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1. Photoreversible interconversion of a phytochrome photosensory module in the crystalline state

   https://doi.org/10.1073/pnas.1912041116  

   Burgie, E Sethe; Clinger, Jonathan A; Miller, Mitchell D; Brewster, Aaron S; Aller, Pierre; Butryn, Agata; Fuller, Franklin D; Gul, Sheraz; Young, Iris D; Pham, Cindy C; et al (January 2020, Proceedings of the National Academy of Sciences)

   A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ inThermosynechococcus elongatusassembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on F_obs− F_obsdifference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion. 

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2. Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase

   https://doi.org/10.1107/s2052252520012701  

   Lee, Ming-Yue; Geiger, James; Ishchenko, Andrii; Han, Gye Won; Barty, Anton; White, Thomas A; Gati, Cornelius; Batyuk, Alexander; Hunter, Mark S; Aquila, Andrew; et al (November 2020, IUCrJ)

   Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A_2Areceptor (A_2AAR) at 2.0 Å resolution is determined and compared with previous A_2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize. 

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3. Rapid and efficient ambient temperature X-ray crystal structure determination at Turkish Light Source

   https://doi.org/10.1038/s41598-023-33989-0  

   Gul, Mehmet; Ayan, Esra; Destan, Ebru; Johnson, J. Austin; Shafiei, Alaleh; Kepceoğlu, Abdullah; Yilmaz, Merve; Ertem, Fatma Betül; Yapici, İlkin; Tosun, Bilge; et al (May 2023, Scientific Reports)

   Abstract High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish light source (Turkish DeLight). The complete dataset was collected in 18.5 min with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme.Turkish DeLightprovides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage. 

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4. High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

   https://doi.org/10.1107/S2052252519011655  

   Moreno-Chicano, Tadeo; Ebrahim, Ali; Axford, Danny; Appleby, Martin V.; Beale, John H.; Chaplin, Amanda K.; Duyvesteyn, Helen M.; Ghiladi, Reza A.; Owada, Shigeki; Sherrell, Darren A.; et al (November 2019, IUCrJ)

   High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX. 

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5. Serial Femtosecond X-Ray Diffraction of HIV-1 Gag MA-IP6 Microcrystals at Ambient Temperature

   https://doi.org/10.3390/ijms20071675  

   I_Ciftci, Halil; G_Sierra, Raymond; Yoon, Chun Hong; Su, Zhen; Tateishi, Hiroshi; Koga, Ryoko; Kotaro, Koiwai; Yumoto, Fumiaki; Senda, Toshiya; Liang, Mengling; et al (April 2019, International Journal of Molecular Sciences)

   The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cell’s plasma membrane. High-resolution structures of the MA domain determined using cryo X-ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first X-ray free-electron laser (XFEL) study of the HIV-1 MA domain’s interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using an XFEL to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of the MA-IP6 complex both diffracted to beyond 3.5 Å resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and will provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation. 

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