key: cord-0913462-zskftqws
authors: Mehra, Rukmankesh; Kepp, Kasper P.
title: Structure and Mutations of SARS-CoV-2 Spike Protein: A Focused Overview
date: 2021-12-02
journal: ACS Infect Dis
DOI: 10.1021/acsinfecdis.1c00433
sha: 4540cd68fe0736d8dfce31b2e2db0d4d23117571
doc_id: 913462
cord_uid: zskftqws

[Image: see text] The spike protein (S-protein) of SARS-CoV-2, the protein that enables the virus to infect human cells, is the basis for many vaccines and a hotspot of concerning virus evolution. Here, we discuss the outstanding progress in structural characterization of the S-protein and how these structures facilitate analysis of virus function and evolution. We emphasize the differences in reported structures and that analysis of structure–function relationships is sensitive to the structure used. We show that the average residue solvent exposure in nearly complete structures is a good descriptor of open vs closed conformation states. Because of structural heterogeneity of functionally important surface-exposed residues, we recommend using averages of a group of high-quality protein structures rather than a single structure before reaching conclusions on specific structure–function relationships. To illustrate these points, we analyze some significant chemical tendencies of prominent S-protein mutations in the context of the available structures. In the discussion of new variants, we emphasize the selectivity of binding to ACE2 vs prominent antibodies rather than simply the antibody escape or ACE2 affinity separately. We note that larger chemical changes, in particular increased electrostatic charge or side-chain volume of exposed surface residues, are recurring in mutations of concern, plausibly related to adaptation to the negative surface potential of human ACE2. We also find indications that the fixated mutations of the S-protein in the main variants are less destabilizing than would be expected on average, possibly pointing toward a selection pressure on the S-protein. The richness of available structures for all of these situations provides an enormously valuable basis for future research into these structure–function relationships.

Since the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1−3 the evolution of the virus has been of increasing concern due to the risk of more infectious, lethal, or antibody-or vaccineresistant variants. 4−9 Of central interest is the spike glycoprotein (S-protein), the heavily glycosylated homotrimeric protein on the surface of coronaviruses giving them their characteristic appearance. 9−11 This protein is responsible for binding to the human cell-surface receptor angiotensinconverting enzyme 2 (ACE2) to promote cell entry. 12−14 Specific presentation of the S-protein to the human immune system is the mode of function of several major vaccines. 15, 16 Considering its central and urgent importance, it is not surprising that many structures of the S-protein have been published. The number of these and the speed at which they have arrived document an outstanding achievement in modern structural biology. A recent database provides an excellent overview for analysis of these structures. 17 Without the recent technical breakthroughs in cryoelectron microscopy of macro-molecules, 18−21 many of these structures would not have been possible, as should be appropriately honored in the context below. Structures have been solved for most relevant conformational states of the receptor binding domain (RBD) without or with (parts of) ACE2 or antibodies bound, at resolutions typically at 2−4 Å.

While sequence-based evolution studies are very helpful and informative, evolution occurs in the context of a folded protein structure. 22−26 Because function is structure-dependent, the structure is the platform on which the amino acid evolution occurs, affecting both the extent of positive and neutral evolution. Accordingly, evolution rates tend to depend on the structural context, e.g., solvent exposure, of the evolving site. 27−29 Of particular importance is the need for the arising mutation to not compromise the overall fold stability of the protein, and thus, the evolution of new mutations often occurs with a tradeoff not to undermine stability. 24,30−33 This trade-off may be the underlying force causing the prefusion S-protein to be metastable in its continuous selection for new mutations that evade antibodies and enhance ACE2 binding. 34 The structure context will aid the understanding of the evolution and function of the S-protein in relation to both immune evasion, development of treatments such as antibodies, and infection via ACE2 binding. S-protein structures from other viruses, notably other human coronaviruses, 35−37 can further strengthen such analysis.

In this Review, we aim to facilitate the use of the wealth of structural information by providing an extensive but datafocused critical overview of more than 200 published structures of the S-protein, including the context of concerning mutations in new emerging variants that may affect ACE2 binding (and thus increase transmission) or reduce antibody binding (and thus impair the efficiency of vaccines). 5,38−40

During natural fusion with ACE2 of the host cell, the S-protein is cleaved and irreversibly changes conformation into a more stable postfusion open conformation state (Figure 1a ). 41 The prefusion S-protein is in a metastable closed state constituted in a lipid surface as a type-I membrane protein with an associated need to be stabilized, both upon fusion with the host cell, and also in the lab to characterize the protein. Strategies employed to this end have relied on mutation to proline in S2 (K986P and V987P), mutation of the furine cleavage site, stabilizing the C-terminal and transmembrane domain by mutation, 34, 42 or employing cystine bridges to stabilize the prefusion trimer. 43 Strategies to improve stabilization and expression of the S-protein are progressing. 44, 45 The S-protein covers the virus lipid surface as a trimer protein, containing the subunit S1 with the N-terminal domain (NTD), RBD, subdomains 1 and 2 (SD1 and SD2), and S2, with heptad repeat 1 (HR1), the central helix (CH), a connecting domain (CD), heptad repeat 2 (HR2), a transmembrane domain (TM), and the C-terminal part of the protein (Figure 1b) . 46 In the closed conformation with all three RBDs in more compact, so-called "down" conformations ("closed" state), the S-protein is not strongly accessible to the human cell, but cleavage into S1 and S2 subunits enables the virus particle to merge with the human host cell membrane, after binding to ACE2 in the "open" conformation where one or more RBDs have changed to the "up" conformation ( Figure  1a) . 42, 46, 47 Mutations may increase infectivity by strengthening amino acid interactions (and thereby the affinity) with ACE2, possibly while also favoring the more open, ACE2-favoring conformations, whereas antibodies will tend to prevent fusion by shielding interaction between the S-protein and ACE2. 47 Recent innovations in cryoelectron microscopy of proteins provide a central underpinning for the success in elucidating the structural biology of SARS-CoV-2 [18] [19] [20] [21] 48 and an important reminder of the importance of basic science enabling later, The first furin S1/S2 cleavage site is shown with yellow balls (R685−S686; fragment is missing in the structure) and the second S2̀cleavage site in magenta (R815−S816). The S1 N-terminal domain (NTD) is followed by the S1 C-terminal domain (S1-CTD). The S1-CTD comprises the receptor binding domain (RBD) region. The second cleavage (S2) forms a fusion peptide upon ACE2 binding. Heptad repeats (HR1 and HR2) are located toward the C-terminal. sometimes urgent applied science. These breakthroughs include advancements in sample handling, electron detectors, and image processing. 19, 20, 48 The structures are obtained with samples of protein molecules typically deposited with a thin layer of vitrified ice on a carbon grid and rapidly cooled using a cryoagent. 19, 20, 49 Subsequent 3D-reconstruction of the protein structure is typically done by single particle analysis 48 or subtomogram averaging of multiple particle data to inform on, e.g., conformational ensembles. 19 Proteins that are embedded in lipids in vivo (such as the spike protein or human membrane proteins) are often difficult to stabilize due their special hydrophobic parts, and various techniques to stabilize them by mutations or with detergents, amphipols, and nanodiscs are common. 50−52 It is important to note for the following that the structures discussed at low temperature may not reflect the real conformations of the S-protein at physiological temperature (37°C): freezing, which is fundamentally important to reduce thermal rearrangements due to ionization-induced radical reactions, 53 tends to remove some conformational dynamics of the protein. 54−58 Freezing-out important conformations and "cryo-contraction" have been observed in X-ray diffraction studies at variable temperature for ribonuclease 56 and myoglobin 57 and in molecular simulations. 54 Conformational changes play a major role in the function of the S-protein upon fusion with the human host cell, and one can expect the conformations to be temperature-dependent, as has indeed recently been observed. 59 Table 1 shows a list of nearly full length (defined here as more than 900 residue coordinates) S-protein structures that we identified from searching the literature, UniProt, and the Protein Data Bank as of June 2021. Structures in the PDB that were not fully documented in the form of a full paper either published or in preprint were not included. The number of structures published during 1.5 years of pandemic is notable; we are not aware of any similar effort in modern structural biology. In addition, several structures of important natural variants have also been elucidated, as summarized in Table 2 , and with already published structures of other coronavirus Sproteins (Table 3) , this provides a wealth of structural information for detailed structure-based functional and evolutionary studies.

The many available structures even for presumably same protein states raises a question on the relevance and transferability of conclusions based on different structures. Many of the structures are of excellent resolution considering that the size of the protein and the way the structures are obtained, approaching 3 Å resolution in some cryo-EM structures. Also, many conformation states and both antibody-(Ab)-bound and ACE2-bound structures have been obtained so that the structures can be used comparatively, and we can estimate the heterogeneity between them and how this affects structure−function relationships. For smaller parts of the RBD interacting with antibodies, some crystal structures are available at resolutions approaching 2 Å. ■ STRUCTURAL HETEROGENEITY: WHICH STRUCTURE SHOULD BE USED?

The protein structures listed in Tables 1−3 were produced by different research groups and sometimes different protocols and protein states. The large number of studies complicates the choice of structure to use for structure-guided functional or evolutionary analysis. Of concern in this choice is (1) the Table 4 summarizes data for the apoprotein, Table 5 for the protein bound to ACE2, and Tables 6 and 7 for the protein bound to antibodies.

In terms of quality, 2.5−3.5 Å represents the resolution range where amino acid side chain conformations and functionally important surface residues become resolved. 60 A resolution of 3 Å does not enable identification of all atom positions, including water molecules, but provides very good information on the overall backbone conformation and secondary and tertiary structure. 60 We expect many surface residues to be not well-resolved at 3 Å resolution, and some rotamers may be mismodeled. These residues are the functionally important ones in the case of the S-protein, i.e., resolution improvements toward the 2-Å range would be of substantial value. Generally, many of the published structures display very good resolution of the secondary structure and tertiary structure of the S-protein, but the confidence in the orientations of individual surface residues is limited (see below).

The percentage of outliers from Ramachandran plots (torsion angles of the peptide backbone) are also shown in Tables 4−7 as an indicator of the structure's backbone conformations. 60 These were calculated using the Procheck program 61 (version 3.6.2) available via the PDBsum server from EMBL-EBI. 62 A smaller number implies a normal and expected backbone conformation in the structure, whereas a larger number implies more unusual or "strained" backbone conformations in the structure. For the apo-S-protein structures of Table 4 , the values are generally quite as expected, with Ramachandran outlier residues typically constituting only 0.0−0.3% of the structures. The few exceptions to this are the active state 7DWZ (0.5%), 63 the prefusion state 7KJ5 (0.4%), 64 and the 1up conformation structure 7KDH (0.4%), 65 but these numbers are fully reasonable, as 0−0.5% translates to only 0−5 of the approximately 1000 residues in the S-protein having an unusual backbone conformation.

In terms of amino acid coverage, the total number of residue sites resolved by coordinates is listed as "N" in Tables 4−7. For a given resolution of ∼3 Å, a more complete structure seems more suitable for study, or if specific mutations are of interest, they should be at least covered by full coordinates in the structure. For example, P681 is an important mutation site that is typically not present in the structures, and most structures miss many of the disordered N-terminal sites harboring mutations of interest such as S13I, L18F, T20N, and P26S. Thus, for example, structure 7LXY has site T20 but misses P26, whereas 6ZB4 has P26 but misses T20. Many other structures, such as 6VXX and 6XM0, miss both.

In addition to choosing the correct protein state for the analysis of interest (apoprotein or bound to, e.g., ACE2), it is also important to consider that some protein structures may represent different, more or less open states, both in terms of having 0, 1, 2, or 3 RBD in the upward conformation, but also in terms of other features, such as mutated or deleted transmembrane domains that may affect the overall structure. Herrera et al. reported that some structures (they identified 6VXX, 6X29, 6X2C, 6X79, 6ZOX, 6ZOY, 6ZP0, 6ZP1, 6ZWV) display one conformation (called conformation 1), whereas others (e.g., 6XR8, 6ZGE, 6ZGH, 6ZGI, 6ZP2, 7JJI, 7JJJ) display conformation 2 and have missing residues with respect to the region that starts at the notorious site 614 (614− 642). 45 As discussed below, the D614 site has substantial heterogeneity even between structures resembling the supposedly same state.

Evidently, the S-protein is very sensitive to changes in the environment, and it is reasonable to expect the in vivo conformations of the protein in the lipid membrane of the virus to differ from the structures obtained in vitro: pH 66,67 and temperature 59 change the conformational states, and molecular crowding, salt, or other features of the local heterogeneous in vivo environment may be expected to do so as well. Conformational states may be affected by delicate effects in the protocols, 68 e.g., the mutations used to stabilize the protein for characterization 34 and the temperature effects on the more dynamic surface residues of the protein. 54−58 Higher temperature as in the human body is likely to favor more entropic conformations states, which might affect the epitopes and may explain the temperature-dependence of some structures. 59 The extent of specific protein variants to favor certain open or closed conformations should thus be seen in this context. For example, the D614G mutation has been reported to favor a more open conformational state 69 but the tendency to do so is probably temperature-dependent. 70 Accordingly, subtomogram averaging and physiological-temperature molecular dynamics simulations are important for analyzing the conformations of the protein, as increasingly explored. 70−74 To determine the extent of the variation in the published Sprotein structures of Tables 4−6, we have listed the average relative solvent accessible surface area (RSA) per site in the structure as an indicator of conformational openness, using the Naccess algorithm 75 as implemented via the FreeSASA program. 76 This property is of central importance in protein evolution, 77 and is particularly relevant because most of the function of interest of the S-protein relates to surface interaction with other proteins, notably antibodies and ACE2. Mutations that affect virus infectivity and immune ACS Infectious Diseases pubs.acs.org/journal/aidcbc Review evasion are likely to be solvent-exposed, as they will then more directly affect the affinity for antibodies or ACE2, e.g., via electrostatic, hydrophobic, or hydrogen bond interactions. As seen from Tables 4−6, the average exposure per residue of the S-protein tends to be approximately 25−30% (RSA values 0.25−0.30). Considering that the number is an average of approximately 1000 residues, even a difference of 0.01 between two structures (1% difference on average) indicates substantial surface heterogeneity, e.g., 20 residues that change from 0 to 50% exposure. However, we cannot determine which conformation is correct, due to mutations used to stabilize the proteins, the modest resolution and cryo-effects on the conformational dynamics. 54 Still, the total average provides a robust indicator of the surface-specific heterogeneity of the protein structures.

We note that for the apo-S-protein (Table 4) (Table 4) . All of the studies that included both closed and partially open states by Melero et al., 68 Juraszek et al., 79 Henderson et al., 46 Wrobel et al., 80 

The critical step in infection is the fusion of the S-protein with human ACE2, a process that enables the partial opening of the cell membrane and injection of virus genetic material into the cytoplasm. 12, 13 New arising mutations in particular on the surface of the S-protein can affect this crucial interaction and thereby the infection process, with both alpha and beta variants shown to possess higher ACE2 affinity. 81 Figure 2a shows the apo-S-protein (i.e., without any antibody or other protein bound), in its primary closed conformation with all three RBDs in the downward conformation (PDB: 7DF3). 82 Prominent naturally occurring mutations are shown in red and orange. Sites of the RBD shown in red harbor mutations of concern either due to enhanced antibody evasion or effects on transmission. Figure 2b and Figure 2c show the corresponding structure and mutations of the open state involved in ACE2 binding: Figure 2b shows the specific conformations of this state, whereas Figure 2c shows the full context of ACE2binding, based on the published structure 7KMS by Zhou et al. 67 One can envision mutations to increase the infectivity either by increasing affinity toward ACE2 by maintaining higher ACE2 affinity relative to the most important antibodies, possibly favoring open conformation states that associate more strongly with ACE2 ( Figure 2) . The virus particle's lifetime in the host may depend on the relative propensity to bind to ACE2 and infect cells vs binding to prominent antibodies. This relative propensity via competitive binding is quantified by the ratio of chemical association constants K ACE2 /K Ab corresponding to the difference in binding free energy of the S-protein to ACE2 vs antibodies (Ab). These affinities are again defined by the mutations that mostly change the interaction with ACE2 and the antibodies. Competitive binding is a distinct consideration from either binding to antibodies (host immunity evasion) or ACE2 (host cell fusion) that plausibly correlates better with virus fitness. Surface mutations may increase binding nonspecifically, and mutations that bind less to antibodies ("antibody escape") can also bind less to ACE2, which may not change the overall fitness of the variant. These considerations indicate why the relative affinity toward ACE2 and antibodies is of importance to structure-based understanding of SARS-CoV-2 transmission. One may also expect individual variations in e.g., ACE2 expression and surface composition to contribute to the heterogeneity in susceptibility and transmissibility that plays a central role in the epidemiology of the disease. 83, 84 For example, ACE2 expression is age-dependent. 85, 86 and may correlate with mortality, 87 consistent with lower susceptibility being a function of S-protein-ACE2 complex formation.

To understand these interactions, we need to apply the structures of the S-protein bound to ACE2, but also a reasonable structure of the apo-S-protein, to appreciate how the protein structure itself is affected by the binding and how mutations affect the two states. Thus, whereas Table 4 lists properties of the apo-S-protein, Table 5 lists corresponding properties of resolved structures of the S-protein bound to (parts of) ACE2. As already mentioned, the S-protein preferably binds ACE2 in a more open conformation; thus, the RSA values of the ACE2-S-protein complexes are generally of the "open" type, typically 0.28−0.29, with only three exceptions in Table 5 . In general, the resolution of the ACE2 complexes is not as good as for the apo-S-protein, with most R-values >3.5 Å. The notable exceptions are the structures by Yan et al. 63 (2.9−3.6 Å) that also include both the 1-up, 2-up, and 3-up conformation states with one or two ACE2 peptidase domains. Also of note, Zhou et al. 67 studied the complexes with 1, 2, and 3 ACE2 molecules bound at two different pH values, 7.4 and 5.5. The RSA values indicate that pH does not affect the ACE2 complexes as much as the apo-S-protein probably because the ACE2-bound S-protein is always in the open state, regardless of pH.

Some properties of 80 structures published with antibodies (including nanobodies, etc.) bound to the S-protein are summarized in Table 6 . Antigenic drift can be defined as reduced affinity (typically in the picomolar range) of new arising mutations toward important antibodies. 88, 89 New variants may harbor mutations at positions of the S-protein that interact strongly with notable antibodies, including prominent antibodies targeting earlier variants, and these new mutations may reduce the affinity for the antibodies, producing more resistant virus variants. 6,90−92 Understanding this antibody evasion thus requires understanding the (loss of) binding affinity caused by mutation, which has been obtained in several important studies 39,93−95 and is a good structural basis for rationalization of the observed effects.

The 80 structures included in Table 6 cover a large range of resolutions and very distinct complexes, and thus offer a wealth of information on the protein−protein interactions of the Sprotein. Despite the complexity of these structures, the structures are often of comparable resolution to typical structures of the apo-S-protein (Table 4) , with many achieving resolutions near 3 Å, which may be considered a benchmark. Some interesting highlights include structures revealing distinct conformation states even for the same antibody, such as P17 in 1-up and 2-up conformations (7CWM/7CWL), 96 which further testifies to the conformational plasticity of the S-protein. Another example is the study by Lv et al. 97 of H014 binding covering both 1, 2, and 3 molecules binding to the Sprotein trimer at reasonable resolution, with corresponding 1up, 2-up, and 3-up conformations, which provides important systematic insight into the effect of binding stoichiometry on the S-protein structure.

An escape mutation with regards to one antibody may be captured by another antibody due to distinct antibody surfaces and associated binding modes. This is the molecular basis for the promising use of antibody cocktails in vaccines to minimize the potential threat of escape mutations. 93 An illustrative example is the antibody cocktail REGN10987+REGN10933 (casirivimab/imdevimab) combining two antibodies that bind to different parts of the RBD of the S-protein and thus potentially makes antigenic drift more difficult as it requires multiple substitutions. 93 This illustrates well the power of structural biology in providing the basis for interpreting the assay data of the escape mutations and the effect of different antibodies. Figure 3 illustrates some representative structures of the Sprotein associated with ACE2 and antibodies, with emphasis on the different conformations achievable. Antibodies are produced by the immune system to encapsulate the virus and target it for destruction by e.g., macrophages, and therefore the binding to ACE2 may involve residues distinct from those interacting with antibodies. Structural alignment of 10 representative ACE2 complexes (Figure 3a ) reveals conformational heterogeneity. Structural alignment of antibody complexes (7A29, Figure 3b ) reveals distinct conformations obtainable even when the same antibody binds. Such heterogeneity is also evident from the different conformational states attained in the strongly bound miniprotein complex 7JZL 98 (Figure 3c) , where two antibodies bound to S-protein attained similar conformations whereas a third attained a different RBD-conformation. Thus, there is substantial conformational variation in the S-protein bound even to the same antibodies, probably also affected by both temperature, molecular crowding, and pH. 67 Because the resolution is lower in nearly complete cryo-EM structures, it is also of interest to discuss X-ray crystal structures of smaller parts of the S-protein interacting with antibodies, which have been obtained at higher resolution in several cases, and thus provide a more precise structural account of the S-protein−antibody interaction. Illustrative examples of such structures of antibodies interacting with the RBD with resolutions at 2.5 Å and below reported in published papers are compiled in Table 7 . These structures provide in some cases a substantially better detail of individual amino acid conformations, with the resolution of interactions also being improved relative to full cryo-EM structures; however, it is at the expense of only having some parts of the full chemical composition, which may affect precision.

For the discussion here, we emphasize enlightening studies of variant effects on RBD interaction with antibodies ( Figure  4 ). In one notable study, Supasa et al. 99 reported 1.8 and 2.2 Å resolution structures of RBD binding to antibodies with and without the N501Y mutation at the ACE2 interacting surface. Although the alpha variant does not generally show escape from natural or monoclonal antibodies or vaccines, it is not easily neutralized by some antibodies, and interaction can occur with the antibody light chain at position 501. 99 Interaction of the COVOX-269 antibody with S-protein RBD was shown to be affected by the larger tyrosine side chain at position 501 (Figure 4a−c) .

In another insightful study of the beta variant (lineage B.1.351) structure (7NXA; Figure 4d ), three mutations, K417N, E484K, and N501Y, affected binding to two antibodies COVOX-222 and EY6A, providing a structural basis for the chemical change in interaction and reduced antibody affinity. 100 Similarly, the published structure of the RBD of gamma (P.1) (7NXB) (Figure 4e ) comprises three gamma mutations K417T, E484K, and N501Y, two of which are also present in beta variant. 100 These mutation sites interact with COVOX-222, whereas EY6A binds to the peripheral region of the RBD (Figure 4d, 4e) . The gamma variant has displayed less resistance to antibodies produced from vaccine or natural infection as compared to beta, indicating possible neutralization effects outside the RDB. 100 As a final example, Figure 4f represents a high-resolution (1.4 Å) RBD structure complexed with the cross-neutralizing antibody 7D6. 101 This antibody binds to a cryptic site, which is distinct from those typically targeted by other antibodies (e.g., Figure 4a −e), and accordingly, the RBD attains a different conformation (closed) as compared to the structures shown in Figure 4a −e. The side-chain amino acid conformations in these good-resolution crystal structures are substantially more precisely determined, which makes the structures highly complementary to the lower-resolution, but more complete cryo-EM structures.

Due to the conformation states being affected sometimes differently by binding ACE2 and since the interacting residues are not generally the same, the binding energy effect of a mutation is likely to differ for ACE2 and any specific antibody. The most successful variants may not be those that increase the free energy of binding to ACE2 or decrease the free energy of binding to prominent antibodies, but those with the largest dif ference in affinity toward ACE2 and prominent antibodies. Such selectivity is plausibly related to the virion lifetime in the host and could in principle be measured or computed from published affinities of mutants toward antibodies and ACE2, 92−95,102,103 using the structures reviewed in Tables 5−7 as input for such analysis.

SARS-CoV-2 is the seventh coronavirus known to infect Homo sapiens, the others being the common-cold causing HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, as well as the more pathogenic SARS-CoV-1 and MERS-CoV. 104, 105 While some of the milder of these are α-coronaviruses and some β-coronaviruses (OC43, HKU1), all the three most pathogenic viruses are β-coronaviruses with distinct functional properties. 106 Coronaviruses consist of four main structural proteins, with the membrane (M), envelope (E), and a N = residues in structure; chains = chains in structure; res (Å) = resolution in Å; RSA avg = average solvent exposure of all residues. % outl. = outliers of Ramachandran plot in % (from PDB full report). nucleocapsid (N) proteins in addition to the S-protein of focus in the present paper. 107 Compared to some other mRNA viruses, which generally have very high mutation rates, coronaviruses tend to evolve several-fold more slowly due to their proof-reading machinery, 108 of the order of 10 −4 substitutions per site per year. 105,109 SARS-CoV-2 is currently evolving substantially faster than this (albeit still slowly compared to other nonproofed mRNA viruses), estimated at 7 × 10 −4 substitutions per site per year (2 × 10 −6 per day), 8 and has seen a remarkable lineage evolution during the pandemic, as expected from its high prevalence and ongoing adaptation to humans. 9 Since the emergence of SARS-CoV-2, estimated to be Autumn 2019, several hundred recurrent mutations have been identified, 80% of these being nonsynonymous changes in the virus proteins, 4 including many in the S-protein. 11 As with the other highly pathogenic coronaviruses MERS-CoV and SARS-CoV, 104 the closest related lineages are found in bats. 4 The virus is subject to both neutral evolution and positive selection, and both the nonsynonymous 4 and synonymous 110 evolution rates have been found to be high.

We expect SARS-CoV-2 to adapt by accumulating mutations that increase binding to human ACE2, under increased selection pressure from S-protein-based immunity induced by vaccines and therapeutic antibodies, which may lead to mutations that minimize binding to S-protein antibodies frequent in the population. 103, 111 Accordingly, bat S-proteins do not have the same affinity for ACE2. 80 These two fitness effect may not work additively; in fact we could envision many mutations to bind better to both proteins, such that the relative specificity for ACE2 vs prominent antibodies would contribute to the fitness. At the same time, overall S-protein stability is probably a restraining parameter on possible evolution, limiting new groups of mutations to those that do not compromise overall conformational stability of the protein in the virion lipid surface, consistent with the constraining role of stability seen more generally in protein evolution. 24, 30, 33, 112, 113 After the early D614G mutant 114 became quickly dominant plausibly due to a modest fitness advantage, 69,115 a period of 119 These variants are of concern due to the presence of amino acid substitutions such as E484K that are associated with escape from monoclonal antibodies. 120, 121 Importantly, whereas the new variants display reduced sensitivity to some vaccines after first dose, vaccine efficiency remains high toward symptomatic infection after full dosing, and the most important function of the vaccines is to reduce the severity of the disease. 122,123 Representative Sprotein structures of variants of SARS-CoV2 are displayed in Table 8 . The D614G structure has been elucidated by Yurkovetskiy et al. (6XS6), 69 Gobeil et al. 65 (7KDK etc.), Yan et al. 63 (7DX1), and Benton et al. 124 (7BNN, 7BNO) . Yurkovetskiy et al. 69 reported a single D614G structure in an open conformation state which was speculated to be suitable for fusion, although the actual binding affinity to ACE2 was lowered. The authors speculated that this conformation change could explain its fixation in the population. The open conformation is consistent with the relative large RSA (0.29) similar to that of Yan et al. 63 (7DX1, 0.29) and Benton et al. 124 (7BNN, 7BNO, 0.30) and has been supported by molecular dynamics simulations at physiologically relevant temperature. 70 Since then, however, other conformation states have been obtained for D614G. Gobeil et al. 65 have elucidated the D614G in several more closed conformation states, with the variant clearly able to form the same closed and open conformation states as the reference early Wuhan variant (structures summarized in Table 4 ). The more closed and 1-up conformations all have RSA values of 0.26−0.27, indicating reduced solvent exposure. Because D614G has been obtained in both open and closed states, and due to the delicate impact of environmental factors on conformation state, 59, 67 the tendency toward open conformations could also be affected by the deletion of the furin site and proline mutation, as used by e.g., Yurkovetskiy et al. 69 Herrera et al. found major heterogeneity in a motif starting at site 614 (614−642) implying its pronounced variability. 45 Because the in vivo protein conformational state is sensitive to composition and molecular environment, it is not possible to deduce that one variant prefers one conformation state over another except when studied under the same conditions with the same protocol.

In addition to D614G, major achievements in the structural biology of variants of concern have been documented during Summer 2021. Notably, Gobeil et al. 125 published both mink- Table 8 ), and Cai et al. published structures and antibody escape data for alpha and beta (7N1Q, 7N1T, 7N1U, 7N1V, 7N1W, 7N1X, and 7N1Y). 127 All of these structures are of good resolution with excellent metrics and form an essential basis for understanding SARS-CoV-2 evolution consistent with this Review's emphasis on three selection pressures: one to preserve or improve protein stability, one toward enhanced binding to ACE2, and one toward reduced affinity toward antibodies. Each mutation is likely to contribute differently to these three terms and thus the overall fitness effect.

Taking the wider perspective of other human-host coronaviruses (Table 9 ) and other spike protein structures (Table 10) , 128 several mutations in the SARS-CoV-2 RBD have contributed to a stronger affinity toward ACE2. 47 Indeed, by far the most sequence variation between SARS-CoV-1 and SARS-CoV-2 occurs in the S1 domain that includes the RBD. 107, 129 In addition, the highly positively charged amino acid sequence RRAR represents a new furin-like cleavage site that is not seen in other related β-coronaviruses. 80, 130 Many sequence parts of the RBD of these viruses show similarity both to nonhuman but also human protein motifs, which may help to understand protein−protein interactions with the Sprotein more broadly. 131 Whereas the S-protein structures of MERS-CoV and SARS-CoV-1 display RBD-up conformations as typically associated with strong ACE2 binding by SARS-CoV-2, these ACE2adapting conformations were not seen in the S-protein structures from HCoV-OC43 and HCoV-HKU1. 132 We note that the two latter coronaviruses mainly use 9-O-acetylated sialic acids for fusion, 107 which could possibly relate to this observation. The structure 6NZK in Table 9 provides a structural basis for this type of interaction. 132 Figure 5 shows the electrostatic potential maps of the Sprotein RBD of the three most pathogenic coronaviruses, SARS-CoV-2 (Figure 5a ), SARS-CoV-1 (Figure 5b) , and MERS-CoV (Figure 5c ), indicating clearly different electrostatic patterns on the surfaces. Electrostatic interactions are dominating interactions in proteins 133−137 and thus particularly likely to give rise to non-neutral functional effects. Whereas MERS-CoV has substantial buried positive charge and less positive charge on the surface, the SARS-CoV-1 and SARS-CoV-2 S-protein RBDs both have considerable positive charge on the surface. This may perhaps relate to MERS-CoV Sprotein interacting with the distinct receptor dipeptidyl peptidase 4, 138 whereas SARS-CoV-1 and SARS-CoV-2 preferably use ACE2. 13, 129, 139, 140 As shown in Figure 6 , electrostatic potential analysis indicates a high negative charge potential on the surface of ACE2 pointing toward the area interacting with the S-protein.

Considering that positive charge introductions in prominent mutations of concern occur on the exact interface of this interaction, it is plausible that this will generate favorable 

In the following, we take a closer look at prominent natural mutations, with relevant structures summarized in Table 8 .

Variants of interest and concern, summarized in Table 11 , harbor mutations that are likely to increase transmission, e.g., by causing the S-protein to bind more strongly to ACE2, or evading the binding of human antibodies or vaccines. 103, 111, 141, 142 Among these mutations, deletions have been associated with substantial escape tendency. 6 Most mutations of concern are, however, missense substitutions where one amino acid has been changed for another. 4,11 Some properties of mutations in the main natural variants are listed in Figure 7a , including the change in polarity (measured by the Grantham scale 143 ) of the amino acid, the change in side-chain volume, ΔV, and the RSA average of the site from eight structures (7LXY, 6XM0, 6ZB4, 7DDD, 6ZGE, 6VXX, 6VYB, and 7DWY). These properties represent three of the five important patterns needed to quantify proteindifference metrics and substitution tendencies (considering hydrophobicity and polarity inversely related, the remaining being secondary structure and codon use), 144 and are important for the general fold stability of protein structures. 145 As seen, the polarity and volume changes tend to be randomly affected, as expected for neutral mutations with no chemical property under selection. On average, the mutations tend to occur in sites of typical exposure (∼0.25−0.30, Table 4 ). However, the mutation sites of concern, such as sites E484 and P681, are more exposed than typical mutated sites, consistent with selection for interaction with other proteins. Figure 7b displays the change in polarity and volume of the same natural mutations, indicating that they spread quite randomly around the average change of all possible mutations in the protein (these properties are obtained from the simple amino acids and are thus not structure-dependent). We note that, except for E484K and E484Q, mutations of concern tend a PDB = PDB code; N = number of residues in structure; chains = number of chains in structure; res (Å) = resolution in Å; RSA avg = average solvent exposure of all residues; reference = primary citation; % outl. = outliers of Ramachandran plot in % (from PDB full report).

pubs.acs.org/journal/aidcbc Review to be farther away from the centroid, i.e. have larger changes in volume and polarity than average, consistent with a nonneutral chemical effect on protein function, e.g., association with ACE2 and antibodies. D614G is itself an unusual mutation involving both a change in charge and introduction of glycine (the smallest and sometimes structure-breaking amino acid), yet was fixated early and probably associated with evolutionary advantage. N501Y, which is characteristic of alpha, beta, and gamma variants, changes from a small polar to an aromatic tyrosine, which increases binding to ACE2. 102 Changes to arginine (P681R, L452R) are also conspicuous. Larger chemical changes are typically less likely to occur in protein evolution and are more often associated with nonneutral evolution. 146, 147 Indeed, L452R is a known antibody escape mutation. 126, 148 In contrast, mutations with typical a PDB = PDB code; N = number of residues in structure; chains = number of chains in structure; res (Å) = resolution in Å; RSA avg = average solvent exposure of all residues; reference = primary citation;. % outl. = outliers of Ramachandran plot in % (from PDB full report). changes in chemical properties that are not very exposed are expected (but not guaranteed) to be neutral. Mutations E484K and E484Q, which do not drastically change polarity and size, instead change the full charge of a very exposed site. Very many of the most prominent mutations change the charge of exposed residues in the S-protein. Of the 33 mutations listed in Figure 7a , 18 change the charge further than already shown for the reference genome S-protein: D80A, D138Y, G142D, E154K, R190S, D215G, R246I, K417N/ K417T, N439K, N440K, L452R, T478K, E484K/E484Q, A570D, D614G, and P681R. Twelve of these change the surface charge toward more positive, the most notably being the early D614G substitution, introduction of lysine (K) at positions 439 or 440 (N439K, N440K), gain of arginine at position 452 (L452R), and loss of glutamate at position 484 (E484K/E484Q), whereas the remaining 6 contribute to more negative surface charge. Functional effects of all three positive charge introductions have been seen for N439K, 149 E484K, 38, 150 and L452R. 126, 148 The delta variant has two positive charge introductions in the RBD, L452R and T478K. 8 Interestingly, mutations that reduce positive charge at position K417 (K417N/T) tend to reduce ACE2 affinity; 102, 149 together, these data could suggest that positive charge in the RBD is a main adaptation to human ACE2 interaction.

We can also estimate the impact on the S-protein stability using the structures available and computational methods. Fold stability (free energy of folding the protein) is the main tradeoff in protein evolution of new functionality 30,31,113,151−153 and also plays a role in many diseases driven by point mutations. 154−156 Such calculations should be of substantial interest, as they may provide information on whether fold stability of the S-protein is a desirable trait in the human host or whether it is relatively easily traded for improved function (ACE2 interaction, antibody evasion). Many such programs exist that use different machine-learning or energy-based forcefields. 157−165 To illustrate this point we computed the change in folding free energy (in kcal/mol) estimated by the recently developed method, Simba; 145 all these methods are inherently subject to limitations 166−169 so these results should only be considered expected estimates; however, Simba has the advantage in contrast to many machine-learning methods of interpretable residue-specific chemical contributions to stability, at generally similar accuracy. 145 These free energy changes are summarized in Figure 7c plotted against the RSA of each mutated site.

The mutations in variants of concern are notably more stabilizing and notably more solvent-exposed than the "average" mutation in the S-protein. It is often expected that evolution will be faster on the surface due to less functional selection pressure and also smaller risk of disturbing the protein structure. 77,170,171 Accordingly, despite the functionality manifesting at the surface of the S-protein, the relation in Figure 7c is not surprising by itself. However, it is remarkable and a possible signature of positive selection that so many of the prominent natural mutations are in the upper right rather than lower left on this trend line, i.e. that they tend to be more solvent exposed and impair S-protein stability less than expected for a random mutation (estimated −1.2 kcal/mol), with several mutations being predicted to be stabilizing, although such considerations need more detailed analysis of evolution rates. Recent nanomechanical stress studies have indicated that the S-protein has evolved enhanced robustness. 172 A particularly concerning mutation in the spike-protein, E484K seen in e.g., beta (B.1.351) and gamma (lineage P.1), 117 is known to evade antibodies 39 and has been reported to have about 10-fold higher escape than the normal variant. 120 Immune plasma from individuals infected with early variants of the virus may not be equally effective against infection with virus variants harboring the E484K mutation so that new antibody cocktails are desirable. 39 The net positive surface potential is already markedly more positive when comparing the reference variant to SARS-CoV and MERS-CoV ( Figure 5 ) and has been supplemented by additional charge addition with E484K, which could suggest an evolutionary advantage of positive surface charge relating to ACE2 interaction and antibody escape. Because full charges contribute the largest electrostatic effects on interaction energies, 134, 135 such charge changes may be expected to also produce large perturbations of interaction also with antibodies.

Other exposed charge-changing mutations of note include K417T and K417N seen in gamma (P.1) and beta (B.1.351), respectively. K417N, like E484K, reduces the binding of the antibody families IGHV3-53/3-66 and IGHV1-2. 91 N439K, a relatively common mutation, displays reduced binding of some antibodies, 148 Finally, it is important to stress that the effects of single amino acid changes, as analyzed above and in the single mutant scan assays for ACE2 binding and antibody binding, 88, 94 are not expected to be completely linear, due to amino acid correlation effects (within-protein epistasis), and possibly epistasis between genes. 173−176 This point is very important in the context of SARS-CoV-2 variants. 9 The currently circulating variants are all multisite mutants, i.e. the virulence, transmission, or antibody evasion cannot be assumed simply a linear function of the individual amino acid effects. Influenza evolution has been shown to be highly dependent on epistasis in relation to maintaining protein stability, 177 and such structure−function trade-offs could be speculated to also cause the apparent avoidance of destabilization of many SARS-CoV-2 mutations in Figure 7c . This should be remembered in the context of Table 11 , where some data are available both for the full variant and for individual mutations.

In the analysis in Figure 7 , we used averages of eight structures (7LXY, 6XM0, 6ZB4, 7DDD, 6ZGE, 6VXX, 6VYB, and 7DWY) to calculate the RSA and stability effects. The reason is that most structural heterogeneity in the S-protein structures tends to occur on the surface of the proteins, which is generally less ordered than the buried parts of the protein, and structural heterogeneity is known to affect protein energies. 167, 178 This is directly seen in the different RSA of the same residues in some reported structures. Because the local environment of the residue is very important for deducing its properties and role, this concern is relevant to any theoretical analysis of mutation structure−function relationships.

To further quantify the structural heterogeneity of the Sprotein surface sites, we calculated the RSA of the 33 mutation sites discussed above, using ten different apo-S-protein structures (6XM0, 6VYB, 7DDD, 6ZGE, 6VXX, 7DWY, 6X6P, 6ZOX, 7CAB, and 6XF5) produced by different groups (Figure 8a ; note that not all sites are available in all the structures, and S13 and P681 are not in any of these structures). The standard deviation in RSA was 0.12, i.e. we expect the solvent exposure of a specific site to have (Table 4) .

As another example of site heterogeneity, the important site K417 (with mutations K417N, K417T which reduce antibody binding 91 ) has only RSA = 0.01 in 7DDD and 0.07 in 7DWY (essentially buried site), but 0.33 in 6XM0, and 0.41 in 6VYB, despite all these structures being of the same chemical composition as apo-S proteins. Analysis that draws conclusions based on the local environment of these important sites, e.g., computational estimates of ACE2-binding and stability, will be very sensitive to this heterogeneity. Larger heterogeneity is also seen in the N-terminal part of the S-protein, such as D80 (D80A) and D138 (D138Y), which are highly exposed in 6XM0 and 6VXX (RSA = 0.52−0.74) but almost fully buried in 7DDD (0.03−0.17) and 6ZGE (0.11−0.13). Part of this is due to the different open and closed conformation states they represent, but a large part is what can be considered experimental noise, since 6VXX, 6XM0, and 6VXX all represent closed conformation states. Because of the large heterogeneity of some sites and the known conformational sensitivity of the protein, 41, 67 it is strongly recommended to account for this heterogeneity for the mutation of interest before deducing its potential effects.

The corresponding standard deviation in the estimated free energy effect of each mutation was 0.2 kcal/mol, which gives a more chemical quantification of the structural heterogeneity (many structural variations occur in disordered low-energy modes which may overemphasize differences). This variation does not change the overall conclusions from Figure 7c that most mutations of concern tend to be solvent-exposed and probably less destabilizing than typical random mutations in the protein. However, the structural heterogeneity of published cryoelectron microscopy structures reflect the real conformational plasticity of the protein as a functional requirement for its conversion from the metastable prefusion state. 34 Conformational plasticity in the prefusion state may even confer evolutionary advantage by providing dynamic variation of the epitopes accessible to antibodies; although this remains to be investigated, such "conformational masking" has been seen in relation to other, e.g., HIV. 179 As a further indication of structural heterogeneity, we aligned 10 apo-S-protein structures (6XM0, 6VYB, 7DDD, 6ZGE, 6VXX, 7DWY, 6X6P, 6ZOX, 7CAB, and 6XF5). The structures differed in their relative RMSDs from 0.5 to 3.6 Å, ranging from close resemblance to substantial differences ( Figure 8b) . Thus, the overall structural heterogeneity is as large for the apo-S-structures as for the different ACE2 complexes (Figure 3a) , i.e. the higher plasticity in the apo-state produces as much heterogeneity as the different conformations induced by ACE2 binding. We conclude that subtle effects in the laboratory may lead to distinct local conformations even for the same protein states, consistent with the conformational sensitivity implied by temperature-and pH-dependent effects; 59,67 on top comes additional cooling effects on the structure and dynamics that may underemphasize entropic states of lipid-embedded proteins. 54 

SARS-CoV-2 is likely to become endemic and evolve into a milder virus due to intense efforts in clinical treatments and vaccination, yet the continued protein evolution, especially of the S-protein, will remain a concern to be monitored and countered. 180−182 The structural biology of the S-protein is a central cornerstone of this future, serving as the structural basis for rationalizing and predicting impacts of new mutations and treatments against them. We have summarized the current state of the art of structures of the S-protein of SARS-CoV-2, emphasizing the importance of completeness of the structures, their resolution, the conformations they represent, and the structural heterogeneity and plasticity especially of functionally important surface residues and its relation to evolutionary and functional analysis.

While many of the published structures are of very good (∼3 Å) resolution allowing secondary structure to be wellaccounted for, surface residue conformations are not generally resolved at 3 Å and this leads to substantial structural heterogeneity in the surface sites, which unfortunately are the sites of main functional interest in the S-protein. Even for protein structures supposedly representing the same conformational state, individual mutation sites can be situated in quite different environments, with solvent exposure varying up to 50% in some cases. These differences resonate with an overall tendency of conformational plasticity in the S-protein, 46, 82 being affected also by temperature and pH. 59, 67 Because of this heterogeneity, we recommend using averages of a group of high-quality protein structures rather than a single structure before reaching conclusions on specific structure−function relationships.

The most important topic for the future of SARS-CoV-2 management is arguably to predict new virus evolution and counter it by, e.g., annual mutation-optimized boosters, to minimize mortality in the endemic state as is also attempted in many countries with influenza. 183−186 A large part of the antigenic drift in the endemic state is also likely to occur in the S-protein, making the structures of the S-protein and their mutations critical to a rational prediction and understanding of this evolution. 180 We expect SARS-CoV-2 to adapt by accumulating mutations that optimize binding to human ACE2 under selection from Sprotein-based immunity induced by vaccines and therapeutic antibodies, which favors mutations that minimize binding to Sprotein antibodies frequent in the population. 103, 111 Indeed, the structures suggest that large chemical changes, notably affecting exposed surface charges, are recurring in the mutations of concern, possibly related to non-neutral effects on ACE2 or antibody interaction. These two fitness contributions may not work additively; many mutations may bind better to both proteins, such that the relative specificity for ACE2 vs prominent antibodies would determine fitness.

We also expect overall protein stability to be a constraint on this evolution: mutations of the S-protein may be less destabilizing than randomly expected, consistent with a constraining selection pressure on the S-protein to not lose overall stability while adapting to the human host. These various selection pressures can be used to predict the part of the virus evolution that is adaptive, including antigenic drift, but not the random, neutral drift. We envision that the many experimental data on mutation effects on ACE2 binding and antibody escape and thermodynamic stability estimates of proteins (experimental and computational) will be used together with features of the mutation in its structural context to train computer models that can rationally predict fitness effects of new arising mutations in the S-protein. The analysis above suggests that mutations in surface-exposed sites with larger chemical effects, typically volume or charge-changing, will drive the adaptive evolution, although precise predictive models need to be semiquantitative. The heroic efforts in elucidating S-protein structures for all of the main conformation states bound to diverse antibodies and ACE2 will provide an unprecedented basis for analyzing the functional evolution of SARS-CoV-2 in its appropriate structural context. 

The Origins and Potential Future of SARS-CoV-2 Variants of Concern in the Evolving COVID-19 Pandemic

COVID-19, the First Pandemic in the Post-Genomic Era

SARS-CoV-2 Variants, Spike Mutations and Immune Escape

Human SARS CoV-2 Spike Protein Mutations

Functional Assessment of Cell Entry and Receptor Usage for SARS-CoV-2 and Other Lineage B Betacoronaviruses

An Overview of Their Replication and Pathogenesis

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases

COVID-19 Vaccines: Where We Stand and Challenges Ahead

SARS-CoV-2 3D Database: Understanding the Coronavirus Proteome and Evaluating Possible Drug Targets

The Resolution Revolution in X-Ray Diffraction, Cryo-EM and Other Technologies

Cryo-Electron Microscopy for Structural Analysis of Dynamic Biological Macromolecules

Unravelling Biological Macromolecules with Cryo-Electron Microscopy

Cryo-Electron Microscopy Methodology: Current Aspects and Future Directions

Universal Distribution of Protein Evolution Rates as a Consequence of Protein Folding Physics

Positively Selected Sites in Cetacean Myoglobins Contribute to Protein Stability

The Interface of Protein Structure

Evolution and the Tertiary Structure of Proteins

A Biophysical Protein Folding Model Accounts for Most Mutational Fitness Effects in Viruses

Mistranslation-Induced Protein Misfolding as a Dominant Constraint on Coding-Sequence Evolution

Structural Determinants of the Rate of Protein Evolution in Yeast

The Relationship between Relative Solvent Accessibility and Evolutionary Rate in Protein Evolution

The Evolution and Evolutionary Consequences of Marginal Thermostability in

Survival of the Cheapest: How Proteome Cost Minimization Drives Evolution

Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins

The SARS-CoV-2 Spike Protein: Balancing Stability and Infectivity

Pre-Fusion Structure of a Human Coronavirus Spike Protein

Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen

Cryo-EM Structures of MERS-CoV and SARS-CoV Spike Glycoproteins Reveal the Dynamic Receptor Binding Domains

Covid-19: The E484K Mutation and the Risks It Poses

Resistance of SARS-CoV-2 Variants to Neutralization by Monoclonal and Serum-Derived Polyclonal Antibodies

Mutation Signatures and In Silico Docking of Novel SARS-CoV-2 Variants of

Distinct Conformational States of SARS-CoV-2 Spike Protein

Cryo-EM Structure of the 2019-NCoV Spike in the Prefusion Conformation

Structure-Based Design of Prefusion-Stabilized SARS-CoV-2 Spikes

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Cryogenic Electron Microscopy and Single-Particle Analysis

Origin and Temperature Dependence of Radiation Damage in Biological Samples at Cryogenic Temperatures

Arrangement of Electron Transport Chain Components in Bovine Mitochondrial Supercomplex I 1 III 2 IV 1

A Saposin-Lipoprotein Nanoparticle System for Membrane Proteins

CryoEM Reconstructions of Membrane Proteins Solved in Several Amphipathic Solvents, Nanodisc, Amphipol and Detergents, Yield Amphipathic Belts of Similar Sizes Corresponding to a Common Ordered Solvent Layer

Single-Particle Cryo-EM-How Did It Get Here and Where Will It Go

Cryo-Temperature Effects on Membrane Protein Structure and Dynamics

Cryogenic Temperature Effects and Resolution upon Slow Cooling of Protein Preparations in Solid State NMR

Effects of Temperature on Protein Structure and Dynamics: X-Ray Crystallographic Studies of the Protein Ribonuclease-A at Nine Different Temperatures from 98 to 320K

Thermal Properties of Water in Myoglobin Crystals and Solutions at Subzero Temperatures

Pros and Cons of Cryocrystallography: Should We Also Collect a Room-Temperature Data Set?

Cold Sensitivity of the SARS-CoV-2 Spike Ectodomain

Protein Crystallography for Non-Crystallographers, or How to Get the Best (but Not More) from Published Macromolecular Structures

PROCHECK: A Program to Check the Stereochemical Quality of Protein Structures

Structural Summaries of PDB Entries

Structural Basis for the Different States of the Spike Protein of SARS-CoV-2 in Complex with ACE2

A Trimeric Human Angiotensin-Converting Enzyme 2 as an Anti-SARS-CoV-2 Agent

Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction

Cryo-EM Structures Delineate a PH-Dependent Switch That Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains

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

Continuous Flexibility Analysis of SARS-CoV-2 Spike Prefusion Structures

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

SARS-CoV-2 Structure and Replication Characterized by in Situ Cryo-Electron Tomography

Conformational Transition of SARS-CoV-2 Spike Glycoprotein between Its Closed and Open States

Situ Structural Analysis of SARS-CoV-2 Spike Reveals Flexibility Mediated by Three Hinges

Correlative Multi-Scale Cryo-Imaging Unveils SARS-CoV-2 Assembly and Egress

FreeSASA: An Open Source C Library for Solvent Accessible Surface Area Calculations

Structure-Based Design with Tag-Based Purification and in-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Stabilizing the Closed SARS-CoV-2 Spike Trimer

SARS-CoV-2 and Bat RaTG13 Spike Glycoprotein Structures Inform on Virus Evolution and Furin-Cleavage Effects

SARS-CoV-2 B. 1.1.7 and B. 1.351 Spike Variants Bind Human ACE2 with Increased Affinity

Conformational Dynamics of SARS-CoV-2 Trimeric Spike Glycoprotein in Complex with Receptor ACE2 Revealed by

Networks of SARS-CoV-2 Transmission

A Mathematical Model Reveals the Influence of Population Heterogeneity on Herd Immunity to SARS-CoV-2

Nasal Gene Expression of Angiotensin-Converting Enzyme 2 in Children and Adults

SARS-CoV-2 Receptor ACE2 Protein Expression in Serum Is Significantly Associated with Age

Individual Variation of the SARS-CoV-2 Receptor ACE2

Complete Map of SARS-CoV-2 RBD Mutations That Escape the Monoclonal Antibody LY-CoV555 and Its Cocktail with LY-CoV016

Ultrapotent Human Antibodies Protect against SARS-CoV-2 Challenge via Multiple Mechanisms

A Systematic Review of Antibody Mediated Immunity to Coronaviruses: Kinetics, Correlates of Protection, and Association with Severity

Structural and Functional Ramifications of Antigenic Drift in Recent SARS-CoV-2 Variants

N-Terminal Domain Antigenic Mapping Reveals a Site of Vulnerability for SARS-CoV-2

Antibody Cocktail to SARS-CoV-2 Spike Protein Prevents Rapid Mutational Escape Seen with Individual Antibodies

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain That Escape Antibody Recognition

Prospective Mapping of Viral Mutations That Escape Antibodies Used to Treat COVID-19

Rational Development of a Human Antibody Cocktail That Deploys Multiple Functions to Confer Pan-SARS-CoVs Protection

Structural Basis for Neutralization of SARS-CoV-2 and SARS-CoV by a Potent Therapeutic Antibody

De Novo Design of Picomolar SARS-CoV-2 miniprotein Inhibitors

Cross-Neutralizing Antibodies Bind a SARS-CoV-2 Cryptic Site and Resist Circulating Variants

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Identification of SARS-CoV-2 Spike Mutations That Attenuate Monoclonal and Serum Antibody Neutralization

Origin and Evolution of Pathogenic Coronaviruses

Genetic Recombination, and Pathogenesis of Coronaviruses

SARS-CoV-2, SARS-CoV, and MERS-CoV Viral Load Dynamics, Duration of Viral Shedding, and Infectiousness: A Systematic Review and Meta-Analysis

An Overview of Spike Surface Glycoprotein in Severe Acute Respiratory Syndrome

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting

Van der Hoek, L. Mosaic Structure of Human Coronavirus NL63, One Thousand Years of Evolution

On the Origin and Continuing Evolution of SARS-CoV-2

The Great Escape? SARS-CoV-2 Variants Evading Neutralizing Responses

A Model of Proteostatic Energy Cost and Its Use in Analysis of Proteome Trends and Sequence Evolution

The Influence of Selection for Protein Stability on DN/DS Estimations

Tracking Changes in SARS-CoV-2 Spike: Evidence That D614G Increases Infectivity of the COVID-19 Virus

SARS-CoV-2 D614G Variant Exhibits Efficient Replication Ex Vivo and Transmission in Vivo

Estimated Transmissibility and Impact of SARS-CoV-2 Lineage B. 1.1.7 in England

Emergence and Rapid Spread of a New Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Lineage with Multiple Spike Mutations in South Africa

SARS-CoV-2 Lineage in Manaus

SARS-CoV-2 B.1.617.2 Delta Variant Replication and Immune Evasion

Antibody Resistance of SARS-CoV-2 Variants B. 1.351 and B. 1.1.7

SARS-CoV-2 Variants B. 1.351 and P. 1 Escape from Neutralizing Antibodies

Effectiveness of COVID-19 Vaccines against the B. 1.617. 2 (Delta) Variant

Neutralising Antibody Activity against SARS-CoV-2 VOCs B. 1.617. 2 and B. 1.351 by BNT162b2 Vaccination

The Effect of the D614G Substitution on the Structure of the Spike Glycoprotein of SARS-CoV-2

Effect of Natural Mutations of SARS-CoV-2 on Spike Structure, Conformation and Antigenicity

Structural Basis for Enhanced Infectivity and Immune Evasion of SARS-CoV-2 Variants

Structure, Function, and Evolution of Coronavirus Spike Proteins

Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop

The Spike Glycoprotein of the New Coronavirus 2019-NCoV Contains a Furin-like Cleavage Site Absent in CoV of the Same Clade

Predicted Structural Mimicry of Spike Receptor-Binding Motifs from Highly Pathogenic Human Coronaviruses

Structural Basis for Human Coronavirus Attachment to Sialic Acid Receptors

Protein Stability and Surface Electrostatics: A Charged Relationship

Improved Interaction Potentials for Charged Residues in Proteins

Electrostatic Interactions in Macromolecules: Theory and Applications

Protein Stability: Electrostatics and Compact Denatured States

Electrostatic Contributions to Protein Stability and Folding Energy

Dipeptidyl Peptidase 4 Is a Functional Receptor for the Emerging Human Coronavirus-EMC

Angiotensin-Converting Enzyme 2 Is a Functional Receptor for the SARS Coronavirus

Interaction of the Spike Protein RBD from SARS-CoV-2 with ACE2: Similarity with SARS-CoV, Hot-Spot Analysis and Effect of the Receptor Polymorphism

Studies in Humanized Mice and Convalescent Humans Yield a SARS-CoV

Escape from Neutralizing Antibodies by SARS-CoV-2 Spike Protein Variants

Amino Acid Difference Formula to Help Explain Protein Evolution

Solving the Protein Sequence Metric Problem

Three Simple Properties Explain Protein Stability Change upon Mutation

Functional Specificity Lies within the Properties and Evolutionary Changes of Amino Acids

Effectiveness of Correlation Analysis in Identifying Protein Residues Undergoing Correlated Evolution

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

SARS-CoV-2 Spike E484K Mutation Reduces Antibody Neutralisation

Protein Stability Imposes Limits on Organism Complexity and Speed of Molecular Evolution

Superoxide Dismutase 1 Is Positively Selected to Minimize Protein Aggregation in Great Apes

Relation Between Protein Stability, Evolution and Structure, as Probed by Carboxylic Acid Mutations

Structural and Physico-Chemical Effects of Disease and Non-Disease NsSNPs on Proteins

On Human Disease-Causing Amino Acid Variants: Statistical Study of Sequence and Structural Patterns

Loss of Protein Structure Stability as a Major Causative Factor in Monogenic Disease

Prediction of the Stability of Protein Mutants Based on Structural Environment-Dependent Amino Acid Substitution and Propensity Tables

Computational Approaches for Predicting Mutant Protein Stability

On the Biases in Predictions of Protein Stability Changes upon Variations: The INPS Test Case

Quantification of Biases in Predictions of Protein Stability Changes upon Mutations

Assessing Computational Methods for Predicting Protein Stability upon Mutation: Good on Average but Not in the Details

SDM-a Server for Predicting Effects of Mutations on Protein Stability and Malfunction

Predicting the Effects of Mutations in Proteins Using Graph-Based Signatures

Fast and Accurate Predictions of Protein Stability Changes upon Mutations Using Statistical Potentials and Neural Networks: PoPMuSiC-2.0

0: Predicting Stability Changes upon Mutation from the Protein Sequence or Structure

Systematic Investigation of the Data Set Dependency of Protein Stability Predictors

A Base Measure of Precision for Protein Stability Predictors: Structural Sensitivity

Performance of Protein Stability Predictors

Protein Variant Stability Predictor. Importance of Training Data Quality

Contact Density Affects Protein Evolutionary Rate from Bacteria to Animals

Causes of Evolutionary Rate Variation among Protein Sites

Quantitative Determination of Mechanical Stability in the Novel Coronavirus Spike Protein

The Importance of Additive and Non-additive Mutational Effects in Protein Engineering

Epistasisthe Essential Role of Gene Interactions in the Structure and Evolution of Genetic Systems

Epistasis as the Primary Factor in Molecular Evolution

Mutation Effects Predicted from Sequence Co-Variation

Stability-Mediated Epistasis Constrains the Evolution of an Influenza Protein

Towards a "Golden Standard" for Computing Globin Stability: Stability and Structure Sensitivity of Myoglobin Mutants

HIV-1 Evades Antibody-Mediated Neutralization through Conformational Masking of Receptor-Binding Sites

After the Pandemic: Perspectives on the Future Trajectory of COVID-19

Endemic SARS-CoV-2 Will Maintain Post-Pandemic Immunity

Immune Life History, Vaccination, and the Dynamics of SARS-CoV-2 over the next 5 Years

Universal Influenza Vaccines: Shifting to Better Vaccines

Influenza VaccineOutmaneuvering Antigenic Shift and Drift

Influenza Vaccine: The Challenge of Antigenic Drift

Vaccination and Antigenic Drift in Influenza

Cross-Neutralization of SARS-CoV-2 by a Human Monoclonal SARS-CoV Antibody

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients; B Cells

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

A Neutralizing Human Antibody Binds to the N-Terminal Domain of the Spike Protein of SARS-CoV-2

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Structural Basis for the Neutralization of SARS-CoV-2 by an Antibody from a Convalescent Patient

Potent Neutralizing Antibodies against Multiple Epitopes on SARS-CoV-2 Spike

Structure-Guided Covalent Stabilization of Coronavirus Spike Glycoprotein Trimers in the Closed Conformation

A Carbohydrate-Binding Protein from the Edible Lablab Beans Effectively Blocks the Infections of Influenza Viruses and SARS-CoV-2

Structural Analysis of Full-Length SARS-CoV-2 Spike Protein from an Advanced Vaccine Candidate

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy

Receptor Binding and Priming of the Spike Protein of SARS-CoV-2 for Membrane Fusion

An Alpaca Nanobody Neutralizes SARS-CoV-2 by Blocking Receptor Interaction

Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein. Science

Bi-Paratopic and Multivalent VH Domains Block ACE2 Binding and Neutralize SARS-CoV-2

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

SARS-CoV-2 Neutralizing Antibody Structures Inform Therapeutic Strategies

Biophysical and Structural Analysis of Synthetic Nanobodies That Effectively Neutralize SARS-CoV-2

Engineered Trimeric ACE2 Binds Viral Spike Protein and Locks It in "Three-up" Conformation to Potently Inhibit SARS-CoV-2 Infection

Development and Structural Basis of a Two-MAb Cocktail for Treating SARS-CoV-2 Infections

Structure-Based Development of Human Antibody Cocktails against SARS-CoV-2

Dimerized Glycan-Reactive Antibodies Are a Structural Category of Natural Antibodies

Structure-Guided Multivalent Nanobodies Block SARS-CoV-2 Infection and Suppress Mutational Escape

The Functions of SARS-CoV-2 Neutralizing and Infection-Enhancing Antibodies in Vitro and in Mice and Nonhuman Primates. bioRxiv 2021

The Neutralizing Antibody, LY-CoV555, Protects against SARS-CoV-2 Infection in Nonhuman Primates

Paired Heavy and Light Chain Signatures Contribute to Potent SARS-CoV-2 Neutralization in

Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

Sahin, U. BNT162b Vaccines Protect Rhesus Macaques from SARS-CoV-2

Structural Basis for Accommodation of Emerging B. 1.351 and B. 1.1. 7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies

Modular Basis for Potent SARS-CoV-2 Neutralization by a Prevalent VH1−2-Derived Antibody Class

A Potently Neutralizing Anti-SARS-CoV-2 Antibody Inhibits Variants of Concern by Binding a Highly Conserved Epitope

Structural Insights into the Cross-Neutralization of SARS-CoV and SARS-CoV-2 by the Human Monoclonal Antibody 47D11

Structural Insight into SARS-CoV-2 Neutralizing Antibodies and Modulation of Syncytia

Structures of Synthetic Nanobody-SARS-CoV-2 Receptor-Binding Domain Complexes Reveal Distinct Sites of Interaction

An Infectivity-Enhancing Site on the SARS-CoV-2 Spike Protein Targeted by Antibodies

Humoral Immune Response to Circulating SARS-CoV-2 Variants Elicited by Inactivated and RBD-Subunit Vaccines

Nanobodies from Camelid Mice and Llamas Neutralize SARS-CoV-2 Variants

Increased Resistance of SARS-CoV-2 Variant P. 1 to Antibody Neutralization

Cryo-Electron Microscopy Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and 2 Potent Neutralizing Antibodies

Glycan Shield and Epitope Masking of a Coronavirus Spike Protein Observed by Cryo-Electron Microscopy

Structures of MERS-CoV Spike Glycoprotein in Complex with Sialoside Attachment Receptors

Cryo-Electron Microscopy Structures of the SARS-CoV Spike Glycoprotein Reveal a Prerequisite Conformational State for Receptor Binding

Stabilized Coronavirus Spikes Are Resistant to Conformational Changes Induced by Receptor Recognition or

Cryo-EM Structure of the SARS Coronavirus Spike Glycoprotein in Complex with Its Host Cell Receptor ACE2

The Human Coronavirus HCoV-229E S-Protein Structure and Receptor Binding

Cryo-EM Structures of HKU2 and SADS-CoV Spike Glycoproteins Provide Insights into Coronavirus Evolution

A Potent SARS-CoV-2 Neutralising Nanobody Shows Therapeutic Efficacy in the Syrian Golden Hamster Model of COVID-19

A SARS-CoV-2 Neutralizing Antibody with Extensive Spike Binding Coverage and Modified for Optimal Therapeutic Outcomes

SARS-CoV-2 Evolution in an Immunocompromised Host Reveals Shared Neutralization Escape Mechanisms

A Noncompeting Pair of Human Neutralizing Antibodies Block COVID-19 Virus Binding to Its Receptor ACE2

A High-Affinity RBD-Targeting Nanobody Improves Fusion Partner's Potency against SARS-CoV-2

Landscape of Human Antibody Recognition of the SARS-CoV-2 Receptor Binding Domain

Cryo-Electron Microscopy Structure of the Swine Acute Diarrhea Syndrome Coronavirus Spike Glycoprotein Provides Insights into Evolution of Unique Coronavirus Spike Proteins

Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State

Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections

Structure and Immune Recognition of the Porcine Epidemic Diarrhea Virus Spike Protein

The 3.1-Angstrom Cryo-Electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation

Cryo-EM Structure of Infectious Bronchitis Coronavirus Spike Protein Reveals Structural and Functional Evolution of Coronavirus Spike Proteins

Bat and Pangolin Coronavirus Spike Glycoprotein Structures Provide Insights into SARS-CoV-2 Evolution

Structure of Mouse Coronavirus Spike Protein Complexed with Receptor Reveals Mechanism for Viral Entry

Cryo-Electron Microscopy Structure of a Coronavirus Spike Glycoprotein Trimer

Cryo-EM Analysis of a Feline Coronavirus Spike Protein Reveals a Unique Structure and Camouflaging Glycans

Structure and Binding Properties of Pangolin-CoV Spike Glycoprotein Inform the Evolution of SARS-CoV-2

South Africa

Evidence of Escape of SARS-CoV-2 Variant B. 1.351 from Natural and Vaccine-Induced Sera

SARS-CoV-2 B.1.617.2 Delta Variant Emergence

Early Epidemiological Signatures of Novel SARS-CoV-2 Variants: Establishment of B.1.617.2 in England

Reduced Sensitivity of SARS-CoV-2 Variant Delta to Antibody Neutralization

Transmission, Infectivity, and Neutralization of a Spike L452R SARS-CoV-2 Variant

1.429 and B.1.351

SARS-CoV-2 B.1.617 Mutations L452R and E484Q

Are Not Synergistic for Antibody Evasion

Impact of SARS-CoV-2 Variant-Associated RBD Mutations on the Susceptibility to Serum Antibodies Elicited by COVID-19 Infection or Vaccination

ACS Infectious Diseases pubs.acs.org/journal/aidcbc Review