Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

Abstract

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Recent Advances in Ultrafast Structural Techniques

“…Therefore, SFX measurements in combination with simulations offer a way to characterize the order-to-disorder dynamics, correct for Bragg termination, find optimal X-ray pulse parameters, and perform structural refinement. The success of SFX is highlighted by the determination of a multitude of protein structures from (typically) sub-10 µm crystals [74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92]. It should be mentioned that the necessity of fs-X-ray pulses for attaining high-resolution structures of radiation-sensitive specimens is questionable.…”

A Bright Future for Serial Femtosecond Crystallography with XFELs

“…Most recently, a novel structure of a mosquito larvicide BinAB has been solved by multiple isomorphous replacement with anomalous scattering (MIRAS) using three different derivatives: mercury, gadolinium and iodine [100]. Another approach successfully used for experimental phasing was based on the use of iodine-containing detergent to purify and crystallize membrane proteins, and serve as a source of anomalous signal for SAD, SIR or SIRAS phase retrieval methods [25]. …”

Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

“…In this experiment, bR crystals in bicelle were loaded with a liquid carrier to the droplet injector, which is synchronized to XFEL pulses (Mafuné et al, 2016). We crystallized bR in bicelle, prepared by mixing DMPC/CHAPSO with a precipitant solution composed of 3.2 M NaH 2 PO 4 , 3.5% triethylene glycol and 180 mM 1,6-hexandiol (Faham et al, 2005;Nakane et al, 2016b). bR crystals including the precipitant mixture were ejected from the droplet injector equipped with a 80 mm nozzle.…”