小分子量膜蛋白冷冻电镜结构解析的通用工程化工具

小分子量膜蛋白冷冻电镜结构解析的通用工程化工具

王  煦，屈前辉^*，周子璇*

（复旦大学生物医学研究院，上海 200032）

摘　要　　膜蛋白作为细胞功能的重要承担者，其结构解析对相关基础研究与药物开发具有重要意义。冷冻电镜技术的革新推动了膜蛋白结构的研究，但单颗粒冷冻电镜技术在小分子量膜蛋白（<100 kDa）的结构解析中仍面临信噪比低、颗粒对准困难等挑战。本文系统总结了近年来发展的多种工程化策略，包括基于纳米抗体的复合物支架（Legobody、Pro-macrobody）、螺旋短肽融合表达（MPER/Fab、ALFA-tag）、刚性结构域融合表达（MBP/DARPin、GFP/Nb、BRIL/Fab）等。这些策略通过提高复合物刚性与尺寸，有效解决了小分子量膜蛋白结构解析中的难题，为包括GPCR、跨膜转运蛋白等小分子量膜蛋白的结构解析提供了通用性解决方案，帮助推动膜蛋白功能机制与药物靶点的研究。

关键词　　冷冻电镜；结构生物学；膜蛋白

中图分类号：Q6-33    文献标识码：A Doi:10.3969/j.issn.1000-6281.2025.05.012

Versatile engineering tools for Cryo-EM structural determination of small transmembrane proteins

WANG Xu，QU Qianhui^*，ZHOU Zixuan^*

（Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China）

Abstract　　Membrane proteins play vital roles in cellular functions and are key targets in biological research and pharmaceutical development. Advances in Cryo-electron microscopy have significantly advanced membrane protein structure studies. However, determining the structures of small membrane proteins ( < 100 kDa ) remains challenging due to low signal-to-noise ratio and difficulties in particle alignment. This paper systematically reviews recent engineering strategies designed to overcome these obstacles, including nanobody-based scaffolds (Legobody, Pro-macrobody), helical peptide fusion expression (MPER / Fab, ALFA-tag), rigid domain fusion expression (MBP / DARPin, GFP / Nb, BRIL / Fab). By improving the rigidity and size of the complex, these strategies improve structural determination of small molecular membrane proteins such as GPCRs and transmembrane transporters. These approaches provide a versatile framework to elucidate membrane protein functional mechanisms and advance pharmaceutical target discovery.

Keywords　　Cryo-electron microscopy; structural biology; membrane protein

[1]BOULOS I, JABBOUR J, KHOURY S, et al. Exploring the world of membrane proteins: Techniques and methods for understanding structure, function, and dynamics [J]. Molecules, 2023, 28(20): 7176.

[2]ZHANG M, CHEN T, LU X, et al. G protein-coupled receptors (GPCRs): Advances in structures, mechanisms and drug discovery [J]. Signal Transduction and Targeted Therapy, 2024, 9(1): 1-43.

[3]FUKUDA Y, STAPLETON K, KATO T. Progress in spatial resolution of structural analysis by Cryo-EM [J]. Microscopy (Oxford, England), 2023, 72(2): 135-143.

[4]刘铮, 沈庆涛, 隋森芳.  AI时代中的电子显微学研究：严峻挑战、无穷机遇与壮阔前景[J]. 电子显微学报, 2025, 44(1): 124-135.

[5]LIAO M, CAO E, JULIUS D, et al. Structure of the TRPV1 ion channel determined by electron cryo-microscopy [J]. Nature, 2013, 504(7478): 107-112.

[6]YEATES T O, AGDANOWSKI M P, LIU Y. Development of imaging scaffolds for Cryo-electron microscopy [J]. Current Opinion in Structural Biology, 2020, 60: 142-149.

[7]HENDERSON R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules [J]. Quarterly Reviews of Biophysics, 1995, 28(2): 171-193.

[8]NYGAARD R, KIM J, MANCIA F. Cryo-electron microscopy analysis of small membrane proteins [J]. Current Opinion in Structural Biology, 2020, 64: 26-33.

[9]NOBLE A J, DANDEY V P, WEI H, et al. Routine single particle Cryo-EM sample and grid characterization by tomography[J]. eLife, 2018, 7: e34257.

[10]MUKHERJEE S, ERRAMILLI S K, AMMIRATI M, et al. Synthetic antibodies against BRIL as universal fiducial marks for single-particle Cryo-EM structure determination of membrane proteins [J]. Nature Communications, 2020, 11(1): 1598.

[11]YEATES T O, AGDANOWSKI M P, LIU Y. Development of imaging scaffolds for Cryo-electron microscopy [J]. Current Opinion in Structural Biology, 2020, 60: 142-149.

[12]WU X, RAPOPORT T A. Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies) [J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(41): e2115001118.

[13]HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains [J]. Nature, 1993, 363(6428): 446-448.

[14]MUYLDERMANS S. Nanobodies: natural single-domain antibodies [J]. Annual Review of Biochemistry, 2013, 82: 775-797.

[15]DE GENST E, SILENCE K, DECANNIERE K, et al. Molecular basis for the preferential cleft recognition by dromedary heavy chain antibodies [J]. Proceedings of the National Academy of Sciences, 2006, 103(12): 4586-4591.

[16]STEYAERT J, KOBILKA B K. Nanobody stabilization of G protein-coupled receptor conformational states [J]. Current Opinion in Structural Biology, 2011, 21(4): 567-572.

[17]ZIMMERMANN I, EGLOFF P, HUTTER C A, et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins [J]. eLife, 2018, 7: e34317.

[18]ZIMMERMANN I, EGLOFF P, HUTTER C A, et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins [J]. eLife, 2018, 7: e34317.

[19]MCMAHON C, BAIER A S, PASCOLUTTI R, et al. Yeast surface display platform for rapid discovery of conformationally selective nanobodies [J]. Nature Structural & Molecular Biology, 2018, 25(3): 289-296.

[20]ZIMMERMANN I, EGLOFF P, HUTTER C A J, et al. Generation of synthetic nanobodies against delicate proteins [J]. Nature Protocols, 2020, 15(5): 1707-1741.

[21]ACKLE F, THAVARASAH S, EARP J C, et al. Rigid enlargement of sybodies with antibody fragments for Cryo-EM analyses of small membrane proteins [J]. Scientific Reports, 2025, 15(1): 9460.

[22]BRUNNER J D, JAKOB R P, SCHULZE T, et al. Structural basis for ion selectivity in TMEM175 K+ channels [J]. eLife, 2020, 9: e53683.

[23]BOTTE M, NI D, SCHENCK S, et al. Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation [J]. Nature Communications, 2022, 13(1): 1826.

[24]UCHAŃSKI T, MASIULIS S, FISCHER B, et al. Megabodies expand the nanobody toolkit for protein structure determination by single-particle Cryo-EM [J]. Nature Methods, 2021, 18(1): 60-68.

[25]KANG Y, CHEN L. Structural basis for the binding of DNP and purine nucleotides onto UCP1 [J]. Nature, 2023, 620(7972): 226-231.

[26]MCILWAIN B C. N-terminal Transmembrane-Helix Epitope Tag for X-ray crystallography and electron microscopy of small membrane proteins [J]. Journal of Molecular Biology, 2021.

[27]LI P, ZHU Z, WANG Y, et al. Substrate transport and drug interaction of human thiamine transporters SLC19A2/A3 [J]. Nature Communications, 2024, 15(1): 10924.

[28]GÖTZKE H, KILISCH M, MARTÍNEZ-CARRANZA M, et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications [J]. Nature Communications, 2019, 10(1): 4403.

[29]WANG Y, ZHANG P, CHAO Y, et al. Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines [J]. Cell Research, 2024, 34(1):47-57.

[30]DEUPI X, KOBILKA B K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function [J]. Physiology (Bethesda, Md.), 2010, 25(5): 293-303.

[31]PARK J H, SCHEERER P, HOFMANN K P, et al. Crystal structure of the ligand-free G-protein-coupled receptor opsin [J]. Nature, 2008, 454(7201): 183-187.

[32]KOEHL A, HU H, MAEDA S, et al. Structure of the µ-opioid receptor-Gi protein complex [J]. Nature, 2018, 558(7711): 547-552.

[33]TSUTSUMI N, MUKHERJEE S, WAGHRAY D, et al. Structure of human Frizzled5 by fiducial-assisted Cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling [J]. eLife, 2020, 9: e58464.

[34]CHE T, ENGLISH J, KRUMM B E, et al. Nanobody-enabled monitoring of kappa opioid receptor states [J]. Nature Communications, 2020, 11(1): 1145.

[35]CHE T, MAJUMDAR S, ZAIDI S A, et al. Structure of the nanobody-stabilized active state of the kappa opioid receptor [J]. Cell, 2018, 172(1-2): 55-67.e15.

[36]ROBERTSON M J, PAPASERGI-SCOTT M M, HE F, et al. Structure determination of inactive-state GPCRs with a universal nanobody [J]. Nature Structural & Molecular Biology, 2022, 29(12): 1188-1195.

[37]HU J, QIN H, GAO F P, et al. A systematic assessment of mature MBP in membrane protein production: Overexpression, membrane targeting and purification [J]. Protein Expression and Purification, 2011, 80(1): 34-40.

[38]PIDATHALA S, LIAO S, DAI Y, et al. Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2 [J]. Nature, 2023, 623(7989): 1086-1092.

[39]BINZ H K, AMSTUTZ P, KOHL A, et al. High-affinity binders selected from designed ankyrin repeat protein libraries [J]. Nature Biotechnology, 2004, 22(5): 575-582.

[40]KRATZ P A, BÖTTCHER B, NASSAL M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids [J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(5): 1915-1920.

[41]NIU Y, LIU R, GUAN C, et al. Structural basis of inhibition of the human SGLT2–MAP17 glucose transporter [J]. Nature, 2022, 601(7892): 280-284.

[42]WU D, CHEN Q, YU Z, et al. Transport and inhibition mechanisms of human VMAT2 [J]. Nature, 2024, 626(7998): 427-434.

[43]CHUN E, THOMPSON A A, LIU W, et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors [J]. Structure (London, England: 1993), 2012, 20(6): 967-976.

[44]VASILIAUSKAITÉ-BROOKS I, HEALEY R D, ROCHAIX P, et al. Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy [J]. Nature Communications, 2018, 9(1): 5437.

[45]GUO Q, HE B, ZHONG Y, et al. A method for structure determination of GPCRs in various states [J]. Nature Chemical Biology, 2024, 20(1): 74-82.

[46]DANG Y, ZHOU D, DU X, et al. Molecular mechanism of substrate recognition by folate transporter SLC19A1 [J]. Cell Discovery, 2022, 8(1): 1-11.