Applied Microscopy

  • 제51권
  • /
  • Pages.13.1-13.7
  • /
  • 2021
  • /
  • 2287-5123(pISSN)
  • /
  • 2287-4445(eISSN)
한국현미경학회 (Korean Society of Microscopy)
권호 표지
DOI QR코드

DOI QR Code

Cryo-EM as a powerful tool for drug discovery: recent structural based studies of SARS-CoV-2

  • Han‑ul Kim (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Hyun Suk Jung (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • 투고 : 2021.07.20
  • 심사 : 2021.09.15
  • 발행 : 2021.12.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has arisen as a global pandemic affecting the respiratory system showing acute respiratory distress syndrome (ARDS). However, there is no targeted therapeutic agent yet and due to the growing cases of infections and the rising death tolls, discovery of the possible drug is the need of the hour. In general, the study for discovering therapeutic agent for SARS-CoV-2 is largely focused on large-scale screening with fragment-based drug discovery (FBDD). With the recent advancement in cryo-electron microscopy (Cryo-EM), it has become one of the widely used tools in structural biology. It is effective in investigating the structure of numerous proteins in high-resolution and also had an intense influence on drug discovery, determining the binding reaction and regulation of known drugs as well as leading the design and development of new drug candidates. Here, we review the application of cryo-EM in a structure-based drug design (SBDD) and in silico screening of the recently acquired FBDD in SARS-CoV-2. Such insights will help deliver better understanding in the procurement of the effective remedial solution for this pandemic.

키워드

과제정보

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0344), the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2021R1A2C1009404 to HSJ) and the Korea Basic Science Institute(KBSI) National Research Facilities &Equipment Center(NFEC) grant funded by the Korea government(Ministry of Education) (2019R1A6C1010006).

참고문헌

  1. K.R. Acharya, M.D. Lloyd, The advantages and limitations of protein crystal structures. Trends Pharmacol. Sci. 26(1), 10-14 (2005). https://doi.org/10.1016/j.tips.2004.10.011
  2. P.V. Afonine, B.K. Poon, R.J. Read, O.V. Sobolev, T.C. Terwilliger, A. Urzhumtsev, P.D. Adams, Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74(Pt 6), 531-544 (2018). https://doi.org/10.1107/S2059798318006551
  3. A. Asai, M. Konno, M. Ozaki, C. Otsuka, A. Vecchione, T. Arai, T. Kitagawa, K. Ofusa, M. Yabumoto, T. Hirotsu, M. Taniguchi, H. Eguchi, Y. Doki, H. Ishii, COVID-19 drug discovery using intensive approaches. Int. J. Mol. Sci. 21(8), 2839 (2020). https://doi.org/10.3390/ijms21082839
  4. K.A. Baker, R.E. Dutch, R.A. Lamb, T.S. Jardetzky, Structural basis for paramyxo‑ virus-mediated membrane fusion. Mol. Cell 3(3), 309-319 (1999). https://doi.org/10.1016/s1097-2765(00)80458-x
  5. J.M. Bell, M. Chen, T. Durmaz, A.C. Fluty, S.J. Ludtke, New software tools in EMAN2 inspired by EMDatabank map challenge. J. Struct. Biol. 204(2), 283-290 (2018). https://doi.org/10.1016/j.jsb.2018.09.002
  6. H.J. Berendsen, S. Hayward, Collective protein dynamics in relation to function. Curr. Opin. Struct. Biol. 10(2), 165-169 (2000). https://doi.org/10.1016/s0959-440x(00)00061-0
  7. S. Boopathi, A.B. Poma, P. Kolandaivel, Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. J. Biomol. Struct. Dyn. 39(9), 3409-3418 (2021). https://doi.org/10.1080/07391102.2020.1758788
  8. B.J. Bosch, R. van der Zee, C.A. de Haan, P.J. Rottier, The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J. Virol. 77(16), 8801-8811 (2003). https://doi.org/10.1128/jvi.77.16.8801-8811.2003
  9. Y. Cai, J. Zhang, T. Xiao, H. Peng, S.M. Sterling, R.M. Walsh Jr., S. Rawson, S. Rits-Volloch, B. Chen, Distinct conformational states of SARS-CoV-2 spike protein. Science 369(6511), 1586-1592 (2020). https://doi.org/10.1126/science.abd4251
  10. L. Caly, J.D. Druce, M.G. Catton, D.A. Jans, K.M. Wagstaff, The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res. 178, 104787 (2020). https://doi.org/10.1016/j.antiviral.2020.104787
  11. Z. Chagla, The BNT162b2 (BioNTech/Pfizer) vaccine had 95% efficacy against COVID-19 >/=7 days after the 2nd dose. Ann. Intern. Med. 174(2), JC15 (2021). https://doi.org/10.7326/ACPJ202102160-015
  12. N. Chazal, D. Gerlier, Virus entry, assembly, budding, and membrane rafts. Microbiol. Mol. Biol. Rev. 67(2), 226-237 (2003). https://doi.org/10.1128/MMBR.67.2.226-237.2003, table of contents
  13. T.F. Chen, Y.C. Chang, Y. Hsiao, K.H. Lee, Y.C. Hsiao, Y.H. Lin, Y.E. Tu, H.C. Huang, C.Y. Chen, H.F. Juan, DockCoV2: a drug database against SARS-CoV-2. Nucleic Acids Res. 49(D1), D1152-D1159 (2021). https://doi.org/10.1093/nar/gkaa861
  14. S. Choudhary, Y.S. Malik, S. Tomar, Identification of SARS-CoV-2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach. Front. Immunol. 11, 1664 (2020). https://doi.org/10.3389/fmmu.2020.01664
  15. B.D. Dorsey, R.B. Levin, S.L. McDaniel, J.P. Vacca, J.P. Guare, P.L. Darke, J.A. Zugay, E.A. Emini, W.A. Schleif, J.C. Quintero et al., L-735,524: the design of a potent and orally bioavailable HIV protease inhibitor. J. Med. Chem. 37(21), 3443-3451 (1994). https://doi.org/10.1021/jm00047a001
  16. A. Dubrovsky, S. Sorrentino, J. Harapin, K.T. Sapra, O. Medalia, Developments in cryo-electron tomography for in situ structural analysis. Arch. Biochem. Biophys. 581, 78-85 (2015). https://doi.org/10.1016/j.abb.2015.04.006
  17. E.H. Egelman, The current revolution in cryo-EM. Biophys. J. 110(5), 1008-1012 (2016). https://doi.org/10.1016/j.bpj.2016.02.001
  18. S. Ekins, J. Mestres, B. Testa, In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Br. J. Pharmacol. 152(1), 9-20 (2007). https://doi.org/10.1038/sj.bjp.0707305
  19. J. Erickson, D.J. Neidhart, J. VanDrie, D.J. Kempf, X.C. Wang, D.W. Norbeck, J.J. Plattner, J.W. Rittenhouse, M. Turon, N. Wideburg et al., Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science 249(4968), 527-533 (1990). https://doi.org/10.1126/science.2200122
  20. D.A. Erlanson, Many small steps towards a COVID-19 drug. Nat. Commun. 11(1), 5048 (2020). https://doi.org/10.1038/s41467-020-18710-3
  21. J. Frank, Advances in the field of single-particle cryo-electron microscopy over the last decade. Nat. Protoc. 12(2), 209-212 (2017). https://doi.org/10.1038/nprot.2017.004
  22. S. Ghosh, A. Nie, J. An, Z. Huang, Structure-based virtual screening of chemical libraries for drug discovery. Curr. Opin. Chem. Biol. 10(3), 194-202 (2006). https://doi.org/10.1016/j.cbpa.2006.04.002
  23. B.S. Graham, M.S.A. Gilman, J.S. McLellan, Structure-based vaccine antigen design. Annu. Rev. Med. 70, 91-104 (2019). https://doi.org/10.1146/annurev-med-121217-094234
  24. T. Grant, A. Rohou, N. Grigorieff, cisTEM, user-friendly software for single-particle image processing. Elife 7, e35383 (2018). https://doi.org/10.7554/eLife.35383
  25. M.A.A. Ibrahim, A.H.M. Abdelrahman, T.A. Hussien, E.A.A. Badr, T.A. Mohamed, H.R. El-Seedi, P.W. Pare, T. Eferth, M.F. Hegazy, In silico drug discovery of major metabolites from spices as SARS-CoV-2 main protease inhibitors. Comput. Biol. Med. 126, 104046 (2020). https://doi.org/10.1016/j.compbiomed.2020.104046
  26. Y.P. Jiang, X.X. Zhao, H.Q. Lv, C.P. Wen, Drug screening and development from the affinity of S protein of new coronavirus with ACE2. Eur. J. Clin. Microbiol. Infect. Dis. 40(4), 715-723 (2021). https://doi.org/10.1007/s10096-020-04048-7
  27. S. Jojoa-Cruz, K. Saotome, S.E. Murthy, C.C.A. Tsui, M.S. Sansom, A. Patapoutian, A.B. Ward, Cryo-EM structure of the mechanically activated ion channel OSCA1.2. Elife 7, e41845 (2018). https://doi.org/10.7554/eLife.41845
  28. D. Kampjut, J. Steiner, L.A. Sazanov, Cryo-EM grid optimization for membrane proteins. iScience 24(3), 102139 (2021). https://doi.org/10.1016/j.isci.2021.102139
  29. M.L. Kelly, C.C. Chu, H. Shi, L.R. Ganser, H.P. Bogerd, K. Huynh, Y. Hou, B.R. Cullen, H.M. Al-Hashimi, Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays. RNA 27(1), 12-26 (2021). https://doi.org/10.1261/rna.076257.120
  30. R.N. Kirchdoerfer, N. Wang, J. Pallesen, D. Wrapp, H.L. Turner, C.A. Cottrell, K.S. Corbett, B.S. Graham, J.S. McLellan, A.B. Ward, Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis. Sci. Rep. 8(1), 15701 (2018). https://doi.org/10.1038/s41598-018-34171-7
  31. D.B. Kitchen, H. Decornez, J.R. Furr, J. Bajorath, Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug. Discov. 3(11), 935-949 (2004). https://doi.org/10.1038/nrd1549
  32. M.D. Knoll, C. Wonodi, Oxford-AstraZeneca COVID-19 vaccine efficacy. Lancet 397(10269), 72-74 (2021). https://doi.org/10.1016/S0140-6736(20)32623-4
  33. W. Kuhlbrandt, Biochemistry. The resolution revolution. Science 343(6178), 1443-1444 (2014). https://doi.org/10.1126/science.1251652
  34. E. Kwon, D. Pathak, H.U. Kim, P. Dahal, S.C. Ha, S.S. Lee, H. Jeong, D. Jeoung, H.W. Chang, H.S. Jung, D.Y. Kim, Structural insights into stressosome assembly. IUCrJ 6(Pt 5), 938-947 (2019). https://doi.org/10.1107/S205225251900945X
  35. C.C. Lai, T.P. Shih, W.C. Ko, H.J. Tang, P.R. Hsueh, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): the epidemic and the challenges. Int. J. Antimicrob. Agents 55(3), 105924 (2020). https://doi.org/10.1016/j.ijantimicag.2020.105924
  36. A.R. Leach, B.K. Shoichet, C.E. Peishoff, Prediction of protein-ligand interactions. Docking and scoring: successes and gaps. J. Med. Chem. 49(20), 5851-5855 (2006). https://doi.org/10.1021/jm060999m
  37. F. Li, Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3(1), 237-261 (2016). https://doi.org/10.1146/annurev-virology-110615-042301
  38. J. Li, J. Sun, Application of X-ray diffraction and electron crystallography for solving complex structure problems. Acc. Chem. Res. 50(11), 2737-2745 (2017). https://doi.org/10.1021/acs.accounts.7b00366
  39. C. Liu, Y. Yang, Y. Gao, C. Shen, B. Ju, C. Liu, X. Tang, J. Wei, X. Ma, W. Liu, S. Xu, Y. Liu, J. Yuan, J. Wu, Z. Liu, Z. Zhang, P. Wang, Liu L, Viral architecture of SARS-CoV-2 with post-fusion spike revealed by Cryo-EM. bioRxiv. 2020.2003.2002.972927 (2020). https://doi.org/10.1101/2020.03.02.972927
  40. E. Mahase, Covid-19: Moderna vaccine is nearly 95% effective, trial involving high risk and elderly people shows. BMJ 371, m4471 (2020). https://doi.org/10.1136/bmj.m4471
  41. L. Maveyraud, L. Mourey, Protein X-ray crystallography and drug discovery. Molecules 25(5), 1030 (2020). https://doi.org/10.3390/molecules25051030
  42. A. Merk, A. Bartesaghi, S. Banerjee, V. Falconieri, P. Rao, M.I. Davis, R. Pragani, M.B. Boxer, L.A. Earl, J.L.S. Milne, S. Subramaniam, Breaking cryo-EM resolution barriers to facilitate drug discovery. Cell 165(7), 1698-1707 (2016). https://doi.org/10.1016/j.cell.2016.05.040
  43. Method of the year 2015. Nat. Methods. 13(1), 1 (2016). https://doi.org/10.1038/nmeth.3730.
  44. Z.T. Muhseen, A.R. Hameed, H.M.H. Al-Hasani, M. Tahir Ul Qamar, G. Li, Promising terpenes as SARS-CoV-2 spike receptor-binding domain (RBD) attachment inhibitors to the human ACE2 receptor: Integrated computational approach. J. Mol. Liq. 320, 114493 (2020). https://doi.org/10.1016/j.molliq.2020.114493
  45. T. Nakane, A. Kotecha, A. Sente, G. McMullan, S. Masiulis, P. Brown, I.T. Grigoras, L. Malinauskaite, T. Malinauskas, J. Miehling, T. Uchanski, L. Yu, D. Karia, E.V. Pechnikova, E. de Jong, J. Keizer, M. Bischof, J. McCormack, P. Tiemeijer, S.W. Hardwick, D.Y. Chirgadze, G. Murshudov, A.R. Aricescu, S.H.W. Scheres, Single-particle cryo-EM at atomic resolution. Nature 587(7832), 152-156 (2020). https://doi.org/10.1038/s41586-020-2829-0
  46. E. Nogales, The development of cryo-EM into a mainstream structural biology technique. Nat. Methods 13(1), 24-27 (2016). https://doi.org/10.1038/nmeth.3694
  47. J. Pallesen, N. Wang, K.S. Corbett, D. Wrapp, R.N. Kirchdoerfer, H.L. Turner, C.A. Cottrell, M.M. Becker, L. Wang, W. Shi, W.P. Kong, E.L. Andres, A.N. Kettenbach, M.R. Denison, J.D. Chappell, B.S. Graham, A.B. Ward, J.S. McLellan, Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl. Acad. Sci. U. S. A. 114(35), E7348-E7357 (2017). https://doi.org/10.1073/pnas.1707304114
  48. A. Pan, L. Liu, C. Wang, H. Guo, X. Hao, Q. Wang, J. Huang, N. He, H. Yu, X. Lin, S. Wei, T. Wu, Association of public health interventions with the epidemiology of the COVID-19 outbreak in Wuhan, China. JAMA 323(19), 1915-1923 (2020a). https://doi.org/10.1001/jama.2020.6130
  49. Y. Pan, Z. Ren, S. Gao, J. Shen, L. Wang, Z. Xu, Y. Yu, P. Bachina, H. Zhang, X. Fan, A. Laganowsky, N. Yan, M. Zhou, Structural basis of ion transport and inhibition in ferroportin. Nat. Commun. 11(1), 5686 (2020b). https://doi.org/10.1038/s41467-020-19458-6
  50. P.K. Panda, M.N. Arul, P. Patel, S.K. Verma, W. Luo, H.G. Rubahn, Y.K. Mishra, M. Suar, R. Ahuja, Structure-based drug designing and immunoinformatics approach for SARS-CoV-2. Sci. Adv. 6(28), eabb8097 (2020). https://doi.org/10.1126/sciadv.abb8097
  51. E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25(13), 1605-1612 (2004). https://doi.org/10.1002/jcc.20084
  52. A.I. Petushkova, A.A. Zamyatnin Jr., Papain-like proteases as coronaviral drug targets: current inhibitors, opportunities, and limitations. Pharmaceuticals (Basel) 13(10), 277 (2020). https://doi.org/10.3390/ph13100277
  53. A. Punjani, J.L. Rubinstein, D.J. Fleet, M.A. Brubaker, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14(3), 290-296 (2017). https://doi.org/10.1038/nmeth.4169
  54. S. Rawson, M.G. Iadanza, N.A. Ranson, S.P. Muench, Methods to account for movement and flexibility in cryo-EM data processing. Methods 100, 35-41 (2016). https://doi.org/10.1016/j.ymeth.2016.03.011
  55. J.P. Renaud, A. Chari, C. Ciferri, W.T. Liu, H.W. Remigy, H. Stark, C. Wiesmann, Cryo-EM in drug discovery: achievements, limitations and prospects. Nat. Rev. Drug Discov. 17(7), 471-492 (2018). https://doi.org/10.1038/nrd.2018.77
  56. L. Riva, S. Yuan, X. Yin, L. Martin-Sancho, N. Matsunaga, L. Pache, S. Burgstaller-Muehlbacher, P.D. De Jesus, P. Teriete, M.V. Hull, M.W. Chang, J.F. Chan, J. Cao, V.K. Poon, K.M. Herbert, K. Cheng, T.H. Nguyen, A. Rubanov, Y. Pu, C. Nguyen, A. Choi, R. Rathnasinghe, M. Schotsaert, L. Miorin, M. Dejosez, T.P. Zwaka, K.Y. Sit, L. Martinez-Sobrido, W.C. Liu, K.M. White, M.E. Chapman, E.K. Lendy, R.J. Glynne, R. Albrecht, E. Ruppin, A.D. Mesecar, J.R. Johnson, C. Benner, R. Sun, P.G. Schultz, A.I. Su, A. Garcia-Sastre, A.K. Chatterjee, K.Y. Yuen, S.K. Chanda, Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing. Nature 586(7827), 113-119 (2020). https://doi.org/10.1038/s41586-020-2577-1
  57. N.A. Roberts, J.A. Martin, D. Kinchington, A.V. Broadhurst, J.C. Craig, I.B. Duncan, S.A. Galpin, B.K. Handa, J. Kay, A. Krohn et al., Rational design of peptide-based HIV proteinase inhibitors. Science 248(4953), 358-361 (1990). https://doi.org/10.1126/science.2183354
  58. D. Rognan, The impact of in silico screening in the discovery of novel and safer drug candidates. Pharmacol. Ther. 175, 47-66 (2017). https://doi.org/10.1016/j.pharmthera.2017.02.034
  59. A. Rohou, N. Grigorieff, CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192(2), 216-221 (2015). https://doi.org/10.1016/j.jsb.2015.08.008
  60. G. Scapin, C.S. Potter, B. Carragher, Cryo-EM for small molecules discovery, design, understanding, and application. Cell Chem. Biol. 25(11), 1318-1325 (2018). https://doi.org/10.1016/j.chembiol.2018.07.006
  61. S.H. Scheres, Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125-157 (2016). https://doi.org/10.1016/bs.mie.2016.04.012
  62. C. Schmidli, S. Albiez, L. Rima, R. Righetto, I. Mohammed, P. Oliva, L. Kovacik, H. Stahlberg, T. Braun, Microfluidic protein isolation and sample preparation for high-resolution cryo-EM. Proc. Natl. Acad. Sci. U. S. A. 116(30), 15007-15012 (2019). https://doi.org/10.1073/pnas.1907214116
  63. N. Sepay, A. Sekar, U.C. Halder, A. Alarif, M. Afzal, Anti-COVID-19 terpenoid from marine sources: a docking, admet and molecular dynamics study. J. Mol. Struct. 1228, 129433 (2021). https://doi.org/10.1016/j.molstruc.2020.129433
  64. C. Sun, L. Chen, J. Yang, C. Luo, Y. Zhang, J. Li, J. Yang, J. Zhang, L. Xie, SARS-CoV-2 and SARS-CoV spike-RBD structure and receptor binding comparison and potential implications on neutralizing antibody and vaccine development. bioRxiv. 2020.2002.2016.951723 (2020). https://doi.org/10.1101/2020.02.16.951723
  65. J.H. Van Drie, L. Tong, Cryo-EM as a powerful tool for drug discovery. Bioorg. Med. Chem. Lett. 30(22), 127524 (2020). https://doi.org/10.1016/j.bmcl.2020.127524
  66. J.A. Walker, S.S. Molloy, G. Thomas, T. Sakaguchi, T. Yoshida, T.M. Chambers, Y. Kawaoka, Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus. J. Virol. 68(2), 1213-1218 (1994). https://doi.org/10.1128/JVI.68.2.1213-1218.1994
  67. W. Wong, X.C. Bai, B.E. Sleebs, T. Triglia, A. Brown, J.K. Thompson, K.E. Jackson, E. Hanssen, D.S. Marapana, I.S. Fernandez, S.A. Ralph, A.F. Cowman, S.H.W. Scheres, J. Baum, Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis. Nat. Microbiol. 2, 17031 (2017). https://doi.org/10.1038/nmicrobiol.2017.31
  68. D. Wrapp, N. Wang, K.S. Corbett, J.A. Goldsmith, C.L. Hsieh, O. Abiona, B.S. Graham, J.S. McLellan, Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367(6483), 1260-1263 (2020). https://doi.org/10.1126/science.abb2507
  69. Y. Wu, F. Wang, C. Shen, W. Peng, D. Li, C. Zhao, Z. Li, S. Li, Y. Bi, Y. Yang, Y. Gong, H. Xiao, Z. Fan, S. Tan, G. Wu, W. Tan, X. Lu, C. Fan, Q. Wang, Y. Liu, C. Zhang, J. Qi, G.F. Gao, F. Gao, L. Liu, A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science 368(6496), 1274-1278 (2020). https://doi.org/10.1126/science.abc2241
  70. S. Xia, Q. Lan, S. Su, X. Wang, W. Xu, Z. Liu, Y. Zhu, Q. Wang, L. Lu, S. Jiang, The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin. Signal Transduct. Target. Ther. 5(1), 92 (2020). https://doi.org/10.1038/s41392-020-0184-0
  71. X. Xiong, K. Qu, K.A. Ciazynska, M. Hosmillo, A.P. Carter, S. Ebrahimi, Z. Ke, S.H.W. Scheres, L. Bergamaschi, G.L. Grice, Y. Zhang, C.-N.C.-B. Collaboration, J.A. Nathan, S. Baker, L.C. James, H.E. Baxendale, I. Goodfellow, R. Doffinger, J.A.G. Briggs, A thermostable, closed SARS-CoV-2 spike protein trimer. Nat. Struct. Mol. Biol. 27(10), 934-941 (2020). https://doi.org/10.1038/s41594-020-0478-5
  72. K.M. Yip, N. Fischer, E. Paknia, A. Chari, H. Stark, Breaking the next Cryo-EM resolution barrier - atomic resolution determination of proteins! bioRxiv. 2020.2005.2021.106740 (2020). https://doi.org/10.1101/2020.05.21.106740
  73. S. Zafar, M.S. Arshad, S. Fatima, A. Ali, A. Zaman, E. Sayed, M.W. Chang, Z. Ahmad, COVID-19: current developments and further opportunities in drug delivery and therapeutics. Pharmaceutics 12(10), 945 (2020). https://doi.org/10.3390/pharmaceutics12100945
  74. K. Zhang, Gctf: real-time CTF determination and correction. J. Struct. Biol. 193(1), 1-12 (2016). https://doi.org/10.1016/j.jsb.2015.11.003
  75. Y. Zhang, F. Yang, S. Ling, P. Lv, Y. Zhou, W. Fang, W. Sun, L. Zhang, P. Shi, C. Tian, Single-particle cryo-EM structural studies of the beta2AR-Gs complex bound with a full agonist formoterol. Cell Discov. 6, 45 (2020a). https://doi.org/10.1038/s41421-020-0176-9
  76. Z.R. Zhang, Y.N. Zhang, X.D. Li, H.Q. Zhang, S.Q. Xiao, F. Deng, Z.M. Yuan, H.Q. Ye, B. Zhang, A cell-based large-scale screening of natural compounds for inhibitors of SARS-CoV-2. Signal Transduct. Target. Ther. 5(1), 218 (2020b). https://doi.org/10.1038/s41392-020-00343-z
  77. M. Zhang, M. Gui, Z.F. Wang, C. Gorgulla, J.J. Yu, H. Wu, Z.J. Sun, C. Klenk, L. Merklinger, L. Morstein, F. Hagn, A. Pluckthun, A. Brown, M.L. Nasr, G. Wagner, Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs. Nat. Struct. Mol. Biol. 28(3), 258-267 (2021). https://doi.org/10.1038/s41594-020-00554-6
  78. S.Q. Zheng, E. Palovcak, J.P. Armache, K.A. Verba, Y. Cheng, D.A. Agard, Motion‑Cor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14(4), 331-332 (2017). https://doi.org/10.1038/nmeth.4193
  79. J. Zivanov, T. Nakane, B.O. Forsberg, D. Kimanius, W.J. Hagen, E. Lindahl, S.H. Scheres, New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018). https://doi.org/10.7554/eLife.42166