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Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbileet al.(2021),Lab Chip,21, 4831–4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for bothin situcrystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals. 

Supercooled water droplets are widely used to study supercooled water [1,2], ice nucleation [3-5], and droplet freezing [6-11]. Their freezing in the atmosphere impacts the dynamics and the climate feedback of clouds [12,13], and can accelerate cloud freezing via secondary ice production [14-17]. Droplet freezing occurs at multiple time and length scales [14,18], and is sufficiently stochastic to make it unlikely that two frozen drops are identical. Here we use optical microscopy and X-ray laser diffraction to investigate the freezing of tens of thousands of water microdrops in vacuum after homogeneous ice nucleation around 234–235 K. Based on drop images, we developed a seven-stage model of freezing and used it to time the diffraction data. Diffraction from ice crystals showed that long-range crystalline order formed in less than 1 ms after freezing, while diffraction from the remaining liquid became similar to the one from quasiliquid layers on premelted ice [19,20]. The ice had a strained hexagonal crystal structure just after freezing, which is an early metastable state that likely precedes the formation of ice with stacking defects [8,9,18]. The techniques reported here could help determine the dynamics of freezing in other conditions, such as drop freezing in clouds, or help understand rapid solidification in other materials. 

Abstract Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics. 

Abstract Oligomerization of Pr55^Gagis a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55^Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55^Gagto membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18–33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles’ membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain’s 2D layers during assembly and budding. 

Significance Light-driven rhodopsin proteins pump ions across cell membranes. They have applications in optogenetics and can potentially be used to develop solar energy–harvesting devices. A detailed understanding of rhodopsin dynamics and functions may therefore assist research in medicine, health, and clean energy. This time-resolved crystallography study carried out with X-ray free-electron lasers reveals detailed dynamics of chloride ion–pumping rhodopsin (ClR) within 100 ps of light activation. It shows the dissociation of Cl⁻from the Schiff base binding site upon light-triggered retinal isomerization. This Cl⁻dissociation is followed by diffusion toward the intracellular direction. The results hint at a common ion-pumping mechanism across rhodopsin families. 

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.