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-

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The g-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergen

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals

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

Development of a new in situ x-ray diffraction technique for characterising embedded nanoparticles

A new non-destructive, high resolution X-ray diffraction technique is developed for the characterisation of ensembles of embedded nanoparticles. The method is based on reciprocal space mapping using an analyser crystal, making it sensitive to very low diffraction contrast between nanoparticles and their surrounds, and capable of encompassing a large volume, representative of the bulk material. The robustness of the technique is demonstrated by its lack of dependence on the X-ray coherence volume and optical stability. In addition, the use of a counting detector provides the necessary high dynamic range, and avoids the restrictions imposed by the finite pixel size of a direct space detector and loss of information due to a beamstop. We review the most widely used techniques for imaging on the nanometre scale and highlight their unique capabilities. We then demonstrate that no single technique alone is sufficient for model independent, non-destructive, nanoscale characterisation of embedded nanoparticles in a bulk material sample. In situ and real-time investigations are imperative for the understanding, and ultimately the control of nanoparticle nucleation and growth in technologically important alloys, colloidal suspensions and various nanomaterial specimens. In this thesis we make significant progress in addressing this crucial omission. We begin by presenting the particulars of scalar diffraction theory that enable us to mathematically describe kinematic diffraction from large ensembles of nanoparticles embedded in a matrix. The requirements of X-ray optics are then discussed, from the pertinent properties of synchrotron X-ray sources through high quality analysing and monochromating crystals. A method of simulating Fraunhofer diffraction and reciprocal space maps from a large, sparse ensemble of weakly diffracting Al-Cu nanoparticles is deduced from elementary coherence considerations. We then demonstrate that quantitative information regarding the nanoparticle ensemble polydispersity can be extracted from the reconstructions of nanoparticles from the Fraunhofer diffraction patterns of numerous such ensembles. In simulated reciprocal space maps we examine the effects of nanoparticle ensemble polydispersity and nanoparticle orientation with respect to the diffraction plane. Experimentally obtained reciprocal space maps of diffracted intensity from nanoparticles in an Al-Cu alloy are then presented, demonstrating the sensitivity of the technique to weakly diffracting embedded nanoparticles and their orientation relative to the diffraction plane. Here we also present the results of an iterative algorithm applied to reconstruct, with <10nm resolution, a two dimensional nanoparticle, representative of the ensemble. Practical considerations for an in situ, real-time X-ray diffraction investigation of the initial growth dynamics of embedded nanoparticles in a bulk material sample are explored in a pilot experiment. Finally, the results from an experimental demonstration of the first, real-time, in situ X-ray diffraction investigation of the early stages of nanoparticle growth (in Al-Cu alloys) are then presented and analysed in the context of clustering and dynamic strain in the sample. Simulations involving a simplified model of local strain are shown to be well correlated with the X-ray diffraction data, and a modal, representative nanoparticle size is determined, which agrees with data from transmission electron micrographs of the sample. In conclusion, current ongoing nanoparticle reconstruction efforts are discussed alongside the future directions suggested as an extension of the nanoparticle characterisation technique

Development of a new in situ x-ray diffraction technique for characterising embedded nanoparticles

A new non-destructive, high resolution X-ray diffraction technique is developed for the characterisation of ensembles of embedded nanoparticles. The method is based on reciprocal space mapping using an analyser crystal, making it sensitive to very low diffraction contrast between nanoparticles and their surrounds, and capable of encompassing a large volume, representative of the bulk material. The robustness of the technique is demonstrated by its lack of dependence on the X-ray coherence volume and optical stability. In addition, the use of a counting detector provides the necessary high dynamic range, and avoids the restrictions imposed by the finite pixel size of a direct space detector and loss of information due to a beamstop. We review the most widely used techniques for imaging on the nanometre scale and highlight their unique capabilities. We then demonstrate that no single technique alone is sufficient for model independent, non-destructive, nanoscale characterisation of embedded nanoparticles in a bulk material sample. In situ and real-time investigations are imperative for the understanding, and ultimately the control of nanoparticle nucleation and growth in technologically important alloys, colloidal suspensions and various nanomaterial specimens. In this thesis we make significant progress in addressing this crucial omission. We begin by presenting the particulars of scalar diffraction theory that enable us to mathematically describe kinematic diffraction from large ensembles of nanoparticles embedded in a matrix. The requirements of X-ray optics are then discussed, from the pertinent properties of synchrotron X-ray sources through high quality analysing and monochromating crystals. A method of simulating Fraunhofer diffraction and reciprocal space maps from a large, sparse ensemble of weakly diffracting Al-Cu nanoparticles is deduced from elementary coherence considerations. We then demonstrate that quantitative information regarding the nanoparticle ensemble polydispersity can be extracted from the reconstructions of nanoparticles from the Fraunhofer diffraction patterns of numerous such ensembles. In simulated reciprocal space maps we examine the effects of nanoparticle ensemble polydispersity and nanoparticle orientation with respect to the diffraction plane. Experimentally obtained reciprocal space maps of diffracted intensity from nanoparticles in an Al-Cu alloy are then presented, demonstrating the sensitivity of the technique to weakly diffracting embedded nanoparticles and their orientation relative to the diffraction plane. Here we also present the results of an iterative algorithm applied to reconstruct, with <10nm resolution, a two dimensional nanoparticle, representative of the ensemble. Practical considerations for an in situ, real-time X-ray diffraction investigation of the initial growth dynamics of embedded nanoparticles in a bulk material sample are explored in a pilot experiment. Finally, the results from an experimental demonstration of the first, real-time, in situ X-ray diffraction investigation of the early stages of nanoparticle growth (in Al-Cu alloys) are then presented and analysed in the context of clustering and dynamic strain in the sample. Simulations involving a simplified model of local strain are shown to be well correlated with the X-ray diffraction data, and a modal, representative nanoparticle size is determined, which agrees with data from transmission electron micrographs of the sample. In conclusion, current ongoing nanoparticle reconstruction efforts are discussed alongside the future directions suggested as an extension of the nanoparticle characterisation technique

SPIND: a reference-based auto-indexing algorithm for sparse serial crystallography data

SPIND (sparse-pattern indexing) is an auto-indexing algorithm for sparse snapshot diffraction patterns (`stills') that requires the positions of only five Bragg peaks in a single pattern, when provided with unit-cell parameters. The capability of SPIND is demonstrated for the orientation determination of sparse diffraction patterns using simulated data from microcrystals of a small inorganic molecule containing three iodines, 5-amino-2,4,6-triiodoisophthalic acid monohydrate (I3C) [Beck & Sheldrick (2008), Acta Cryst. E64, o1286], which is challenging for commonly used indexing algorithms. SPIND, integrated with CrystFEL [White et al. (2012), J. Appl. Cryst. 45, 335–341], is then shown to improve the indexing rate and quality of merged serial femtosecond crystallography data from two membrane proteins, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2 and the NTQ chloride-pumping rhodopsin (CIR). The study demonstrates the suitability of SPIND for indexing sparse inorganic crystal data with smaller unit cells, and for improving the quality of serial femtosecond protein crystallography data, significantly reducing the amount of sample and beam time required by making better use of limited data sets. SPIND is written in Python and is publicly available under the GNU General Public License from https://github.com/LiuLab-CSRC/SPIND