Crystal-on-crystal chips for <i>in situ</i> serial diffraction at room temperature

Ren, Zhong; Ayhan, Medine; Bandara, Sepalika; Bowatte, Kalinga; Kumarapperuma, Indika; Gunawardana, Semini; Shin, Heewhan; Zeng, Xiaobo; Yang, Xiaojing

doi:10.1039/c8lc00489g

Cited by 29 publications

(36 citation statements)

References 37 publications

(58 reference statements)

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“…Monochromatic datasets collected at 100 K were used to obtain an initial model for 2551g3 at 3.1 Å resolution by the molecular replacement method (PHENIX) using the crystal structure of the two-Cys CBCR TePixJ (PDB: 4GLQ) (8) as a search model. To improve the resolution and map quality, we employed an in situ room-temperature (RT) data collection method developed by our laboratory (53), which yielded a complete Laue dataset at 2.7 Å resolution from >800 crystals of 2551g3. Our serial RT crystallography strategy not only alleviates X-ray radiation damage but also evades crystal freezing, particularly beneficial given a high solvent content of ~70% in 2551g3 crystals.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Bandara

Rockwell

Zeng

et al. 2020

Preprint

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Author contributionsXY designed the project; SB, SSM and XZ carried out cloning, purification, and crystallization; SB, XZ, SSM and NR conducted mutagenesis and spectroscopic experiments; SB, XZ, ZR, HS and XY collected monochromatic diffraction data; HS, CW and ZR collected Laue diffraction data; ZR processed the Laue data and carried out singlecrystal spectroscopic experiments; SB and XY determined, refined and analyzed crystal structures; NR carried out phylogenetic analysis; SB, XY, NR, MVM, and JCL interpreted the data; XY, NR, and JCL wrote the paper with inputs from all authors. Abstract (156 words)Cyanobacteriochromes are small, panchromatic photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. The molecular basis of far-red (FR) light perception by cyanobacteriochromes is currently unknown. Here we report the crystal structure of a far-red-sensing cyanobacteriochrome from Anabaena cylindrica PCC 7122, which exhibits a reversible far-red/orange photocycle.The 2.7 Å structure of its FR-absorbing dark state, determined by room temperature serial crystallography and cryo-crystallography, reveals an all-Z,syn configuration of its bound linear tetrapyrrole (bilin) chromophore that is less extended than the bilin chromophores of all known phytochromes. Based on structural comparisons with other bilin-binding proteins and extensive spectral analyses on mutants, we identify key proteinchromophore interactions that enable far-red sensing in bilin-binding proteins. We propose that FR-CBCRs employ two distinct tuning mechanisms, which work together to produce a large batho-chromatic shift. Findings of this work have important implications for development and improvement of photoproteins with far-red absorption and fluorescence. Significance Statement (99 words)Phytochromes are well known far-red-light sensors found in plants that trigger adaptive responses to facilitate competition for light capture with neighboring plants. Red-and farred-sensing are critical to cyanobacteria living in the far-red-enriched shade of plants.Here we report the crystal structure of a far-red-sensing cyanobacteriochrome, a distant cyanobacterial relative of phytochrome. These studies shed insight into the poorly understood molecular basis of far-red-sensing by phytobilin-based photoreceptors.Owing to the deep tissue penetration of far-red light, far-red-sensing photoreceptors offer promising protein scaffolds for developing gene-based photoswitches, optoacoustic contrast agents and fluorescent probes for in situ imaging and optogenetic applications.

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Section: Resultsmentioning

confidence: 99%

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Bandara

Rockwell

Zeng

et al. 2020

Preprint

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“…As a consequence, the crystals are not accessible to any chemical reactant after sealing, preventing, for example, micro-diffusion and other time-resolved experiments. A similar approach for on-chip microbatch crystallization between two micro-structured single-crystalline quartz plates has recently been reported by Ren et al (2018).…”

Section: Introductionmentioning

confidence: 80%

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies

Lieske

Cerv

Kreida

et al. 2019

IUCrJ

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Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, `naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

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“…First, it is not practical to produce and inject an astronomical number of nano or microcrystals of these proteins into an XFEL beam to support data collections of a time series. The current serial protocol used to obtain the MbCO and other data has a diminishingly small yield of useful diffraction data, based on the ratio between the number of diffracting crystals and the total number of crystals produced (Ren et al, 2018). That is to say, the vast majority of the crystals have no chance to diffract X-ray even when they are injected into the X-ray beam.…”

Section: Discussionmentioning

confidence: 99%

“…Such a yield, defined differently from the hit rate, is not sustainable for other photosensitive protein crystals. Several “fixed target” implementations of crystal delivery systems have been reported recently, including one that we have recently proposed (Ren et al, 2018). However, it remains to be seen how effective they are in achieving a higher yield while maintaining the diffraction quality.…”

Section: Discussionmentioning

confidence: 99%

Ultrafast Structural Changes Decomposed from Serial Crystallographic Data

Ren

2019

Preprint

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Direct imaging of ultrafast and subtle structural events during a biochemical 13 reaction, such as a single electronic transition from one atomic or molecular 14 orbital to another, is highly desirable but has been beyond the reach of protein 15 crystallography. It entails the capability of observing changes in electronic 16 distributions at both an ultrafast time scale and an ultrahigh spatial resolution 17 (Itatani et al., Nature 432, 867, 2004). The recent developments in femtosecond 18 serial crystallography at X-ray free electron lasers (XFELs) have brought the 19 achievable temporal resolution within a striking distance. This paper presents 20 the electron density map decomposed from the XFEL data that shows the 21 formation of several 3d atomic orbitals of the heme iron tens of femtoseconds 22 after the laser-triggered photolysis. A key strategy that has enabled the findings 23 here is a numerical deconvolution to resolve concurrent variations in a series of 24 time-resolved electron density maps so that the image depicting an electron 25 transfer event can be isolated as a partial change from the overwhelming 26 presence of the bulk electrons that are not directly involved in bonding. The 27 subtle changes in electron distribution due to a spin crossover can be decoupled 28 from far greater changes due to atomic displacements. Direct observations of 29 electronic orbitals could offer unprecedented mechanistic insights into a myriad 30 of chemical and biochemical reactions such as electron transfer in redox 31 reactions, and formation, rupture, and isomerization of chemical bonds. 32 33

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Crystal-on-crystal chips for in situ serial diffraction at room temperature

Cited by 29 publications

References 37 publications

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

Molecular Basis of Far-red Sensing in Cyanobacteriochrome

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies

Ultrafast Structural Changes Decomposed from Serial Crystallographic Data

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