Go BIG! - Purification and Structural Determination of RyR1, 2.3MDa membrane protein
Release Time:
2023-02-17 14:20
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With our state-of-the-art CryoEM facility and gene-to-structure pipeline, Biortus’s experienced team has solved a 2.8 Å CryoEM structure of RyR1, the largest known ion channel at 2.3 MDa.
Ryanodine Receptor (RyR) channels are recognized as a high-value target for drug discovery to treat severe skeletal and cardiac myopathies, diabetes, and neurodegeneration. Their dysregulation causes elevated cytoplasmic calcium, characteristic of these pathologies [1]. RyR channels are the largest known ion channels, usually homotetramers with each protomer at about 5000 residues (565 kDa) [2]. The Type 1 Ryanodine Receptor (RyR1) mediates excitation-contraction coupling in skeletal muscle.
Biortus’s team purified this membrane protein with high enzymatic activity, high purity (> 95%), and high concentration (> 5 mg/ml, 1.6mg) from 100 g of rabbit skeletal muscles [3]. Using our new Refeyn Two Mass Photometer, our team confirmed the functional tetrameric state of RyR1. The final structure was resolved at an overall resolution of a 2.8 Å with the transmembrane domain at a local resolution of a 2.5 Å – majority with clear side chain density. From rabbit skeletal muscles to structure, this project took 2 weeks.
Biortus is your dedicated partner for your protein needs with protein production, assay development, fragment screening, and structure determination (MicroED, X-ray Crystallography, and CryoEM) all under one roof. For new inquiries, reach out to info@wuxibiortus.com.
Citations:
[1] Rebbeck, Robyn T., et al. "RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays." Scientific reports 10.1 (2020): 1-13.
[2] Iyer, Kavita A., et al. "Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM." Proceedings of the National Academy of Sciences 119.30 (2022): e2122140119.
[3] Hu, Yifan, et al. "Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells." Bio-protocol 11.15 (2021): e4112-e4112.
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