Serial macromolecular crystallography at ALBA Synchrotron Light Source

12 pags., 4 figs., 2 tabs. -- Addenda and errata: https://journals.iucr.org/s/issues/2022/03/00/rv5160/rv5160.pdfThe increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 μm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 μm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.The following funding is acknowledged: Ayuda de Atracciony Retencion de Talento Investigador" from the Community of Madrid (scholarship No. 2019-T1/BMD-15552); STC Programof the National Science Foundation through BioXFEL (awardNo. 1231306); the Centre for Applied Structural Discovery(CASD) at the Biodesign Institute at Arizona State University; the Spanish Ministry of Science and Innovation, grants EQC2021-007532-P, PID2020-117028GB-I00, BIO2016-77883-C2-2-

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

15 pags., 6 figs., 1 tab.With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.Financial support from the STC Program of the National Science Foundation through BioXFEL under agreement no. 1231306, NSF ABI Innovations award no. 1565180, and the National Institutes of Health award no. R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687.Peer reviewe

Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals

Effects of self-seeding and crystal post-selection on the quality of Monte Carlo-integrated SFX data

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here

Towards phasing using high X-ray intensity

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed

Towards phasing using high X-ray intensity

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol

Light-induced Trpin/Metout switching during BLUF domain activation in ATP-bound photoactivatable adenylate cyclase OaPAC

Chretien A, Nagel M, Botha S, et al. Light-induced Trpin/Metout switching during BLUF domain activation in ATP-bound photoactivatable adenylate cyclase OaPAC. bioRxiv. Unpublished.**Abstract** The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase fromOscillatoria acuminata(OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in a non-productive conformation. However, FTIR spectroscopic experiments confirm that the non-productive conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the initial events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Initial changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metouttransition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed