Multi-strategy Synergy Enhances the Diffraction Resolution of Protein Crystals

doi:10.13560/j.cnki.biotech.bull.1985.2025-0975

Abstract

Abstract:

Objective Protein crystallographic diffraction is an essential technique in structural biology research. The diffraction resolution of crystals directly determines the accuracy of atomic models and their practical applicability. Therefore, a combination of multiple strategies is required to optimize protein crystallization quality and enhance diffraction resolution. Method The P450 enzyme, which catalyzes aryl coupling reactions, was chosen as the research focus. A prokaryotic expression system was first constructed via molecular biology techniques. The protein was purified using nickel-affinity chromatography and size-exclusion chromatography. During the crystallization stage, 1,632 initial conditions were systematically screened through the vapor diffusion method. Based on preliminary results, key variables including buffer pH, precipitant type and concentration were finely optimized, and various additives were introduced to improve the crystallization environment. Furthermore, rational truncation of flexible regions was designed according to structural predictions, and SUMO tag fusion was attempted to enhance protein stability. Result The P450 protein was successfully expressed solubly at high levels in E. coli and purified to high purity via a two-step purification process, making it suitable for crystallization screening. Through large-scale crystallization condition screening and multiple rounds of optimization, combined with synergistic strategies including sequence truncation and SUMO fusion, crystal morphology was significantly improved—transitioning from initial microcrystals or amorphous precipitates to well-defined single crystals with regular shapes and sharp edges. The diffraction resolution of the crystals was markedly enhanced from an initial 10 Å to 2.86 Å, yielding high-quality crystals suitable for high-resolution structural analysis. Conclusion By employing a multi-strategy synergy, the protein crystal morphology and diffraction resolution of protein crystals is significantly enhanced.

Key words: protein crystallization, X-ray diffraction, protein purification, protein expression, cytochrome P450

REN Hong-yu, PANG Cui-ping, GU Yang, ZHOU Jia-hai. Multi-strategy Synergy Enhances the Diffraction Resolution of Protein Crystals[J]. Biotechnology Bulletin, 2026, 42(1): 76-85.

Figures/Tables 12

Fig. 1 Replacing expression tags and co-expressing molecular chaperones of P450A-C: The expression of P450KstB, P450MciB, and P450ScnB, respectively, with molecular chaperone co-expression and fusion tag swapping. 1: Uninduced strain, 2: SUMO-P450, 3-5: the SUMO-P450 strain was introduced with the molecular chaperones pGro7, pTF16, and pKJE7, respectively, 6: TF-P450, 7: MBP-P450. D: The expression after replacing the GST tag. 1: Uninduced strain, 2-4: P450KstB, P450ScnB, and P450MciB fused with GST tag, respectively

Fig. 2 Ni-NTA analysis of soluble proteins results by Ni-affinity chromatographyA-C: The purification results of affinity chromatography using nickel columns for pCold-TF-KstB, pRSF-sumo-KstB+pGro7, and pMALc6T-MBP-MciB, respectively

Fig. 3 Analysis of P450GpeC protein expressionA: The cell lysate of P450GpeC and the SDS-PAGE analysis of precipitate and supernatant. B: The UV absorption spectrum of P450GpeC with a characteristic absorption peak at 425 nm

Fig. 4 Analysis of P450GpeC results by Ni-affinity chromatography

Fig. 5 Elution chromatogram and SDS-PAGE analysis results of P450GpeC purified by SEC

Fig. 6 Structural analysis of the P450GpeC protein predicted by AlphaFold 3

Fig. 7 Crystal images and diffraction patterns of P450GpeC under various initial crystallization conditionsThe arrow indicates the crystal used for diffraction

Fig. 8 Crystal images and diffraction patterns of P450GpeC optimized by ammonium sulfate addition and seeding methodThe arrow indicates the crystal used for diffraction

Fig. 9 Analysis of SUMO-P450GpeC-ΔC8 results by Ni-affinity chromatography

Fig. 10 Elution chromatogram and SDS-PAGE analysis results of SUMO-P450GpeC-△C8 purified by SEC

Fig. 11 Crystal images and diffraction patterns of optimized SUMO-P450GpeC-△C8 crystalsThe arrow indicates the crystal used for diffraction

Table 1 Crystal optimization methods and effects

优化方式 Optimization method	优化效果 Optimization result	分辨率 Resolution （Å）
结晶初筛	无法获得蛋白晶体	-
C端loop截短	晶体过小或者表面粗糙	10
溶剂优化	改善晶体形态	5-8
添加硫酸铵	依附盐晶生长	3-4
添加晶种	脱离盐晶	3-4
添加SUMO标签	外观规则且棱角清晰的高质量集体	2.86

References 34

[1]	Papageorgiou AC, Poudel N, Mattsson J. Protein structure analysis and validation with X-ray crystallography [M]//Protein Downstream Processing. New York, NY: Springer US, 2020: 377-404.
[2]	Banari A, Samanta AK, Munke A, et al. Advancing time-resolved structural biology: latest strategies in cryo-EM and X-ray crystallography [J]. Nat Meth, 2025, 22(7): 1420-1435.
[3]	Nogales E, Mahamid J. Bridging structural and cell biology with cryo-electron microscopy [J]. Nature, 2024, 628(8006): 47-56.
[4]	Abramson J, Adler J, Dunger J, et al. Addendum: Accurate structure prediction of biomolecular interactions with AlphaFold 3 [J]. Nature, 2024, 636(8042): E4.
[5]	Li FC, Liu RZ, Li WJ, et al. Synchrotron radiation: a key tool for drug discovery [J]. Bioorg Med Chem Lett, 2024, 114: 129990.
[6]	Heras B, Edeling MA, Byriel KA, et al. Dehydration converts DsbG crystal diffraction from low to high resolution [J]. Structure, 2003, 11(2): 139-145.
[7]	McPherson A, Gavira JA. Introduction to protein crystallization [J]. Acta Crystallogr F Struct Biol Commun, 2014, 70(1): 2-20.
[8]	Budziszewski GR, Stojanoff V, Bowman SEJ. Preparing for successful protein crystallization experiments [J]. Acta Crystallogr F Struct Biol Commun, 2025, 81(7): 272-280.
[9]	Zhang XF, Xu ZT, Zhou JH, et al. Enhancement of protein crystallization using nano-sized metal-organic framework [J]. Crystals, 2022, 12(5): 578.
[10]	Maki S, Hagiwara M. Contactless crystallization method of protein by a magnetic force booster [J]. Sci Rep, 2022, 12: 17287.
[11]	Han Q, Brown SJ, Drummond CJ, et al. Protein aggregation and crystallization with ionic liquids: Insights into the influence of solvent properties [J]. J Colloid Interface Sci, 2022, 608: 1173-1190.
[12]	Chen TE, Hiramatsu H, Toyouchi S, et al. Laser-polarization-induced anisotropy enhances protein crystallization [J]. Angew Chem Int Ed, 2025, 64(21): e202501827.
[13]	Dessau MA, Modis Y. Protein crystallization for X-ray crystallography [J]. JoVE, 2011(47).
[14]	Chayen NE, Saridakis E. Protein crystallization: from purified protein to diffraction-quality crystal [J]. Nat Meth, 2008, 5(2): 147-153.
[15]	McPherson A, Cudney B. Searching for silver bullets: an alternative strategy for crystallizing macromolecules [J]. J Struct Biol, 2006, 156(3): 387-406.
[16]	de Wijn R, Hennig O, Ernst FGM, et al. Combining crystallogenesis methods to produce diffraction-quality crystals of a psychrophilic tRNA-maturation enzyme [J]. Acta Crystallogr F Struct Biol Commun, 2018, 74(11): 747-753.
[17]	Khurshid S, Saridakis E, Govada L, et al. Porous nucleating agents for protein crystallization [J]. Nat Protoc, 2014, 9(7): 1621-1633.
[18]	Chernov AA. Protein crystals and their growth [J]. J Struct Biol, 2003, 142(1): 3-21.
[19]	Zhao MZ, Ma JS, Li M, et al. Cytochrome P450 enzymes and drug metabolism in humans [J]. Int J Mol Sci, 2021, 22(23): 12808.
[20]	Sang RY, Jiang WZ, Zhang C, et al. Effect of food components on cytochrome P450 expression and activity [J]. Hum Nutr Metab, 2025, 40: 200304.
[21]	Song YR, Li CX, Liu GZ, et al. Drug-metabolizing cytochrome P450 enzymes have multifarious influences on treatment outcomes [J]. Clin Pharmacokinet, 2021, 60(5): 585-601.
[22]	Esteves F, Rueff J, Kranendonk M. The central role of cytochrome P450 in xenobiotic metabolism—a brief review on a fascinating enzyme family [J]. J Xenobiot, 2021, 11(3): 94-114.
[23]	Greule A, Stok JE, De Voss JJ, et al. Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism 1 [J]. Nat Prod Rep, 2018, 35(8): 757-791.
[24]	Rudolf JD, Chang CY, Ma M, et al. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function [J]. Nat Prod Rep, 2017, 34(9): 1141-1172.
[25]	Zhang XW, Guo JW, Cheng FY, et al. Cytochrome P450 enzymes in fungal natural product biosynthesis [J]. Nat Prod Rep, 2021, 38(6): 1072-1099.
[26]	He BB, Liu J, Cheng Z, et al. Bacterial cytochrome P450 catalyzed post-translational macrocyclization of ribosomal peptides [J]. Angew Chem Int Ed, 2023, 62(46): e202311533.
[27]	Hu YL, Yin FZ, Shi J, et al. P450-modified ribosomally synthesized peptides with aromatic cross-links [J]. J Am Chem Soc, 2023, 145(50): 27325-27335.
[28]	Coe LJ, Zhao YW, Padva L, et al. Reassignment of the structure of a tryptophan-containing cyclic tripeptide produced by the biarylitide crosslinking cytochrome P450blt [J]. Chem, 2024, 30(38): e202400988.
[29]	Kandy SK, Pasquale MA, Chekan JR. Aromatic side-chain crosslinking in RiPP biosynthesis [J]. Nat Chem Biol, 2025, 21(2): 168-181.
[30]	Zdouc MM, Alanjary MM, Zarazúa GS, et al. A biaryl-linked tripeptide from Planomonospora reveals a widespread class of minimal RiPP gene clusters [J]. Cell Chem Biol, 2021, 28(5): 733-739.e4.
[31]	Hug JJ, Dastbaz J, Adam S, et al. Biosynthesis of cittilins, unusual ribosomally synthesized and post-translationally modified peptides from Myxococcus xanthus [J]. ACS Chem Biol, 2020, 15(8): 2221-2231.
[32]	Wang F, Zhou HY, Olademehin OP, et al. Insights into key interactions between vancomycin and bacterial cell wall structures [J]. ACS Omega, 2018, 3(1): 37-45.
[33]	Hu BD, Yu HB, Zhou JW, et al. Whole-cell P450 biocatalysis using engineered Escherichia coli with fine-tuned heme biosynthesis [J]. Adv Sci, 2023, 10(6): 2205580.
[34]	Panavas T, Sanders C, Butt TR. SUMO fusion technology for enhanced protein production in prokaryotic and eukaryotic expression systems [J]. Methods Mol Biol, 2009, 497: 303-317.