Abstract
Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
Highlights
Serial femtosecond crystallography (SFX) is a novel structural biology technique that allows challenging protein structures to be solved from submicrometre/micrometre crystals at room temperature (Chapman et al, 2011)
Most SFX experiments far have been based on protein crystals delivered using a gas dynamic virtual nozzle (GDVN), where the crystals are delivered to the X-ray beam in their mother liquor (DePonte et al, 2008)
We have shown that the agarose medium might be suitable as a general delivery system for SFX of both soluble and membrane protein crystals and that it is compatible with a wide range of crystallization conditions as well as temperatures
Summary
Serial femtosecond crystallography (SFX) is a novel structural biology technique that allows challenging protein structures to be solved from submicrometre/micrometre crystals at room temperature (Chapman et al, 2011). The majority of membrane protein structures solved by X-ray crystallography have been determined from crystals of protein–detergent micelles grown in solution, which have been successfully used for SFX experiments (Chapman et al, 2011; Aquila et al, 2012; Johansson et al, 2012, 2013; Kupitz et al, 2014) These membrane protein crystals were delivered either with the GDVN liquid injector (DePonte et al, 2008), requiring large. The grease mixture (Sugahara et al, 2015) has been used to deliver crystals to the XFEL beam for SFX data collection of soluble model proteins at the SPring-8 Compact Free Electron Laser (SACLA XFEL), while petroleum jelly (Botha et al, 2015) has been used to deliver lysozyme at the Swiss Light Source (SLS) Both of these delivery methods have so far only been demonstrated at ambient pressure and they produce significant and undesirable Debye–Scherrer rings in the region of 3.77–5 A. Monomer is composed of two subunits, and (Schirmer et al, 1985)
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