Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.

Danny Axford,Juan F Bada Juarez,James Birch,Anthony Watts,Isabel Moraes,Peter J Judge,Javier Vinals,Tristan O C Kwan

doi:10.1107/s2059798321011220

Abstract

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Å resolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.

Highlights

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes (Shoemaker & Ando, 2018; Ren et al, 2020)
For membrane-protein crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; this method requires a considerable amount of protein sample, the production of which may not be feasible for many membrane proteins (Weierstall et al, 2014; Ren et al, 2020)
All of the major structural features visible in the high-resolution cryo crystal structures were resolved at room temperature, including the retinylidene chromophore, which is formed from the covalent conjugation of retinal to Lys226, and the conversion of the

Summary

Introduction

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes (Shoemaker & Ando, 2018; Ren et al, 2020). For membrane-protein crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; this method requires a considerable amount of protein sample, the production of which may not be feasible for many membrane proteins (Weierstall et al, 2014; Ren et al, 2020) New approaches, including those using thin polymer films (Huang et al, 2015; Axford et al, 2016; Doak et al, 2018; Lieske et al, 2019), are required to allow complete data sets to be obtained from small sample volumes, especially if the quantity. This seven transmembrane helices and a retinylidene chromophore research demonstrates that AR3 can convert from created by the covalent conjugation of retinal to Lys226 via a LA to DA in a crystalline environment, and that

Methods

Results

Conclusion