structure of a bacterium’s ribosome mutates to differ from the normal structure, making it harder for the antibiotic to recognize and target it. Dr. Jogl aims to use cryoEM to obtain images of the abnormal ribosome structures, including those not suitable for X-ray crystallography, to help advance understanding of antibiotic resistance.

To gain the necessary expertise to conduct cryoEM studies, Dr. Jogl applied to the cross-training program offered at the Common Fund-supported National Center for CryoEM Access and Training (NCCAT). The intensive training program consists of instruction from NCCAT staff, access to equipment for sample preparation, and access to microscopes for screening and data collection, all of which was performed remotely due to the pandemic. An advantage of the remote training format was the increased flexibility for distributing and scheduling the lessons on cryoEM theory, allowing Dr. Jogl to better prepare for, review, and absorb the information. Remote demonstrations of preparing the grids that hold the sample and using the specialized cryoEM microscopes still allowed Dr. Jogl to achieve most of the training goals. Once institutional policies allow for post-pandemic in-person interactions, Dr. Jogl plans to meet the final training goals addressing practical equipment use. Dr. Jogl’s experience is one example of how the CryoEM program is broadening access to high-resolution cryoelectron microscopy through cross-training, even as the pandemic limits on-site access for biomedical researchers.
 

In late 2019, the virus SARS-CoV-2 was identified as the cause of the novel respiratory disease COVID-19. Many biomedical research groups shifted their focus to SARS-CoV-2 at unprecedented speeds, including the National Center for CryoEM Access & Training (NCCAT) in New York City, one of three national centers for cryoelectron microscopy (cryoEM) supported by the NIH Common Fund’s Transformative High-Resolution Cryoelectron Microscopy (CryoEM) program. CryoEM is a structural biology technique that can obtain high-resolution (highly detailed) images of molecules, such as parts of a virus. It is also becoming an established method in the development of vaccines and therapeutics, because visualizing the structures of the molecules can provide critical insights. Instruments used for cryoEM can be expensive and require specialized training, which often prohibits their use by researchers. As part of the CryoEM program’s goal to broaden access to high-resolution cryoEM, NCCAT granted multiple research groups studying COVID-19 rapid access to its cutting-edge cryoEM instruments and resources.

For researchers with cryoEM experience, the largest barrier to completing their projects was the amount of time needed on the instruments to collect multiple, large data sets. The state-of-the-art microscopes and cameras available at NCCAT allowed for speedy data acquisition, with some research groups obtaining high-resolution image reconstructions the same day data collection started. Additionally, research groups without cryoEM expertise were able to work with NCCAT staff to overcome technical challenges with instrumentation use that would have otherwise slowed data collection and interpretation.

Amidst a global pandemic, foundational knowledge uncovered by researchers accessing NCCAT is accelerating strategies to respond to the virus and to lessen the impact of COVID-19. For example, data collected on SARS-CoV-2 focused on characterizing the S-protein found on the virus surface. The S-protein binds to human cells, allowing for infection, and antibodies that defend the body from infection attach to the S-protein. Researchers using NCCAT’s resources characterized antibodies from COVID-19 patients and provided cryoEM images of the S-protein attached to these antibodies. When combined with new data on the structure of a SARS-CoV-2 variant’s S-protein, this shed important new light on how antibodies bind to SARS-CoV-2 variants. Other researchers using NCCAT described the structure of the viral helicase, a protein the virus uses to replicate and spread. Data collected at NCCAT have also been combined with other types of data to advance the development of reagents and test kits (for diagnosis), laboratory assays (for basic research), and vaccines and therapeutics (to prevent and treat the disease). Thus, the quick pivot of NCCAT to COVID-related research has led to important contributions against the pandemic on multiple fronts.

The specific research projects listed here were not financially supported by the Common Fund's CryoEM program.

References:

Controlling the SARS-CoV-2 spike glycoprotein conformation. Rory Henderson, Robert J. Edwards, Katayoun Mansouri, Katarzyna Janowska, Victoria Stalls, Sophie M. C. Gobeil, Megan Kopp, Dapeng Li, Rob Parks, Allen L. Hsu, Mario J. Borgnia, Barton F. Haynes, Priyamvada Acharya. Nature Structure & Molecular Biology, 2020, 27, 925–933.

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Lihong Liu, Pengfei Wang, Manoj S. Nair, Jian Yu, Micah Rapp, Qian Wang, Yang Luo, Jasper F.-W. Chan, Vincent Sahi, Amir Figueroa, Xinzheng V. Guo, Gabriele Cerutti, Jude Bimela, Jason Gorman, Tongqing Zhou, Zhiwei Chen, Kwok-Yung Yuen, Peter D. Kwong, Joseph G. Sodroski, Michael T. Yin, Zizhang Sheng, Yaoxing Huang, Lawrence Shapiro & David D. Ho. Nature, 2020, 584, 450–456.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex. James Chen, Brandon Malone, Eliza Llewellyn, Michael Grasso, Patrick M.M. Shelton, Paul Dominic B. Olinares, Kashyap Maruthi, Edward T. Eng, Hasan Vatandaslar, Brian T. Chait, Tarun M. Kapoor, Seth A. Darst, Elizabeth A. Campbell. Cell, 2020, 182, 1560-1573.e13.

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes. Tongqing Zhou, I-Ting Teng, Adam S. Olia, Gabriele Cerutti, Jason Gorman, Alexandra Nazzari, Wei Shi, Yaroslav Tsybovsky, Lingshu Wang, Shuishu Wang, Baoshan Zhang, Yi Zhang, Phinikoula S. Katsamba, Yuliya Petrova, Bailey B. Banach, Ahmed S. Fahad, Lihong Liu, Sheila N. Lopez Acevedo, Bharat Madan, Matheus Oliveira de Souza, Xiaoli Pan, Pengfei Wang, Jacy R. Wolfe, Michael Yin, David D. Ho, Emily Phung, Anthony DiPiazza, Lauren A. Chang, Olubukola M. Abiona, Kizzmekia S. Corbett, Brandon J. DeKosky, Barney S. Graham, John R. Mascola, John Misasi, Tracy Ruckwardt, Nancy J. Sullivan, Lawrence Shapiro, Peter D. Kwong. Cell Reports, 2020, 33, 108322.

Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains. Tongqing Zhou, Yaroslav Tsybovsky, Jason Gorman, Micah Rapp, Gabriele Cerutti, Gwo-Yu Chuang, Phinikoula S Katsamba, Jared M Sampson, Arne Schön, Jude Bimela, Jeffrey C Boyington, Alexandra Nazzari, Adam S Olia, Wei Shi, Mallika Sastry, Tyler Stephens, Jonathan Stuckey, I-Ting Teng, Pengfei Wang, Shuishu Wang, Baoshan Zhang, Richard A Friesner, David D Ho, John R Mascola, Lawrence Shapiro, Peter D Kwong. Cell Host & Microbe, 2020, 28, 867-879.e5.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis. Natalia G. Herrera, Nicholas C. Morano, Alev Celikgil, George I. Georgiev, Ryan J. Malonis, James H. Lee, Karen Tong, Olivia Vergnolle, Aldo B. Massimi, Laura Y. Yen, Alex J. Noble, Mykhailo Kopylov, Jeffrey B. Bonanno, Sarah C. Garrett-Thomson, David B. Hayes, Robert H. Bortz III, Ariel S. Wirchnianski, Catalina Florez, Ethan Laudermilch, Denise Haslwanter, J. Maximilian Fels, M. Eugenia Dieterle, Rohit K. Jangra, Jason Barnhill, Amanda Mengotto, Duncan Kimmel, Johanna P. Daily, Liise-anne Pirofski, Kartik Chandran, Michael Brenowitz, Scott J. Garforth, Edward T. Eng, Jonathan R. Lai, Steven C. Almo. ACS Omega, 2021, 6, 85–102.
 

The NIH Common Fund's Transformative High-Resolution Cryoelectron Microscopy (CryoEM) Program has funded the National Network for Cryoelectron Tomography (cryoET) as part of its initiative to advance the application of cryoET. This network will offer the biomedical community access to a specialized cryoelectron microscopy (cryoEM) technique that is uniquely capable of visualizing intact regions of cells and tissues at high resolution and with little perturbation.

The National Network for CryoET will be established with four service centers, including one service center that will also serve as the central network hub. The network hub will be located at University of Wisconsin-Madison and led by Dr. Elizabeth Wright. The network service centers will be located at the New York Structural Biology Center (Dr. Bridget Carragher, contact), SLAC National Accelerator Laboratory-Stanford University (Dr. Wah Chiu), and University of Colorado Boulder (Dr. Andreas Hoenger, contact). The service centers will specialize in cryoET specimen preparation. The hub will perform cryoET data collection for all the user laboratories served by the network, in addition to its service center functions.