A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine ‘nonmodel’ and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip conceptThe following funding is acknowledged: Agence Nationale de la Recherche (contract No. ANR-11-LABX- 0057_MITOCROSS to Claude Sauter, Bernard Lorber; contract No. ANR-10-LABX-0036_NETRN to Claude Sauter, Bernard Lorber; contract No. ANR-13-BS07-0007-01 to Eric Girard, Sylvain Engilberge); Ministère des Affaires Etrangères (contract No. PROCOPE Hubert Curien to Claude Sauter, Mario Mörl); Deutsche Forschungsgemeinschaft (contract No. Mo 634/10-1 to Mario Mörl, Heike Betat); Université de Strasbourg [grant No. Initiative d’excellence (IDEX) to Claude Sauter, Raphaël de Wijn]; Centre National de la Recherche Scientifique (grant No. MRCT- 2012_PTI_UPR9002 to Claude Sauter)

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods

Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering

International audienceThe reproducible preparation of well-diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus, and the lysozyme from hen egg white in the presence of a synthetic chemical nucleant. We were able to (i) detect early nucleation events and (ii) drive the crystallization system (through cycles of dissolution/crystallization) toward growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction

EXtra-Xwiz: A Tool to Streamline Serial Femtosecond Crystallography Workflows at European XFEL

X-ray free electron lasers deliver photon pulses that are bright enough to observe diffraction from extremely small crystals at a time scale that outruns their destruction. As crystals are continuously replaced, this technique is termed serial femtosecond crystallography (SFX). Due to its high pulse repetition rate, the European XFEL enables the collection of rich and extensive data sets, which are suited to study various scientific problems, including ultra-fast processes. The enormous data rate, data complexity, and the nature of the pixelized multimodular area detectors at the European XFEL pose severe challenges to users. To streamline the analysis of the SFX data, we developed the semiautomated pipeline EXtra-Xwiz around the established CrystFEL program suite, thereby processing diffraction patterns on detector frames into structure factors. Here we present EXtra-Xwiz, and we introduce its architecture and use by means of a tutorial. Future plans for its development and expansion are also discussed

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