This cryo-EM protocol was used to determine the B cell epitope map on the CdtB subunit of typhoid toxin, an AB toxin secreted by Typhi during infection. Immunoglobulin G (IgG) was directly mixed with typhoid toxin in this protocol, different from our previous cryo-EM protocol that uses the Fab fragments in place of IgG. This simple approach requires smaller amounts of materials, supporting the broader use of this protocol for determining antibody recognition sites on various antigens. For complete details on the use and execution of this protocol, please refer to Ahn et al. (2021) and Nguyen et al. (2021).
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http://dx.doi.org/10.1016/j.xpro.2021.100852 | DOI Listing |
Protein Expr Purif
January 2025
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address:
E6AP/UBE3A is the founding member of the HECT (Homologous to the E6-AP Carboxyl Terminus) ubiquitin E3 ligase family, which add ubiquitin post-translationally to protein substrates. E6AP has been structurally defined in complex with human papillomavirus (HPV) oncoprotein E6 and its gain-of-function substrate tumor suppressor p53; however, there is currently no report of E6AP being expressed and purified from mammalian cells, as studies to date have isolated E6AP from E. coli or insect cells.
View Article and Find Full Text PDFRSC Med Chem
December 2024
Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara 44121 Ferrara Italy
The N/OFQ-NOP receptor is a fascinating peptidergic system with the potential to be exploited for the development of analgesic drugs devoid of side effects associated with classical opioid signalling modulation. To date, up to four X-ray and cryo-EM structures of the NOP receptor in complex with the endogenous peptide agonist N/OFQ and three small molecule antagonists have been solved and released. Despite the available structural information, the details of selective small molecule agonist binding to the NOP receptor in the active state remain elusive.
View Article and Find Full Text PDFJ Phys Chem B
January 2025
Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, Uppsala SE-75124, Sweden.
Adenosine receptors, particularly AAR, are gaining attention for their role in pathological conditions such as cancer immunotherapy, prompting the exploration for promising therapeutic applications. Despite numerous selective AAR antagonists, the lack of selective full agonists makes the partial agonist BAY60-6583 one of the most interesting activators of this receptor. Recent cryo-EM structures have univocally revealed the binding mode of nonselective ribosidic agonists such as adenosine and its derivative NECA to AAR; however, two independent structures with BAY60-6583 show alternative binding orientations, raising the question of which is the physiologically relevant binding mode.
View Article and Find Full Text PDFBio Protoc
December 2024
School of Bioengineering, Dalian University of Technology, Dalian, China.
Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples.
View Article and Find Full Text PDFbioRxiv
December 2024
Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY 10032.
Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid.
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