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Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne Encephalitis Virus Subtype (Sofjin Strain)

Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne... ISSN 1063-7745, Crystallography Reports, 2022, Vol. 67, No. 4, pp. 581–585. © The Author(s), 2022. This article is an open access publication, corrected publication 2022. Russian Text © The Author(s), 2022, published in Kristallografiya, 2022, Vol. 67, No. 4, pp. 623–627. STRUCTURE OF MACROMOLECULAR COMPOUNDS Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne Encephalitis Virus Subtype (Sof jin Strain) a b c b K. M. Dubova , A. V. Vlaskina , D. A. Korzhenevskiy , Yu. K. Agapova , b a,b, T. V. Rakitina , and V. R. Samygina * Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” Russian Academy of Sciences, Moscow, 119333 Russia National Research Centre “Kurchatov Institute,” Moscow, 123182 Russia Federal State Budget Institution “Federal Center for Brain and Neurotechnologies,” of the Federal Medical and Biological Agency of Russia, Moscow, 117513 Russia *e-mail: lera@crys.ras.ru Received February 21, 2022; revised February 21, 2022; accepted February 22, 2022 Abstract—The envelope (E) protein of f laviviruses is an attractive target for the development of antiviral agents because this protein plays an important role in the formation of virus particles and in the virus invasion in host cells. Currently, there is no specif ic antiviral therapy for tick-borne encephalitis. The goal of this study is to determine the crystal structure of the envelope (E) protein ectodomain of Far-Eastern tick-borne encephalitis virus subtype (Sof jin strain). The knowledge of the three-dimensional structure can serve as the basis for the development of specific inhibitors of conformational rearrangements of the (E) protein, which are essential for the initial stages of infection. DOI: 10.1134/S10637745220 4006X INTRODUCTION This protein forms the outer virion surface, provides stability of the virus particle, and is involved in the ini- Flaviviruses belong to the family Flaviviridae and tial stages of infection, such as the binding to cell can be classified into three groups according to the receptors, the entry of the virus into the cell, and the transmission vectors: tick-borne f laviviruses, mosqui- uncoating of the viral RNA [2]. toborne f laviviruses, and f laviviruses with no known There are the following three main subtypes of vectors [1]. Notorious human pathogens, such as Zika TBEV: the Central Europe subtype, the Siberian sub- virus, dengue virus, and West Nile virus, are spread by type, and the Far-Eastern subtype. The Siberian and mosquitoes. Tick-borne encephalitis virus and loup- Far-Eastern subtypes of TBEV are prevalent in Russia. ing ill virus are transmitted by ticks. They can cause Each subtype contains numerous strains, which have encephalitis or encephalomeningitis diseases in the small differences in the amino-acid sequence of the central nervous system. Louping ill virus (LIV) mainly proteins. infects small animals, such as sheep and red grouse. The goal of this study was to grow crystals of the E Occasionally, this virus can also infect humans. protein ectodomain of Far-Eastern tick-borne In contrast, the more widespread tick-borne encepha- encephalitis virus subtype (Sofjin strain), the amino- litis virus (TBEV) usually causes encephalitis in acid sequence of which is shown in Fig. 1, for the sub- humans, although it can infect rodents. Tick-borne sequent analysis of the structure of the apo protein and encephalitis virus is common in Eurasia, causing the detailed structural study of interactions between about 10 000 to 14 000 cases of tick-borne encephalitis the protein and prototypes of inhibitors. annually [2, 3]. Despite available vaccines, the rate of TBEV infection is still high due to low vaccination coverage. Currently, there are no specific therapeutics MATERIALS AND METHODS for the treatment of TBEV. The drug design is one of Isolation and Purification of the E Protein Ectodomain the modern approaches to the development of thera- peutics based on the knowledge of the three-dimen- The strain producing the recombinant E protein sional structures of the targets [4, 5]. In the case of ectodomain was constructed by the transformation of TBEV, the envelope (E) protein serves as the target. the E. coli BL21(DE3) RIPL strain cells with the 581 582 DUBOVA et al. MRCTHLENRDFVTGTQGTTRVTLVLELGGCVTITAEGKPSMDVWLDSIYQENPAKTREYCLHAKLSDTKV AARCPTMGPATLAEEHQSGTVCKRDQSDRGWGNHCGLFGKGSIVTCVKASCEAKKKATGHVYDANKIVYT VKVEPHTGDYVAANETHSGRKTASFTVSSERTILTMGDYGDVSLLCRVASGVDLAQTVILELDKTSEHLP TAWQVHRDWFNDLALPWKHEGAQNWNNAERLVEFGAPHAVKMDVYNLGDQTGVLLKSLAGVPVAHIDGTK YHLKSGHVTCEVGLEKLKMKGLTYTMCDKTKFTWKRIPTDSGHDTVVMEVAFSGTKPCRIPVRAVAHGSP DVNVAMLMTPNPTIENNGGGFIEMQLPPGDNIIYVGELSHQWFQK Fig. 1. Amino-acid sequence (residues 1–395) of the E protein ectodomain of the Far-Eastern tick-borne encephalitis virus sub- type (Sofjin strain; UniProt code P07720). (а) (b) (c) Fig. 2. Crystals of the E protein ectodomain of the Far-Eastern tick-borne encephalitis virus subtype (Sofjin strain): (a) grown under initial conditions; (b, c) grown under the optimized conditions. recombinant pET22 plasmid. The protein was isolated tems. The initial crystallization conditions were as fol- from inclusion bodies by refolding according to a pro- lows: 2.0 M ammonium sulfate in 0.1 M citrate buffer, cedure described in [6]. The supernatant was concen- pH 4.0. Under these conditions, small crystals were trated using VIVAFLOW 200 cassettes with a 10-kDa obtained (Fig. 2a). The crystallization conditions were PES membrane (Sartorius stedim LAB Ltd) and then then optimized by varying the concentration of using a 10-kDa centrifugal filter unit (Millipore, Bur- ammonium sulfate and the pН value of the buffer and lington, MA, USA) followed by the filtration through using the following additives: polyethylene glycol, a filter with a 0.22-μm pore size (PES membrane, glycerol, and 2-methyl-2,4-pentanediol (MPD). The Millipore Millex-GP). The target protein was purif ied conditions suitable for obtaining single crystals with by gel f iltration on a HiLoad 16/600 Superdex 200prep a size of at least 0.1 mm were not found as yet. grade column (GE Healthcare, Sweden). X-ray Diffraction Data Collection and Preliminary Crystallization of the E Protein Ectodomain Е(sE) X-ray Diffraction Analysis The crystallization conditions for the monomer Before X-ray diffraction data collection, the crys- and dimer of the E protein ectodomain were screened tals were picked up with a cryoloop (Hampton by the sitting-drop vapor-diffusion method. Drops Research), transferred to a cryoprotectant solution were composed of 0.7 μL of the protein solution at containing, apart from the components of the reser- a concentration of 5 mg/mL and 0.7 μL of the reser- voir solution, 20% glycerol, and soaked in this solution voir solution. The volume of the reservoir solution was for 15 s. Then the crystals in the loop were placed in 200 μL. The initial crystallization conditions were a metallic storage cassette compatible with an auto- screened in Intelli-Plate 48-3 plates (Hampton mated sample changer for pre-frozen crystals in liquid Research) using commercially available Hampton nitrogen. The X-ray diffraction data were collected Research crystallization screen kits at 20°С. Clearly at 100 К at the ID30B beamline of the European Syn- faceted single crystals were grown in acidic buffer sys- chrotron Radiation Facility (ESRF, Grenoble, CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 PRELIMINARY X-RAY DIFFRACTION ANALYSIS 583 Table 1. X-ray diffraction data collection and processing France) equipped with the PILATUS3 6M detector statistics (Dectris). The X-ray diffraction data were processed using the XDS and XSCALE programs [7]. The X-ray Sp. gr. P4 32 diffraction data collection statistics are given in Table 1. The X-ray diffraction data were collected to 3.2 Å res- a = b = c, Å 165.06 olution. The structure was solved by the molecular- α = β = γ, deg 90 replacement method with the MOLREP program [8]. The structure of the E protein ectodomain of LIV Wavelength, Å 0.899990 (Louping ill virus) ref ined at 3.6 Å resolution (pdb ref- Detector-to-crystal distance, cm 590 code 6J5C) [1] was used as the starting model. This model was refined to an intermediate R factor of Scan step, deg 0.1 22.6% using the Refmac program [9]. The crystal Scan angle, deg 360 structure was analyzed with the Coot molecular- graphics program [10], the PyMol molecular viewer Mosaicity, deg 0.073 [11], and the ССР4 suite of programs [12]. Resolution range, Å 50.00–3.2 (3.42–3.2)* Total number of ref lections 1 285 978 RESULTS AND DISCUSSION Number of unique ref lections 50 365 The initial crystallization conditions were screened by the sitting-drop vapor-diffusion method using Completeness of data, % 100 (100) commercially available Hampton Research crystalli- zation screen kits. The crystallization conditions were I/ϭ (I)32.5 (3.6) then optimized to prepare crystals with a size of 0.1– Redundancy 75.9 (65.4) 0.15 mm suitable for X-ray diffraction (Fig. 2). To achieve this purpose, the concentration of ammonium R 0.023 (0.221) pim sulfate was decreased by 0.2–0.4 M compared to the R 0.145 (1.775) merge initial concentration, the pH value was varied, and MPD was added, which made it possible to decrease * The data for the highest-resolution shell are given in parentheses. the number of crystals that grew in the drop and obtain larger single crystals. Since the solubility of the protein is rather low, the protein concentration was not varied. the structure 1SVB was determined for the E protein The best crystals were obtained in 0.1 M citrate buffer, ectodomain of TBEV Western subtype (Neudoerfl pH 4.5, supplemented with 1.6–1.8 M ammonium strain) cleaved from the surface of virus particles with sulfate and 2% MPD. The crystals with a size of up to trypsin. In this study, we determined the structure of 0.1–0.15 mm appeared within two–three days the recombinant protein expressed in the bacterial (Fig. 2b). expression system. Nevertheless, the conformational The X-ray diffraction data were collected from the similarity between the recombinant and natural pro- frozen crystals at 100 К on the ESRF synchrotron teins shows that the soluble part of the E protein radiation source. The data processing demonstrated expressed in E. coli is suitable for the detailed struc- that the highest spatial resolution was 3.2 Å. The crys- tural study of protein−inhibitor interactions. The tals belong to the cubic sp. gr. Р4 21 with the unit cell structure refinement is currently underway. parameters a = b = c = 165.061 Å, α = β = γ = 90°. It was demonstrated that, despite the utilization of There is one enzyme molecule per asymmetric unit. the bacterial expression system for the production of The polypeptide chain fold is similar to that in the the recombinant E protein, the conformation of the structure of the E protein ectodomain of TBEV Cen- molecule is similar to that observed in the virus parti- tral European subtype (Neudoerf l strain; pdb refcode cle. Therefore, the crystals of this protein can be used 1SVB) [13] and the E protein ectodomain of LIV (pdb refcode 6J5C) (Fig. 3). It should be noted that the for the X-ray diffraction analysis of the complexes with prototypes of inhibitors of the E protein. However, the conformation of the E protein ectodomain of LIV, the found crystallization conditions are not optimal crystals of which suitable for X-ray diffraction were because the crystals of the protein in complexes with obtained under similar crystallization conditions, is less different from the conformation of the structure inhibitors for the subsequent X-ray diffraction analysis under consideration despite the lower homology [1], are usually obtained by soaking because of a low solu- bility of inhibitors. This method generally impairs the particularly in the domain III region. The molecular structure of the title enzyme was superimposed with spatial resolution of the crystals. The resolution the molecular structure of 6J5C using the Сα atoms required for the detailed analysis of the binding of with a root-mean-square deviation (rmsd) of 0.546 Å; inhibitors to the protein should be not lower than 2.5 Å the superposition with the molecular structure of [5]. The soaking of the crystals will apparently lead to 1SVB gives the rmsd of 1.519 Å. It is worth noting that a decrease in the resolution from the crystals and, con- CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 584 DUBOVA et al. (a) (b) Fig. 3. Superposition of the structure of sE, determined in this study, with the structures (a) 6J5C and (b) 1SVB. The structures 6J5C and 1SVB are shown in dark. state assignment for the Federal Scientific Research Centre sequently, the crystals diffracting to a resolution of “Crystallography and Photonics” of the Russian Academy 3.2 Å cannot be used for obtaining crystals of the com- plexes, providing sufficiently detailed data. of Sciences. Therefore, the search for conditions of crystalliza- tion of the protein sE, which will allow the structure OPEN ACCESS determination at a resolution of about 2 Å, is currently underway. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any CONFLICT OF INTEREST medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to The authors declare no conf licts of interest, f inancial or the Creative Commons license, and indicate if changes otherwise. were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the ACKNOWLEDGMENTS material. If material is not included in the article’s Cre- We thank the ESRF staff for help with the X-ray diffrac- ative Commons license and your intended use is not per- tion data collection at the synchrotron radiation source. mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit FUNDING http://creativecommons.org/licenses/by/4.0/. This study was performed within the framework of the thematic plan of the National Research Centre “Kurchatov REFERENCES Institute” and was financially supported in part by the Rus- sian Foundation for Basic Research (project no. 18-02- 1. Xu Yang, Jianxun Qi, Ruchao Peng, et al., J. Virol. 93 40026) and by the Ministry of Science and Higher Educa- (8), e02132 (2019). tion of the Russian Federation within the framework of the https://doi.org/10.1128/JVI.02132-18 CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 PRELIMINARY X-RAY DIFFRACTION ANALYSIS 585 2. T. Fuzik, P. Formanova, D. Růžek, et al., Nat. Com- 8. A. A. Vagin and A. Teplyakov, J. Appl. Crystallogr. 30, mun. 9, 436 (2018). 1022 (1997). https://doi.org/10.1038/s41467-018-02882-0 https://doi.org/10.1107/S0021889897006766 9. G. N. Murshudov, P. Skubák, A. A. Lebedev, et al., 3. T. S. Gritsun, V. A. Lashkevich, and E. A. Gould, An- Acta Crystallogr. D 67, 355 (2011). tiviral Res. 57, 129 (2003). https://doi.org/10.1107/S0907444911001314 https://doi.org/10.1128/JVI.77.1.25-36.2003 10. P. Emsley, B. Lohkamp, W. Scott, et al., Acta Crystal- 4. Jaeyoung Ha, Hankum Park, Jongmin Park, and Seung logr. D 66, 486 (2010). Bum Park, Cell Chem. Biol. 28 (3), 394 (2021). https://doi.org/10.1107/S0907444910007493 https://doi.org/10.1016/j.chembiol.2020.12.001 11. W. L. DeLano and J. W. Lam, Abstr. Papers Am. 5. A. R. Bradley, A. Echalier, M. Fairhead, et al., Essays Chem. Soc. 230, 1371 (2005). Biochem. 61 (5), 495 (2017). 12. Collaborative Computational Project Number 4, Acta https://doi.org/10.1042/EBC20170051 Crystallogr. D 50, 760 (1994). 6. Lianpan Dai, Jian Song, Xishan Lu, et al., Cell Host. https://doi.org/10.1107/S0907444994003112 Microbe 19, 696 (2016). 13. F. A. Rey, F. X. Heinz, C. Mandl, et al., Nature 375, https://doi.org/10.1016/j.chom.2016.0 4.013 291 (1995). 7. W. Kabsch, Acta Crystallogr. D 66, 125 (2010). https://doi.org/10.1107/S0907444909047337 Translated by T. Safonova CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Crystallography Reports Springer Journals

Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne Encephalitis Virus Subtype (Sofjin Strain)

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Copyright © The Author(s) 2022. ISSN 1063-7745, Crystallography Reports, 2022, Vol. 67, No. 4, pp. 581–585. © The Author(s), 2022. This article is an open access publication, corrected publication 2022. Russian Text © The Author(s), 2022, published in Kristallografiya, 2022, Vol. 67, No. 4, pp. 623–627.
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ISSN 1063-7745, Crystallography Reports, 2022, Vol. 67, No. 4, pp. 581–585. © The Author(s), 2022. This article is an open access publication, corrected publication 2022. Russian Text © The Author(s), 2022, published in Kristallografiya, 2022, Vol. 67, No. 4, pp. 623–627. STRUCTURE OF MACROMOLECULAR COMPOUNDS Preliminary X-ray Diffraction Analysis of the Envelope (E) Protein of Far-Eastern Tick-Borne Encephalitis Virus Subtype (Sof jin Strain) a b c b K. M. Dubova , A. V. Vlaskina , D. A. Korzhenevskiy , Yu. K. Agapova , b a,b, T. V. Rakitina , and V. R. Samygina * Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” Russian Academy of Sciences, Moscow, 119333 Russia National Research Centre “Kurchatov Institute,” Moscow, 123182 Russia Federal State Budget Institution “Federal Center for Brain and Neurotechnologies,” of the Federal Medical and Biological Agency of Russia, Moscow, 117513 Russia *e-mail: lera@crys.ras.ru Received February 21, 2022; revised February 21, 2022; accepted February 22, 2022 Abstract—The envelope (E) protein of f laviviruses is an attractive target for the development of antiviral agents because this protein plays an important role in the formation of virus particles and in the virus invasion in host cells. Currently, there is no specif ic antiviral therapy for tick-borne encephalitis. The goal of this study is to determine the crystal structure of the envelope (E) protein ectodomain of Far-Eastern tick-borne encephalitis virus subtype (Sof jin strain). The knowledge of the three-dimensional structure can serve as the basis for the development of specific inhibitors of conformational rearrangements of the (E) protein, which are essential for the initial stages of infection. DOI: 10.1134/S10637745220 4006X INTRODUCTION This protein forms the outer virion surface, provides stability of the virus particle, and is involved in the ini- Flaviviruses belong to the family Flaviviridae and tial stages of infection, such as the binding to cell can be classified into three groups according to the receptors, the entry of the virus into the cell, and the transmission vectors: tick-borne f laviviruses, mosqui- uncoating of the viral RNA [2]. toborne f laviviruses, and f laviviruses with no known There are the following three main subtypes of vectors [1]. Notorious human pathogens, such as Zika TBEV: the Central Europe subtype, the Siberian sub- virus, dengue virus, and West Nile virus, are spread by type, and the Far-Eastern subtype. The Siberian and mosquitoes. Tick-borne encephalitis virus and loup- Far-Eastern subtypes of TBEV are prevalent in Russia. ing ill virus are transmitted by ticks. They can cause Each subtype contains numerous strains, which have encephalitis or encephalomeningitis diseases in the small differences in the amino-acid sequence of the central nervous system. Louping ill virus (LIV) mainly proteins. infects small animals, such as sheep and red grouse. The goal of this study was to grow crystals of the E Occasionally, this virus can also infect humans. protein ectodomain of Far-Eastern tick-borne In contrast, the more widespread tick-borne encepha- encephalitis virus subtype (Sofjin strain), the amino- litis virus (TBEV) usually causes encephalitis in acid sequence of which is shown in Fig. 1, for the sub- humans, although it can infect rodents. Tick-borne sequent analysis of the structure of the apo protein and encephalitis virus is common in Eurasia, causing the detailed structural study of interactions between about 10 000 to 14 000 cases of tick-borne encephalitis the protein and prototypes of inhibitors. annually [2, 3]. Despite available vaccines, the rate of TBEV infection is still high due to low vaccination coverage. Currently, there are no specific therapeutics MATERIALS AND METHODS for the treatment of TBEV. The drug design is one of Isolation and Purification of the E Protein Ectodomain the modern approaches to the development of thera- peutics based on the knowledge of the three-dimen- The strain producing the recombinant E protein sional structures of the targets [4, 5]. In the case of ectodomain was constructed by the transformation of TBEV, the envelope (E) protein serves as the target. the E. coli BL21(DE3) RIPL strain cells with the 581 582 DUBOVA et al. MRCTHLENRDFVTGTQGTTRVTLVLELGGCVTITAEGKPSMDVWLDSIYQENPAKTREYCLHAKLSDTKV AARCPTMGPATLAEEHQSGTVCKRDQSDRGWGNHCGLFGKGSIVTCVKASCEAKKKATGHVYDANKIVYT VKVEPHTGDYVAANETHSGRKTASFTVSSERTILTMGDYGDVSLLCRVASGVDLAQTVILELDKTSEHLP TAWQVHRDWFNDLALPWKHEGAQNWNNAERLVEFGAPHAVKMDVYNLGDQTGVLLKSLAGVPVAHIDGTK YHLKSGHVTCEVGLEKLKMKGLTYTMCDKTKFTWKRIPTDSGHDTVVMEVAFSGTKPCRIPVRAVAHGSP DVNVAMLMTPNPTIENNGGGFIEMQLPPGDNIIYVGELSHQWFQK Fig. 1. Amino-acid sequence (residues 1–395) of the E protein ectodomain of the Far-Eastern tick-borne encephalitis virus sub- type (Sofjin strain; UniProt code P07720). (а) (b) (c) Fig. 2. Crystals of the E protein ectodomain of the Far-Eastern tick-borne encephalitis virus subtype (Sofjin strain): (a) grown under initial conditions; (b, c) grown under the optimized conditions. recombinant pET22 plasmid. The protein was isolated tems. The initial crystallization conditions were as fol- from inclusion bodies by refolding according to a pro- lows: 2.0 M ammonium sulfate in 0.1 M citrate buffer, cedure described in [6]. The supernatant was concen- pH 4.0. Under these conditions, small crystals were trated using VIVAFLOW 200 cassettes with a 10-kDa obtained (Fig. 2a). The crystallization conditions were PES membrane (Sartorius stedim LAB Ltd) and then then optimized by varying the concentration of using a 10-kDa centrifugal filter unit (Millipore, Bur- ammonium sulfate and the pН value of the buffer and lington, MA, USA) followed by the filtration through using the following additives: polyethylene glycol, a filter with a 0.22-μm pore size (PES membrane, glycerol, and 2-methyl-2,4-pentanediol (MPD). The Millipore Millex-GP). The target protein was purif ied conditions suitable for obtaining single crystals with by gel f iltration on a HiLoad 16/600 Superdex 200prep a size of at least 0.1 mm were not found as yet. grade column (GE Healthcare, Sweden). X-ray Diffraction Data Collection and Preliminary Crystallization of the E Protein Ectodomain Е(sE) X-ray Diffraction Analysis The crystallization conditions for the monomer Before X-ray diffraction data collection, the crys- and dimer of the E protein ectodomain were screened tals were picked up with a cryoloop (Hampton by the sitting-drop vapor-diffusion method. Drops Research), transferred to a cryoprotectant solution were composed of 0.7 μL of the protein solution at containing, apart from the components of the reser- a concentration of 5 mg/mL and 0.7 μL of the reser- voir solution, 20% glycerol, and soaked in this solution voir solution. The volume of the reservoir solution was for 15 s. Then the crystals in the loop were placed in 200 μL. The initial crystallization conditions were a metallic storage cassette compatible with an auto- screened in Intelli-Plate 48-3 plates (Hampton mated sample changer for pre-frozen crystals in liquid Research) using commercially available Hampton nitrogen. The X-ray diffraction data were collected Research crystallization screen kits at 20°С. Clearly at 100 К at the ID30B beamline of the European Syn- faceted single crystals were grown in acidic buffer sys- chrotron Radiation Facility (ESRF, Grenoble, CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 PRELIMINARY X-RAY DIFFRACTION ANALYSIS 583 Table 1. X-ray diffraction data collection and processing France) equipped with the PILATUS3 6M detector statistics (Dectris). The X-ray diffraction data were processed using the XDS and XSCALE programs [7]. The X-ray Sp. gr. P4 32 diffraction data collection statistics are given in Table 1. The X-ray diffraction data were collected to 3.2 Å res- a = b = c, Å 165.06 olution. The structure was solved by the molecular- α = β = γ, deg 90 replacement method with the MOLREP program [8]. The structure of the E protein ectodomain of LIV Wavelength, Å 0.899990 (Louping ill virus) ref ined at 3.6 Å resolution (pdb ref- Detector-to-crystal distance, cm 590 code 6J5C) [1] was used as the starting model. This model was refined to an intermediate R factor of Scan step, deg 0.1 22.6% using the Refmac program [9]. The crystal Scan angle, deg 360 structure was analyzed with the Coot molecular- graphics program [10], the PyMol molecular viewer Mosaicity, deg 0.073 [11], and the ССР4 suite of programs [12]. Resolution range, Å 50.00–3.2 (3.42–3.2)* Total number of ref lections 1 285 978 RESULTS AND DISCUSSION Number of unique ref lections 50 365 The initial crystallization conditions were screened by the sitting-drop vapor-diffusion method using Completeness of data, % 100 (100) commercially available Hampton Research crystalli- zation screen kits. The crystallization conditions were I/ϭ (I)32.5 (3.6) then optimized to prepare crystals with a size of 0.1– Redundancy 75.9 (65.4) 0.15 mm suitable for X-ray diffraction (Fig. 2). To achieve this purpose, the concentration of ammonium R 0.023 (0.221) pim sulfate was decreased by 0.2–0.4 M compared to the R 0.145 (1.775) merge initial concentration, the pH value was varied, and MPD was added, which made it possible to decrease * The data for the highest-resolution shell are given in parentheses. the number of crystals that grew in the drop and obtain larger single crystals. Since the solubility of the protein is rather low, the protein concentration was not varied. the structure 1SVB was determined for the E protein The best crystals were obtained in 0.1 M citrate buffer, ectodomain of TBEV Western subtype (Neudoerfl pH 4.5, supplemented with 1.6–1.8 M ammonium strain) cleaved from the surface of virus particles with sulfate and 2% MPD. The crystals with a size of up to trypsin. In this study, we determined the structure of 0.1–0.15 mm appeared within two–three days the recombinant protein expressed in the bacterial (Fig. 2b). expression system. Nevertheless, the conformational The X-ray diffraction data were collected from the similarity between the recombinant and natural pro- frozen crystals at 100 К on the ESRF synchrotron teins shows that the soluble part of the E protein radiation source. The data processing demonstrated expressed in E. coli is suitable for the detailed struc- that the highest spatial resolution was 3.2 Å. The crys- tural study of protein−inhibitor interactions. The tals belong to the cubic sp. gr. Р4 21 with the unit cell structure refinement is currently underway. parameters a = b = c = 165.061 Å, α = β = γ = 90°. It was demonstrated that, despite the utilization of There is one enzyme molecule per asymmetric unit. the bacterial expression system for the production of The polypeptide chain fold is similar to that in the the recombinant E protein, the conformation of the structure of the E protein ectodomain of TBEV Cen- molecule is similar to that observed in the virus parti- tral European subtype (Neudoerf l strain; pdb refcode cle. Therefore, the crystals of this protein can be used 1SVB) [13] and the E protein ectodomain of LIV (pdb refcode 6J5C) (Fig. 3). It should be noted that the for the X-ray diffraction analysis of the complexes with prototypes of inhibitors of the E protein. However, the conformation of the E protein ectodomain of LIV, the found crystallization conditions are not optimal crystals of which suitable for X-ray diffraction were because the crystals of the protein in complexes with obtained under similar crystallization conditions, is less different from the conformation of the structure inhibitors for the subsequent X-ray diffraction analysis under consideration despite the lower homology [1], are usually obtained by soaking because of a low solu- bility of inhibitors. This method generally impairs the particularly in the domain III region. The molecular structure of the title enzyme was superimposed with spatial resolution of the crystals. The resolution the molecular structure of 6J5C using the Сα atoms required for the detailed analysis of the binding of with a root-mean-square deviation (rmsd) of 0.546 Å; inhibitors to the protein should be not lower than 2.5 Å the superposition with the molecular structure of [5]. The soaking of the crystals will apparently lead to 1SVB gives the rmsd of 1.519 Å. It is worth noting that a decrease in the resolution from the crystals and, con- CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 584 DUBOVA et al. (a) (b) Fig. 3. Superposition of the structure of sE, determined in this study, with the structures (a) 6J5C and (b) 1SVB. The structures 6J5C and 1SVB are shown in dark. state assignment for the Federal Scientific Research Centre sequently, the crystals diffracting to a resolution of “Crystallography and Photonics” of the Russian Academy 3.2 Å cannot be used for obtaining crystals of the com- plexes, providing sufficiently detailed data. of Sciences. Therefore, the search for conditions of crystalliza- tion of the protein sE, which will allow the structure OPEN ACCESS determination at a resolution of about 2 Å, is currently underway. 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To view a copy of this license, visit FUNDING http://creativecommons.org/licenses/by/4.0/. This study was performed within the framework of the thematic plan of the National Research Centre “Kurchatov REFERENCES Institute” and was financially supported in part by the Rus- sian Foundation for Basic Research (project no. 18-02- 1. Xu Yang, Jianxun Qi, Ruchao Peng, et al., J. Virol. 93 40026) and by the Ministry of Science and Higher Educa- (8), e02132 (2019). tion of the Russian Federation within the framework of the https://doi.org/10.1128/JVI.02132-18 CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022 PRELIMINARY X-RAY DIFFRACTION ANALYSIS 585 2. T. Fuzik, P. Formanova, D. Růžek, et al., Nat. Com- 8. A. A. Vagin and A. Teplyakov, J. Appl. Crystallogr. 30, mun. 9, 436 (2018). 1022 (1997). https://doi.org/10.1038/s41467-018-02882-0 https://doi.org/10.1107/S0021889897006766 9. G. N. Murshudov, P. Skubák, A. A. Lebedev, et al., 3. T. S. Gritsun, V. A. Lashkevich, and E. A. 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D 66, 125 (2010). https://doi.org/10.1107/S0907444909047337 Translated by T. Safonova CRYSTALLOGRAPHY REPORTS Vol. 67 No. 4 2022

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