Protein-protein interactions are essential in diverse cellular processes including signal transduction. It is not uncommon for protein-protein interactions in the transmembrane signaling to exhibit low binding affinities with a dissociation constant in the μM range or higher. Presumably, membrane proteins can selectively bind with their binding partners via low-affinity interactions because they can only diffuse laterally in the membrane plane and interact with the partners within a limited space on the membrane surface or within the membrane. This review focuses on the crystallographic studies on cell-surface receptors and intramembrane proteases that perform the transmembrane signaling mediated by low-affinity protein-protein interactions.

NMR methods provide information about conformational dynamics of GPCRs over a wide range of frequencies in aqueous media at near-physiological temperature, with minimal modification of the wild-type GPCR covalent structures. Here we review our solution NMR studies of the functionrelated conformational dynamics of GPCRs, including β₂ adrenergic receptor, μ opioid receptor, and adenosine A_2A receptor.

It is difficult to perform the thermodynamic analysis of conformational changes occurring in protein crystals by conventional cryogenic X-ray crystallography because flash-cooled crystals have ambiguous information about temperature. We have developed a humid air and glue-coating（HAG）method in which the crystals are coated with a water-soluble polymer to control humidity. This method realized X-ray crystallographic analysis with controlled temperature and humidity in a non-cryogenic environment. In this paper, we report details of the HAG method and the thermodynamic analysis of conformational changes of copper-containing amine oxidase using the HAG method.

Conventional X-ray protein crystallography captures only “static” structures due to the limitation of temporal resolution. Recently, X-ray free electron lasers（XFEL）enabled to capture dynamic structures at the atomic level with time resolutions of several tenth femtoseconds. We have been working on the development of serial femtosecond crystallography（SFX）, which is a protein structure determination technique, at SACLA and successfully obtained “molecular movies” of various proteins by combining an X-ray free electron laser with a reaction trigger. This technique is suitable for tracking fast reactions such as chemical reactions because of its high temporal resolution. We will describe current advances and future prospects of molecular movies using XFEL.

Cryo-crystallography is usually used at synchrotron macromolecular crystallography（MX）beamlines in order to mitigate radiation damage. However, the structures determined at cryogenic temperatures sometimes hide conformational states observed at room temperature. Serial femtosecond crystallography（SFX）developed at XFELs demonstrated that room temperature structure analysis is possible even using micro-crystals. Following these successes, serial crystallography is also performed at synchrotron MX beamlines aiming at room temperature structure determination and time resolved structure analysis. In this review, synchrotron serial crystallography（SSX）performed at synchrotron MX beamlines in worldwide is summarized, and development of SSX at SPring-8 MX beamlines is introduced together with ongoing project toward time resolved experiments.

Cryo-electron microscopy（cryo-EM）is a powerful method to visualize high resolution structures of biomolecules under physiological condition. Since cryo-EM images represent conformational ensemble of the target biomolecule, cryo-EM is applicable to analyze the biomolecular dynamics such as metastable conformations and/or free-energy landscape in conformational changes. Here, we briefly introduce our method to deduce free-energy landscape of biomolecules from cryo-EM images and compare it with other existing methods.