It is in the lamellar domain that nucleation and crystal growth take place (figure adapted from Li et al. Components of the precipitant solution are proposed to locally stabilize a lamellar domain into which the protein preferentially partitions from the LCP which acts as a reservoir.
( b) Cartoon representation of the T4L-rhodopsin-arrestin complex protein (rhodopsin is in blue, arrestin in green, and T4L is omitted) reconstituted in the LCP of 9.7 MAG, with the rhodopsin embedded in the bilayer of the LCP and the arrestin accommodated in the aqueous channel. Of all the lipids tested, only 9.7 MAG reproducibly facilitated the crystallization of the T4L-rhodopsin-arrestin complex. We thus tested several alternative lipids including 6.9 MAG (Nu Chek), 7.9 MAG (Anatrace), 9.7 MAG, 8.7 MAG (Anatrace) and 10.7 MAG (Nu Chek).
Monoolein (9.9 MAG, Nu Chek), the most widely used host lipid for GPCR crystal growth, was first used as the host lipid for crystallization of the fusion protein with different concentrations of PEG 400 in combination with the StockOption Salt kit (Hampton Research) at various pH levels, but did not support crystal growth of the T4L-rhodopsin-arrestin complex. All-trans-retinal at a molar ratio of 5 retinal:1 protein was added prior to crystallization.LCP crystallization of T4L-rhodopsin-arrestin fusion protein was performed using the monoacylglycerol (MAG) monopalmitolein (9.7 MAG, Nu Chek) containing 10% (w/w) cholesterol as the host lipid. The protein sample was further purified using size exclusion chromatography and was concentrated to about 30 mg/ml for crystallization. In recent years, reports have been increasingly emerging about the successful structural determination by X-ray crystallography, owing to the development and the application of new techniques including protein fusion and the lipidic cubic phase (LCP) crystallization by which protein crystallogenesis occurs in a membrane-mimicking mesophase environment. Crystal structure determination of GPCRs and their complexes has been challenging due to their low expression levels, low stability in detergent micelles, and their recalcitrance to crystallization. Thus, SFX represents an important advancement in protein crystallography.GPCRs comprise a large family of membrane proteins that are involved in many key signal transduction pathways in human physiology, and are targeted by approximately 40% of all approved pharmaceutical drugs. SFX has a high potential for structure determination of challenging proteins such as GPCRs and other membrane proteins that often don’t form crystals of sufficient size for synchrotron data collection. XFEL pulses of shorter than fifty femtosecond duration diffract protein crystals and terminate before significant radiation damage occurs in the protein, thus enabling data collection with reduced radiation damage using a dose higher than tolerable for cryogenically cooled crystals. An XFEL beam delivers extremely intense X-ray laser pulses that allow high resolution diffraction data collection from crystals of micrometer to nanometer size in random orientations.
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Background and SummarySerial femtosecond X-ray crystallography (SFX) is an innovative development for protein structure determination, which uses X-ray free electron lasers (XFELs) as a radiation source to elicit diffraction from crystals. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. To view a copy of this license, visit Metadata associated with this Data Descriptor is available at and is released under the CC0 waiver to maximize reuse.
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