Christopher Kupitz

High-resolution protein structure determination by serial femtosecond crystallography

Size Matters Less X-ray crystallography is a central research tool for uncovering the structures of proteins and other macromolecules. However, its applicability typically requires growth of large crystals, in part because a sufficient number of molecules must be present in the lattice for the sample to withstand x-ray—induced damage. Boutet et al. (p. 362, published online 31 May) now demonstrate that the intense x-ray pulses emitted by a free-electron laser source can yield data in few enough exposures to uncover the high-resolution structure of microcrystals. A powerful x-ray laser source can probe proteins in detail using much smaller crystals than previously required. Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography

Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

Serial femtosecond crystallography of G protein-coupled receptors.

G Structures G protein–coupled receptors (GPCRs) are eukaryotic membrane proteins that have a central role in cellular communication and have become key drug targets. To overcome the difficulties of growing GPCRs crystals, Liu et al. (p. 1521) used an x-ray free-electron laser to determine a high-resolution structure of the serotonin receptor from microcrystals. The structure of a human serotonin receptor was solved using a free-electron laser to analyze microcrystals. X-ray crystallography of G protein–coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.

Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal. Structural changes during a macromolecular reaction are captured at near-atomic resolution by an x-ray free electron laser. Watching a protein molecule in motion X-ray crystallography has yielded beautiful high-resolution images that give insight into how proteins function. However, these represent static snapshots of what are often dynamic processes. For photosensitive molecules, time-resolved crystallography at a traditional synchrotron source provides a method to follow structural changes with a time resolution of about 100 ps. X-ray free electron lasers (XFELs) open the possibility of performing time-resolved experiments on time scales as short as femtoseconds. Tenboer et al. used XFELs to study the light-triggered dynamics of photoactive yellow protein. Electron density maps of high quality were obtained 10 ns and 1 µs after initiating the reaction. At 1 µs, two intermediates revealed previously unidentified structural changes. Science, this issue p. 1242

Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein.

Visualizing a response to light Many biological processes depend on detecting and responding to light. The response is often mediated by a structural change in a protein that begins when absorption of a photon causes isomerization of a chromophore bound to the protein. Pande et al. used x-ray pulses emitted by a free electron laser source to conduct time-resolved serial femtosecond crystallography in the time range of 100 fs to 3 ms. This allowed for the real-time tracking of the trans-cis isomerization of the chromophore in photoactive yellow protein and the associated structural changes in the protein. Science, this issue p. 725 The trans-to-cis isomerization of a key chromophore is characterized on ultrafast time scales. A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

Macromolecular diffractive imaging using imperfect crystals

The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

A novel inert crystal delivery medium for serial femtosecond crystallography

Viscous sample delivery that decreases the net protein consumed in serial femtosecond crystallography is described. The agarose stream has a low background, is compatible with membrane proteins and can be used at a wide range of temperatures.

Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system

Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.