key: cord-0825047-e7wjkjut
authors: Ahmad, Javeed; Jiang, Jiansheng; Boyd, Lisa F.; Zeher, Allison; Huang, Rick; Xia, Di; Natarajan, Kannan; Margulies, David H.
title: Structures of synthetic nanobody–SARS-CoV-2–receptor binding domain complexes reveal distinct sites of interaction
date: 2021-09-16
journal: J Biol Chem
DOI: 10.1016/j.jbc.2021.101202
sha: 8275ef041dd2d94eb7c4247d374b859a3f98da1e
doc_id: 825047
cord_uid: e7wjkjut

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin converting enzyme 2 (ACE2) interface, and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity determining region 2 (CDR2). In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBC/ACE2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike (S) protein. Superposition of the X-ray structures of sybodies onto the trimeric S protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

(residues [27] [28] [29] [30] [31] [32] [33] [34] [35] lie between the CDR2 and CDR3 loops. Superposition of the two structures, based on the RBD, emphasizes the diametrically opposite orientation of the two (Fig. 2, C) , revealing that the CDR2 of Sb16 and CDR3 of Sb45 recognize the same epitopic regions.

Exploring conditions using mixtures of two or three sybodies and the RBD, we obtained crystals and solved the structures of ternary complexes consisting of Sb45-RBD-Sb68 at 2.6 Å resolution (Table 1 and Fig. 2, D) and Sb14-RBD-Sb68 at 1.7 Å resolution (Fig. 2, E) . The refined models revealed that while Sb14 and Sb45 interact with the ACE2 interface of the RBD, Sb68 binds the RBD at a distinct site ( Fig. 2 D and E) . In the ternary complex, Sb45 binds in an identical orientation to that observed in the binary Sb45-RBD structure (RMSD of superposition, 0.491 Å for 1981 atoms), but Sb68 addresses a completely different face of the RBDsimilar to that bound by Fab of CR3022 on RBD of SARS-CoV-2 (25) and by VHH72 on RBD of SARS-CoV-1 (26) .

Of particular interest, whereas Sb45 CDR2 and CDR3 span the RBD saddle as noted above, the distinct contacts of Sb68 to the RBD are through the longer CDR3, with only minor contributions from CDR1 and CDR2. Walter et al visualized similar distinct interactions in cryo-EM maps of two sybodies (Sb15 and Sb68) bound to S protein with local resolution of 6-7 Å (18) . Similarly, Sb14, which interacts via distinct sybody residues with the RBD at the ACE2 site (see description below), still permits Sb68 to bind to its epitope as seen in the Sb45-RBD-Sb68 structure (Fig. 2, 

Scrutiny of the different interfaces provides insights into the distinct ways each sybody exploits its unique CDR residues for interaction with epitopic residues of the RBD (Fig. 3 ).

(Compilation of the contacting residues for each of the four sybodies to the RBD is provided in Table S1 ). Both Sb16 and Sb45 use longer CDR2 and CDR3 to straddle the RBD, positioning CDR1 residues over the central crest of the saddle (Fig. 2, A-C; Fig. 3 , A and B, and Table S1 ).

J o u r n a l P r e -p r o o f 6 Also, several non-CDR residues (Y37, E44, and W47 for Sb16), derived from framework 2 (27) , provide additional contacts to the RBD (see Table S1 ). By contrast with Sb16 and Sb45, Sb14, despite interacting with a large surface area of the RBD, uses both CDR2 and CDR3 on the same side and exploits many non-CDR residues, particularly sheets of -strand as its binding surface ( Fig. 3C and Table S1 ). The interface of Sb68 with RBD ( Fig. 3D) is quite different, predominantly exploiting nine CDR3, four CDR2, and one CDR1 residues at the interface (see Table S1 ).

Sybodies block ACE2-RBD interaction in discrete ways. To evaluate the structural basis for the ability of these four sybodies to block the interaction of RBD with ACE2, we superposed each of three sybody-RBD structures onto the ACE2-RBD structure and examined the steric clashes ( Fig.   4A ). Sb16 and Sb45 directly impinge on the ACE2 binding site, offering a structural rationale for their viral neutralization capacity (18) . Sb68, which also blocks viral infectivity, binds to RBD at a site which appears to be noncompetitive for ACE2 binding. The carbohydrate at ACE2 residues N322 and N546 provides an explanation (Fig. 4A) .

To compare the epitopic areas captured by these sybodies, we evaluated the buried surface area (BSA) interfaces between RBD and ACE2 or the sybodies. The BSA at the ACE2-RBD, Sb14-RBD, Sb16-RBD, Sb45-RBD, and Sb68-RBD interfaces are 844 Å 2 , 1,040 Å 2 , 1,003 Å 2 , 976 Å 2 , and 640 Å 2 , respectively (Fig. 3 , A-E). Sb16 and Sb45 capture more surface area than ACE2 or other published nanobody or sybody-RBD complexes (see Table S2 ). The interface with Sb68 is the smallest (640 Å 2 ) (Fig. 3D ). The total BSA captured by Sb45 and Sb68 in the ternary complex is 1,650 (1,010 plus 640) Å 2 (Table S2 ) and is consistent with the view that a linked bispecific sybody, as described by Walter et al (18) , would exert strong avidity effects. Table S2 summarizes these BSA values and those of other nanobody-RBD interactions. Sb68 reveals the smallest BSA with the RBD and binds at a distinct site, it still   blocks ACE2 binding. A reasonable explanation for the ability of Sb68 to block the ACE2-RBD   interaction arises on inspection of the sites where Sb68, bound to the RBD, might clash with ACE2. Scrutiny of a superposition of Sb68-RBD with ACE2-RBD reveals several areas of steric interference. Sb68 loop 40-44 clashes with amino acid side chains of ACE2 (residues 318-320 and 548-552), loop 61-64 with ACE2 N322 carbohydrate, and loop 87-89 (a 3,10 helix) with ACE2 N546 carbohydrate as well as residues 313 and 316-218 (Fig. 4A) . The ACE2 used in the crystallographic visualization of ACE2-RBD (28) was expressed in Trichoplusia ni insect cells, which produce biantennary N-glycans terminating with N-acetylglucosamine residues (29, 30) .

Electron density was observed only for the proximal N-glycans at residues N322 and N546, but larger, complex, non-sialylated, biantennary carbohydrates have been detected in glycoproteomic analysis of ACE2 in mammalian cells (31) . These carbohydrates are highly flexible, adding greater than 1500 Da at each position, and are larger than the single carbohydrate residues visualized in the crystal structure. Additionally, molecular dynamics simulations of ACE2-RBD implicated the direct interaction of carbohydrate with the RBD (32) . Thus, the ability of Sb68 to impinge on ACE2 interaction with RBD likely involves the steric clash of the N322-and N546-linked glycans.

We also obtained a 1.9 Å structure of free Sb16 (Fig. S3) . Remarkably, the CDR2 of Sb16 shows Y54 in starkly different positions in the unliganded structure as compared to the complex: the Cα carbon is displaced by 6.0 Å, while the Oη oxygen of Y54 is 15.2 Å distant, indicative of dynamic flexibility.

To gain further insight into the interaction of Sb45 with the full S protein, we prepared complexes of Sb45 with HexaPro S (S-6P), a stable spike variant containing six beneficial proline substitutions (33) , and acquired cryo-EM images as described in Experimental procedures. All image processing, 2D class, 3D J o u r n a l P r e -p r o o f 8 reconstruction, and map refinements were performed with cryoSPARC (34) (35) (36) (37) , model fitting with Chimera (38) and refinement with PHENIX (39) . We identified two conformations of S-6P with RBD in either a 1-up, 2-down (7N0G/EMD-24105) or 2-up, 1-down (7N0H/EMD-24106) position as determined by 3D classification (3D Ab-initio reconstruction) (Fig. S4) . We have built in additional loops of the NTD and glycans based on the models of 6XKL, 7KGJ, and 7B62. We used unsharpened maps for the model refinement. The overall correlation coefficients (CC) (mask/volume/peaks) of models for 7N0G and 7N0H are 0.84/0.84/0.77 and 0.83/0.83/0.77 respectively. The model quality is shown in Table 2 Superposition of sybodies on trimeric spike protein models. To gain additional insight into the structural consequences of the interactions of each of these sybodies with a trimeric S protein, we superposed each of the individual sybody-RBD complexes on S-6P of our cryo-EM structures (7N0G and 7N0H) (see Fig. S6 ). Sb16 and Sb45 may dock on all three RBDs in the trimeric S in any of the four configurations, without any apparent clash (Fig. S6, A and B) . Sb14, however, reveals clashes when the Sb14-RBD complex is superposed on trimeric S in any down position J o u r n a l P r e -p r o o f 9 (Fig. S6E ). Sb68 could not be superposed without clashes to any RBD of the 3-down or to the 1up, 2-down position. The only permissible superpositions were to two in the 2-up, 1-down; and to all three in the 3-up position (Fig. S6F ). For paired sybodies, Sb16 and Sb68 (Fig. S6B) , or Sb45 and Sb68 (Fig. S6D) , superposition was possible without clashes, with two or more RBDs in the up conformation. We also observed direct interactions of Sb14 and Sb68 in the Sb14-RBD-Sb68 X-ray crystal structure. Walter et al (18) suggested that a covalent bispecific Sb15-Sb68 reagent could bind S in both the 2-up and 3-up configurations, based on cryo-EM maps of complexes of S with Sb15 and Sb68. It appears that Sb16 binds to S in an orientation similar to but in detail distinct from that of Sb15. This analysis demonstrates an advantage of the small size of sybodies or nanobodies in accessing epitopic regions of S (see Fig. S7C ).

Binding to RBD mutants. The major circulating variants, specifically B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil), contain mutations in the RBD that lead to increased binding affinity to ACE2 and have the potential to reduce vaccine efficacy (4, 14, (40) (41) (42) . Specifically, in addition to other mutations throughout the S protein and viral genome, all three harbor N501Y.

B.1.351 and P.1 also have the E484K substitution, as well as substitution of K417 (to N for B.1.351 and to T for P.1). To assess the effect that substitution at each of these positions exerts on reactivity with Sb14, Sb15, Sb16, Sb45, and Sb68, we engineered individual mutations in the RBD and tested them by SPR (see Fig. 6A ). In general, the five sybodies which interact with the parental (designated wild type (WT)) RBD with KD values of 6.8 x 10 -9 (for Sb15) to 6.3 x 10 -8 M (for Sb68) (see Fig. 1 ), showed different patterns of binding to the K417N, E484K, and N501Y mutants. Sb68 bound each with similar affinity, consistent with its epitope lying outside of the ACE2 binding site on RBD, while each of the others revealed a distinct pattern. Sb14 binding was most affected by K417N. Sb15 bound both K417N and E484K less efficiently than N501Y. Sb16, J o u r n a l P r e -p r o o f 10 largely unaffected in binding to K417N showed decreased recognition of N501Y and failed to interact detectably with E484K. Similar to Sb16, Sb45 also failed to bind E484K and showed decreased recognition of K417N and N501Y as compared to WT. To understand the structural basis of these differences in recognition of the different RBD mutants, we generated models based on the sybody-RBD structures (Fig. 6 , B-E). For Sb16, Sb45, and Sb14, interaction with the N501Y mutant resulted in displacement of its 496-506 loop by 2.0 Å, 1.0 Å, and 1.5 Å respectively.

Nevertheless, R60 of Sb16 and H103 of Sb45 maintained contact with N501Y. This suggests that N501Y mutation would not escape recognition by these sybodies. Other cryo-EM studies indicate modest effects of the N501Y substitution on binding to different antibodies (43) . In contrast to the effects of N501Y, E484K revealed major incompatibilities due to charge repulsion, in the interaction with Sb16 via K32 and of Sb45 via R33 (Fig. 6 , D and E).

Our studies of the X-ray structures of Sb16 alone, Sb16-RBD, Sb45-RBD, the ternary Sb14-RBD-Sb68 and Sb45-RBD-Sb68 complexes, and the cryo-EM structures of Sb45-S provide critical detail describing the basis of the inhibition of S binding to the cell surface ACE2 receptor and the resulting block of viral infectivity. Sybodies and nanobodies, by virtue of their single domain structure and ability to be expressed in E. coli systems, as noted by others (17, 19) , offer advantages over Fab. Our X-ray structures (at resolutions 1.7 to 2.6 Å) are complemented by the recent preliminary report of cryo-EM-based models of Sb15 and Sb68 bound to S (18) . Although competes presumably due to its spatial orientation. Overall, our structural studies not only define the Sb14, Sb16, Sb45, and Sb68 epitopes at high resolution, but also reveal that these sybodies capture a rather large epitopic area (Table S2 ), suggesting that a judicious choice of several sybodies or nanobodies has the potential to effectively saturate the available RBD surface. Based on the design of the sybody libraries, Walter et al (18) considered Sb14 and Sb16 as "concave," Sb45 as "loop," and Sb68 as "convex." The X-ray structures indicate that though Sb14 and Sb16 are of the same group, Sb14 interacts with the RBD primarily through nonCDR residues (Fig. 3C) while Sb16 binds through CDR1, 2 and 3 (Fig. 3A) . Sb45, which is oriented differently at the ACE2-RBD interface, exploits all three CDRs as well as nonCDR residues (Fig. 3B) . Sb68, on the other hand, exploits its long CDR3 to bind at its distinct site. Thus, it is possible that convex sybodies may offer an opportunity for identifying distinct epitopes.

J o u r n a l P r e -p r o o f 12 The significance of the ternary structures of Sb45-RBD-Sb68 (7KLW) and Sb14-RBD-Sb68 (7MFU) is shown in a recent paper (45) . Koenig et al (45) determined a ternary nanobody structure of VHH-E-RBD-VHH-U (7KN5) which illustrates the binding to two distinct epitopic sites. The ternary structure may also be considered as illustrative of the potential behavior of a bispecific construct linking two nanobodies. The bivalent or multivalent binding by antibody or nanobody would be expected to increase neutralization potential (19, (45) (46) (47) . Superposition of Sb14-RBD-Sb68 or Sb45-RBD-Sb68 on VHH-E-RBD-VHH-U indicates that Sb14, Sb45, and VHH-E represent class 1 and class 2 in recognizing the epitopic region but do so in somewhat different orientations (Fig. S7B ). Sb45 exploits its two lengthy CDR2 and CDR3 loops which ride along both sides of the RBD surface, and Sb14 uses both CDR2 and CDR3 on the same side close to Sb68, while VHH-E uses a long CDR3 loop engaging one side of the RBD surface. Furthermore, Sb14 and Sb68 in Sb14-RBD-Sb68 (7MFU) show contacts (Y57-E44, G55-E44, and T54-H108) between two specific sybodies on the RBD surface (Fig. S7C) , which emphases the potential benefit of using complementary, bivalent, or multivalent antibodies/nanobodies against the virus.

Recently, several SARS-CoV-2 spike variants have been isolated and characterized with respect to their infectivity and severity of disease. The UK-SARS-CoV-2 variant has multiple substitutions including N501Y in the RBD (1). The mutation of E484K leads to repulsion of charged residues of antibody/nanobody/sybodies (Fig. 6) . To accommodate such a mutation, the complementary charged residues of the antibody/nanobody/sybody should also reverse their charge. Alternatively, employing another antibody/nanobody/sybody with opposite charge could capture such an escape mutation. Indeed, knowledge of the location of common or recurrent escape mutations and their potential resistance to antibody/nanobody/sybodies would provide a rational basis for either sequential or simultaneous use of reagents with complementary specificity. Thus, J o u r n a l P r e -p r o o f 13 precise mapping of anti-RBD antibody, nanobody, and sybody epitopes, especially for those that are developed for clinical trials, has implications not only for mechanistic understanding of the interactions of the RBD with ACE2, but also for evaluating the potential susceptibility of newly arising viral variants to currently administered vaccines and antibodies. 

Purified sybodies (Sb14, Sb15, Sb16, Sb45 and Sb68) and RBD were mixed in approximate 1:1 molar ratio to a final concentration of 8 mg/ml. The protein mixtures were incubated on ice for 1 hour prior to screening. Initial screening for crystals was carried out using the hanging drop vapor diffusion method using the Mosquito robotic system (sptlabtech.com). Crystals of SB16-RBD and SB45-RBD complexes and Sb16 alone were observed within one week using Protein Complex Table 1 .

Sb45 in a 1:3 molar ratio and repurified by size exclusion chromatography. Negative stain screening was accomplished with a Tecnei T12 120-keV microscope (Thermo Fisher). We screened several sybody-S complexes for good negative staining images, and complexes of Sb45-S gave the best data. The protein complexes were concentrated to 0.7-1 mg/ml and 3 l of the sample was applied onto holey-carbon cryo-EM grids (Cu R1.2/1.3, 300 mesh, Quantifoil), which had been glow discharged for 60 seconds, blotted for 3 seconds, and plunge frozen into liquid ethane with a Vitrobot (Thermo Fisher Scientific) at 4 °C and 95% humidity. Cryo-EM data in selected grid regions were collected on a Titan Krios 300-keV microscope (Thermo Fisher).

Images were acquired automatically with SerialEM (57) Image processing and structure solution. All image processing, 2D class, 3D reconstruction, and map refinements were performed with cryoSPARC v3.1 and v3.2 (34) (35) (36) (37) . A total of 9,725 micrographs was imported into cryoSPARC. Following "patch motion correction" and "patch CTF estimation," the number of micrographs was reduced to 9,703. Micrographs were inspected by "curate exposures," in which outliers of defocus range, defective micrographs, and those with a low-resolution estimation of the CTF fit (>5 Å) were discarded, resulting in 9,237 micrographs.

"Blob picker" was used with the particle diameter between 128 and 256 angstroms for picking J o u r n a l P r e -p r o o f 19 particles. After "inspect particles" with NCC (Normalized Correlation Coefficient) 0.28 and "power threshold" between 500 and 1000 (which removed ice and aggregates), the number of particles was 1,876,941. To determine the "box size," we performed several trials indicating that the box size should be larger than 336 pixels, and finally used a box size of 400 pixels and extracted 1,433,963 particles. After "2D classification" (100 classes), 18 2D classes were selected, retaining 662,994 particles. The particles were submitted to a series of "Ab initio 3D reconstruction" classification and divided into 2 or 4 sub-groups. After removing the particles of un-recognized or "defective" shape, a total of 417,460 particles with shape resembling spike remained. These particles were subjected to "homogeneity refinement," followed by "CTF global and local refinement" and "non-uniform refinement." No symmetry was imposed aside from C1 during the map refinements. The map after refinement could reach 2.84 Å resolution by the gold-standard FSC estimation with a 0.143 cut-off criterion. We then identified further the two conformations of S-6P as previously described (33) . One sub-class of 214,171 particles revealed the conformation of "1-up, 2-down" of RBD (Fig. S4C) , and one sub-class of 61,062 particles showed the conformation of "2-up, 1-down" (Fig. S4C) . The maps of "1-up, 2-down" and "2-up, 1-down" were refined at 3.02 Å and 3.34 Å resolution respectively. Local resolution plots for each map are shown in Fig. S4 , D and E. The maps are deposited in EMDB as EMD-24105 and EMD-24106.

An initial model for S-6P was generated using PDB 6XKL and was fit as a rigid body into the map using Chimera (38) followed by PyMOL. The Sb45-RBD (7KGJ) crystal structure was superimposed onto the S-6P model in PyMOL. We used real space refinement in PHENIX (39) including rigid-body refinement. The model was split into subdomains, NTD (24-289) and RBD (334-528) for rigid-body refinement. Simulated annealing (SA) was performed initially, including a local grid search and ADP refinement, using secondary structure restraints. We noticed that the These two models are deposited in PDB as 7N0G and 7N0H. Data processing, refinement statistics, and model validation are listed in Table 2 . Supporting information. This article contains supporting information. J o u r n a l P r e -p r o o f Table S1 ). CDR1, CDR2, CDR3 regions are painted pink, orange and red respectively. Additional non-CDR region contacting residues are colored lime. On the RBD surface, the epitopic residues that contact the sybodies are colored according to the sybody CDR.

J o u r n a l P r e -p r o o f , and Sb14 are shown. E484, K417 and N501 of RBD (wild type) interact with K32, Y54 and R60 of Sb16 respectively; E484 and N501 of RBD (wild type) interact with R33 and H103 of Sb45 respectively; and E484, K417 and N501 of RBD (wild type) interact with Q39, E35, and Y60 of Sb14 respectively. C, Comparison of complex structures with minimized models involving the N501Y mutation. In silico mutagenesis of N501Y was performed using 7KGK (Sb16+RBD), 7KGJ (Sb45+RBD), and 7MFU (Sb14+RBD+Sb68). Following amino acid substitution in Coot, local energy minimization (within 15 to 20 Å of the mutant residue) was performed through three rounds in PHENIX. For the Sb16-RBD complex, when N501 is mutated to Y501, the loop (496-506, from yellow to wheat) extends about 2.4 Å, but R60 (revealing a double conformation) still forms hydrogen bonds with the Y501 loop; for the Sb45-RBD complex, when N501 is mutated to Y501, the loop (496-506, from yellow to wheat) extends about 1.0 Å, but H103 of Sb45 would still interact with Y501; for the Sb14-RBD complex, when N501 is mutated to Y501, the loop (496-506, from yellow to wheat) is extended about 2.0 Å, but T58 and K65 still the hydrogen bonds with Y501; D, The surface charge of Sb16, K32 forms a hydrogen bond with E484 of RBD with the opposite charge; the surface charge of Sb45, R33 forms a hydrogen bond with E484 of RBD with the opposite charge; the surface charge of Sb14, Q39 (a neutral residue) interacts with E484 of RBD; E, Surface charge of wild type of RBD and surface charge of RBD with the three mutations (E484, K417N, and N501Y). When E484 is mutated to K484, the surface charge is changed from negative to positive, therefore the hydrogen bonds are brokenpushing Sb16 and Sb45 out of contact, while since Q39 of Sb14 is not a charged residue, it still may interact with K484 of the mutated RBD.

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69-2.60) 45.59-2.30 (2.38-2.30) 42

We appreciate the help of Joy (Huaying) Zhao and Peter Schuck, NIBIB, NIH