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Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.
Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, Poleschuk VF, (1983) Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology 20:23-31
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL, (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905-921
Collaborative CP, (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr Sect D, Biol Crystallogr 50:760
Dodson EJ, (2001) Using electron-microscopy images as a model for molecular replacement. Acta Crystallogr D 57:1405-1409. doi:10.1107/S0907444901013415
Guu TS, Liu Z, Ye Q, Mata DA, Li K, Yin C, Zhang J, Tao YJ, (2009) Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. Proc Natl Acad Sci USA 106:12992-12997. doi:10.1107/S0907444901013415
Jiang W, Guo F, Liu Z, (2012) A graph theory method for determination of cryo-EM image focuses. J Struct Biol 180:343-351. doi:10.1016/j.jsb.2012.07.005
Jones TA, Zou JY, Cowan SWT, Kjeldgaard M, (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr Sect A: Found Crystallogr 47:110-119
Kleywegt GJ, Jones TA, (1996) xdlMAPMAN and xdlDATAMAN-programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr D Biol Crystallogr 52:826-828
Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T, (1997) Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 71:7207-7213
Li S, Tang X, Seetharaman J, Yang C, Gu Y, Zhang J, Du H, Shih JW, Hew CL, Sivaraman J, Xia N, (2009) Dimerization of hepatitis E virus capsid protein E2 s domain is essential for virus-host interaction. PLoS Pathog 5:e1000537. doi:10.1371/journal.ppat.1000537
Liang Y, Ke EY, Zhou ZH, (2002) IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database. J Struct Biol 137:292-304
Liu Z, Zhang J-J, (2014) Revolutionary breakthrough of structure determination-recent advances of electron direct detection device application in cryo-EM. Acta Biophys Sin 30:1-12
Liu H, Cheng L, Zeng S, Cai C, Zhou ZH, Yang Q, (2008) Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions. J Struct Biol 161:64-73. doi:10.1016/j.jsb.2007.09.016
Liu Z, Guo F, Wang F, Li TC, Jiang W, (2016) 2.9 Å resolution cryo-EM 3D reconstruction of close-packed virus particles. Structure 24:319-328. doi:10.1016/j.str.2015.12.006
Ludtke SJ, Baldwin PR, Chiu W, (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82-97
Navaza J, (2008) Combining X-ray and electron-microscopy data to solve crystal structures. Acta Crystallogr Sect D, Biol Crystallogr 64:70-75. doi:10.1107/S0907444907053334
Otwinowski Z, Minor W, (1997) Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol 276:307-326
Stein PE, Boodhoo A, Armstrong GD, Cockle SA, Klein MH, Read RJ, (1994) The crystal structure of pertussis toxin. Structure 2:45-57. doi:10.1073/pnas.1101309108
Ten Eyck LF, (1977) Efficient structure-factor calculation for large molecules by the fast Fourier transform. Acta Crystallogr Sect A 33:486-492
Tong L, Rossmann MG, (1997) Rotation function calculations with GLRF program. Methods Enzymol 276:594
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