key: cord-0783743-l3175qxw
authors: Rapp, Micah; Shapiro, Lawrence; Frank, Joachim
title: Contributions of Single-particle Cryo-EM toward Fighting COVID-19
date: 2021-10-30
journal: Trends Biochem Sci
DOI: 10.1016/j.tibs.2021.10.005
sha: 493489232ea8911b4f29b774e4b88fd6870b9e97
doc_id: 783743
cord_uid: l3175qxw

Single-particle cryo-electron microscopy (cryo-EM), whose full capabilities have been realized only within the past decade, has had a pivotal role in the fight against COVID-19. This is due to the technique’s intrinsic power to depict both structure and dynamical features of molecules, in this case, of the spike protein of the SARS-CoV2 virus. By now, numerous cryo-EM studies have furthered our understanding of spike protein-ACE2 receptor interactions, which has informed the design of effective vaccines and to the characterization of neutralizing antibody binding sites, which will lead to the design of novel therapeutics as the virus evolves.

We are now living through a time that will be remembered by historians and by generational family memory for its vast impact on our economy, on the way we live, and on our culture. Much of this is still merely visible in outline and difficult to extrapolate. Because of the worldwide spread of the COVID-19 disease caused by the SARS-CoV-2 virus, the impact is felt by all of humankind. We have learned from history books that, over the past millennium at least, epidemics such as the plague have inevitably brought profound -if not revolutionary -changes in the order of the societies inflicted with them. (The last pandemic on a similar scale, the "Spanish flu" in 1918, was overshadowed by the devastations wrought by the First World War that ended in the same year, and its societal impact has therefore been difficult to tell apart from that of the war).

Our chances of success in fighting this pandemic differ greatly from those in previous times because we are much better prepared. The reason for this is the unprecedented knowledge revolution that has taken place in the middle of the 20th century and onwards: we uncovered the unity of life and much of the molecular basis of life processes in all organisms. Particularly, we know how cells receive their instructions for making proteins and sustaining their metabolism from a genetic blueprint in the form of DNA. We know that the current pandemic is caused by a virus that, as many other viruses, exploits and subverts the molecular apparatus of the host to its own gain following a universal playbook we are able to recognize.

That body of knowledge, accumulated by research in molecular genetics, molecular biology, and structural biology, has transformed and empowered medicine profoundly. What it means is that, unlike the people who lived through the 1918 pandemic, we are no longer powerless in the global fight against a viral disease.

Paramount in this revolution of medicine have been research endeavors we refer to under the rubric of 'structural biology,' endeavors whose aim is to elucidate the structural basis of life processes on the atomic scale. On that sub-light microscopic level of structure, molecular interactions can be understood and even simulated in detail using the laws of Newtonian mechanics.

For decades, starting with the groundbreaking work of Max Perutz on the structure of myoglobin and hemoglobin [1] , X-ray crystallography has been the dominant method for molecular structure research. To date, it has given us more than 140,000 atomic structures of biological molecules, all readily accessible in the public database, the Protein Data Bank (PDB) i, ii . Application of this method of structure research is, however, restricted to molecules that can be induced to form highly ordered crystals. This limitation has resulted in the exclusion of many other molecules important for medicine, particularly membrane-bound channels and receptors. Another limitation lies in the fact that X-ray crystallography seldom captures molecules in their (multiple) native states. Such limitations do not exist in the relatively recent technique of single-particle cryo-electron microscopy, or cryo-EM for short, which was recently highlighted by the award of the 2017 Chemistry Nobel Prize iii [2] .

Although the methods for cryo-preparation of sample and computational methods for data analysis and reconstruction go back all the way to the 1980s, atomic resolution could not be achieved for asymmetric structures until the development of novel single-electron detecting cameras [3] [4] [5] and their commercial introduction in 2012. It is quite fortuitous [6] , in hindsight, that the technique was ready in time to help in the combat against several viruses implicated in recent deadly epidemics -Ebola (2014-2016) [7] [8] [9] , Zika (2015-2016) [10, 11] , Dengue (2019-2020) [12] [13] [14] [15] [16] , MERS-CoV (2012-2015) [17] [18] [19] [20] , and now SARS-CoV-2.

In the following, we wish to highlight the crucial role this technique has played in combating the SARS-CoV-2 virus, by helping elucidate the structures of both the virus and of the host molecules they interact with as the virus seeks entry into the cell to engineer its takeover. Specifically, both the rapid developments of messenger RNA (mRNA)-based vaccinations and effective antibody therapies have J o u r n a l P r e -p r o o f Journal Pre-proof drawn from knowledge gained by single-particle cryo-EM. It will become readily apparent that the most important contributions of the technique have been twofold: first, the capture of the spike protein in its different conformational states and the inference these structures allow to draw on the dynamics of the molecule as it interacts with the host, and second, the characterization of complexes formed by the spike protein with receptors and antibodies.

The key to both the development of vaccines and neutralizing antibodies has been in understanding the way the virus gains entry into the host cell during infection. For this to happen, the viral spike glycoprotein must first recognize and latch on to angiotensin-converting enzyme 2 (ACE2), a receptor on the cell surface [21, 22] .

Knowledge of this interaction has been gained by single-particle cryo-EM studies capturing the structures of the isolated spike protein in its pre-and postfusion configurations, and structure determination of the complex formed by spike protein with ACE2 [18, 19, [23] [24] [25] .

It is remarkable that the structure and dramatic conformational changes of the spike protein preceding the fusion event were already understood before the onset of the COVID-19 pandemic caused by the SARS-CoV-2 virus [18, 19] . This is due to the close similarity of SARS-CoV-2, in structure and mode of infection, to the SARS-CoV and MERS-CoV viruses, as revealed by the studies of the Ward, McLellan, and Veesler groups [18, 19, 22, 23] . Briefly, these studies of coronaviruses have shown that the spike protein is a trimer comprising structurally identical monomers, each composed of two subunits, S1 and S2. The spike protein is a metastable fusion machine. On the surface of the virus, spike exists predominantly in the prefusion form, ready to fuse with host when they bind ACE2. However, due to its metastability, some spikes will dissociate the S1 subunit and trigger the conformational changes in S2 associated with fusion. Critically, it is the prefusion conformation that must be recognized by the immune system to interdict infection.

In the prefusion conformation, the subunit S1 conformation alternates between two ManifoldEM, which employs a geometric machine-learning method, manifold embedding, in the analysis of cryo-EM data from a large ensemble of molecules in thermal equilibrium. It yields a tabulation of occupancies across conformational states and, eventually, a map of the molecule's energy landscape [27] .

The development of the first two successful vaccines against COVID-19, by Pfizer and Moderna, have relied on a strategy that had been devised in the fight against the similar coronaviruses, SARS-CoV and MERS-CoV, several years ago [19, 28] . As noted above, the similarity between the three viruses extends from the structural similarity to the similarity of the way the viruses infect the host. In particular, this strategy of vaccine development makes use of mRNA that encodes the spike protein.

When mRNA coding for spike protein is injected, the body's ribosomes make many copies of this protein, which in turn elicit the body's immune response, making the body equipped to fight off the real virus whenever it tries to enter later on [29] . group then at Dartmouth, first working with the common cold HKU1 coronavirus [28] , and later with MERS-CoV [17] , discovered that a pair of point mutations to proline will prevent formation of a helix associated with the post-fusion conformation [17] . 

Before emergency approval for the first vaccines was given by the U.S. Food and Drug Administration (FDA) [30, 31] , much of the clinical focus was on development of effective treatments for COVID-19. While hydroxychloroquine and Remdesivir dominated the headlines [32] [33] [34] , physicians were, and still are, using monoclonal antibodies to treat high-risk cases of COVID-19 [35] [36] [37] . These treatments involve injecting patients with antibodies identified from convalescent COVID-19 patients that specifically target the spike protein, work made possible by the discovery of stabilizing mutations discussed above [38] [39] [40] [41] [42] [43] [44] [45] . Overall, these studies show that effective neutralizers target either the receptor binding domain (RBD) or the Nterminal domain (NTD) -domains located near the top of the spike (Figure 2) .

The most effective RBD-directed antibodies neutralize SARS-CoV-2 by blocking RBD interactions with ACE2 [38, 41, 42, [44] [45] [46] Several genetic "classes" of antibodies are known to arise in common among humans in response to SARS-CoV-J o u r n a l P r e -p r o o f Journal Pre-proof RBD-directed antibodies bind only with RBD in the 'down' position, thus locking the spike in a conformation that cannot bind ACE2 [46, 49] . Others bind to the 'side' of RBD and can bind with RBD in the 'up' position [41, [49] [50] [51] . In both cases, neutralization is thought to arise through interference with ACE2 interaction.

The other region vulnerable to neutralization by antibodies is the NTD.

Neutralizing antibodies targeting the NTD are just as potent as those targeting the RBD [41] , but they neutralize through an as-yet unknown mechanism. There are several possibilities for this action: (i) it blocks the prefusion to postfusion conformational transition, as was seen with a MERS antibody [52] ; (ii) it blocks interaction with one of the other proposed molecules that the virus may use to infect cells [53] [54] [55] ; (iii) it may interfere with some later step in the infection process, after the virus has entered the cell; or (iv) the binding of the antibody to NTD affects the mobility of the RBD allosterically, i.e., through conformational changes that propagate over a long distance [56] . Cryo-EM has revealed that these antibodies target a single 'supersite' [57, 58] in a highly prevalent response. The presence of a single vulnerable site may be due to the large number of sugars shielding much of the NTD, or it may be due to the need for antibodies to approach the viral spike from a particular angle or to induce or prevent a specific conformational change [57] . Most recent data appear to imply the first of these modes of action, namely inhibition of the fusion process after binding to ACE2 [59] .

Understanding the variety of ways antibodies can bind to and neutralize the virus is of critical importance in designing antibody therapies against emerging variants of concern (VOC). Unfortunately, many mutations in these VOC are within the epitopes of the reproducible antibody classes, consistent with the idea that human immune pressure is the driving force in the generation of mutations [60] .

One approach to mitigating this problem was to develop therapies that are 'cocktails' of multiple antibodies, an approach taken by Regeneron [36] . If the mutation giving rise to the variant reduces the efficacy of one antibody, another antibody in the cocktail may still work.

The study of proliferating variants has driven much of the recent cryo-EM work on SARS-CoV-2-neutralizing antibodies. This work has elucidated the atomic positions of the mutations [61, 62] ; helped explain what is driving these mutations [51] ; and most importantly, solved structures of antibodies that are capable of neutralizing the variants, thus making them ideal candidates for improved therapeutics [51, 63] .

Overall, the advent of cryo-EM has revolutionized our ability to quickly visualize and understand viral proteins, their conformational states, and interactions with receptors and antibodies. The speed at which such information became available for SARS-CoV-2 during the COVID-19 pandemic was breathtaking.

For HIV, by comparison, the first structures of pieces of the viral spike appeared about a decade after the onset of the AIDS pandemic [64] , and those of the complete viral spike appeared a decade later [65] [66] [67] . The advent of cryo-EM has defined a "new normal" in which such structural information can be acquired in weeks or months, rather than decades or years. Cryo-EM has thus become a critical pillar in the protection against pandemic threats. Outstanding questions  Integration of techniques, such as genetic sequencing, cryo-EM, and molecular dynamics described in this article, has enabled rapid understanding of virus-host interactions. How will the increasing toolbox of genomics and structural tools (e.g. by addition of cryo-electron tomography) shed further light on these interactions and viral lifecycles? What new therapeutics can be derived from these observations?  As technologies for cryo-EM continue to develop (i.e. more reproducible sample preparation, enhanced detectors, increased accessibility to equipment and expertise), how quickly can the scientific communities contribute to combatting future pandemics? Further, with these advances, how can the global scientific community contribute to on-going battles with known infectious diseases?

J o u r n a l P r e -p r o o f 

Chapter 1: Building the ground for the first two protein structures: Myoglobin and Haemoglobin

Detective quantum efficiency of electron area detectors in electron microscopy

Characterization of a direct detection device imaging camera for transmission electron microscopy

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

Just in Time": The role of cryo-electron microscopy in combating recent pandemics

Emergence of Zaire Ebola virus disease in Guinea

Cryo-EM structure of the Ebola virus nucleoprotein-RNA complex at 3.6 Å resolution

Structural and molecular basis for Ebola virus neutralization by protective human antibodies

Zika virus outbreak, bahia, brazil

The 3.8 Å resolution cryo-EM structure of Zika virus

Largest dengue outbreak of the decade with high fatality may be due to reemergence of DEN-3 serotype in Dhaka, Bangladesh, necessitating immediate public health attention

The origin and molecular epidemiology of dengue fever in Hainan Province, China

Circulation of dengue serotype 1 viruses during the 2019 outbreak in Dar es Salaam, Tanzania. Pathogens and Global Health

Lying in wait: the resurgence of dengue virus after the Zika epidemic in Brazil

Cryo-EM structure of the mature dengue virus at 3.5-Å resolution

Middle East Respiratory Syndrome

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Structures of Ebola virus GP and sGP in complex with therapeutic antibodies

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structural basis for the recognition of SARS-CoV-2 by fulllength human ACE2

Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 reveal a pH-dependent switch to mediate endosomal positioning of receptorbinding domains

A glycan gate controls opening of the SARS-CoV-2 spike protein

Continuous changes in structure mapped by manifold embedding of single-particle data in cryo-EM

Pre-fusion structure of a human coronavirus spike protein

The race for coronavirus vaccines: a graphical guide

FDA takes key action in fight against COVID-19 by issuing emergency use authorization for first COVID-19 vaccine

FDA takes additional action in fight against COVID-19 by issuing emergency use authorization for second COVID-19 vaccine

Trump says he's taking hydroxychloroquine to prevent COVID-19

Hopes rise for coronavirus drug remdesivir

Coronavirus (COVID-19) Update: FDA issues emergency use authorization for potential COVID-19 treatment

Coronavirus (COVID-19) update: FDA authorizes drug combination for treatment of COVID-19

Coronavirus (COVID-19) update: FDA authorizes monoclonal antibodies for treatment of COVID-19

COVID antibody treatments show promise for preventing severe disease

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with Covid-19

Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Potently neutralizing and protective human antibodies against SARS-CoV-2

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Structural basis of a shared antibody response to SARS-CoV-2

An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain

Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

Structural basis for accommodation of emerging B.1.351 and B.1.1.7 variants by two potent SARS-CoV-2 neutralizing antibodies

Structural definition of a neutralization epitope on the Nterminal domain of MERS-CoV spike glycoprotein

AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells

SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

Leaving no stone unturned: Allosteric targeting of SARS-CoV-2 spike protein at putative druggable sites disrupts human angiotensinconverting enzyme interactions at the receptor binding domain

Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single supersite

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity

Structural insights into the cross-neutralization of SARS-CoV and SARS-CoV-2 by the human monoclonal antibody 47D11

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Crystal structure of a soluble cleaved HIV-1 envelope trimer

Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer

Structure and immune recognition of trimeric prefusion HIV-1 Env