Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

Abstract

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Microfluidics: From crystallization to serial time-resolved crystallography

“…25,31,33,34,37,143,185,[191][192][193][194][195] The simplest mounting strategy involves the deposition of a crystal slurry between two thin silicon nitride wafers. 143 However, most approaches have utilized microfabricated mesh-type structures.…”

“…This process, along with the removal of excess mother liquor to reduce the background, can be facilitated by the application of vacuum to the mesh structure. 37 Mesh structures have been fabricated out of silicon 25,31,37,192,193 and polymers such as SU-8 ( Figure 8(d)) 191 and polycarbonate (Figure 8(e)). 185,195 The material of the mesh itself should not affect the quality of the observed diffraction, provided that the crystal is well located within the center of the well.…”

“…However, care must be taken when crystalline materials such as silicon or metal are used, as intense diffraction from the mesh structure can potentially damage the detector. 37,191,193 One particular advantage of this type of mesh, or well-based approach, is its ability to insulate crystals in one region of the device from the destructive aspects of the intense XFEL beam when applied to a neighboring well. Such isolation strategies would be critical in adapting any of the aforementioned protein crystallization devices for use as fixed-target XFEL delivery platforms.…”

“…Alternatively, micromachining can be used to fabricate ultra-thin (e.g., 20-50 nm-thick) silicon nitride windows, which provide rigidity, sample stability, and excellent X-ray transmission. 34,191,194 These hard materials have the potential to facilitate the analysis of nanocrystals but are more expensive and labor-intensive to manufacture than polymer films. Ultimately, it should be possible to use atomically thin barrier materials such as graphene for this purpose.…”

A Graphene-Based Microfluidic Platform for Electrocrystallization and In Situ X-ray Diffraction

“…Such platforms have been increasingly harnessed to facilitate the diffraction analysis of challenging targets for both static and dynamic structure determination. Various platforms have been developed to improve the growth and subsequent mounting of tiny and fragile crystals for X-ray diffraction analysis [24][25][26][27][28], including dense array-style devices [29][30][31][32][33][34][35][36][37][38][39][40][41], platforms for the lipidic cubic phase crystallization of membrane proteins [42,43], and thin-film sandwich devices [44]. In the meantime, the challenges of such platforms lie in the need to maintain a protected sample environment, as well as minimize the interference of device materials with the subsequent X-ray analysis.…”