MicroED structure of the human vasopressin 1B receptor

Abstract

The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 A structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.

Toward physics‐based precision medicine: Exploiting protein dynamics to design new therapeutics and interpret variants

“…For example, some myosin motors function as cargo transporters while others participate in filaments to drive muscle contraction (Robert‐Paganin et al, 2020 ; Trivedi et al, 2020 ). Structural biologists have solved structures across several protein families with relevance to human disease, like myosins, kinases, and G‐protein‐coupled receptors (GPCRs) (Canagarajah et al, 1997 ; Day et al, 2009 ; Dominguez et al, 1998 ; Shiriaeva et al, 2023 ; Wacker et al, 2017 ). Comparing structures of these closely related proteins could, in principle, provide clues as to why proteins within the same family vary in their function.…”

Reaching the potential of electron diffraction

“…Predicted structures from AF2 have been used for crystallographic phasing over the last several years to great success, and the first novel structures have recently been solved with AF2 models for MicroED. 74 , 75 Fragment-based phasing methods are also being investigated and have allowed ab initio phasing of peptides 76 and proteins, where an electron counting detector contributed to improved resolution. 46 …”