LEAPS-INNOV D5.4 - Definition of European-wide standard for fixed target support sample handling

Deliverable no. D5.4 1 Project information Project full title LEAPS pilot to foster open innovation for accelerator-based light sources in Europe Project acronym LEAPS-INNOV Grant agreement no. 101004728 Instrument Research and Innovation Action (RIA) Duration 01/04/2021 – 31/03/2025 Website Deliverable information Deliverable no. Deliverable title Definition of European-wide standard for fixed target support sample handling Deliverable responsible Related WorkPackage/Task WP5, Task 5.4 Type (e.g. Report; other) Report Author(s) Daniele de Sanctis Dissemination level Document Version 1 Date 26/08/2024 Download page Document information Version no. Date Author(s) Comment 1 29/08/2024 D. de Sanctis 2 17/12/2024 E. Plönjes Deliverable title adapted, editing 3 18/12/2024 U. Lächele editing 4 19/12/2024 D. de Sanctis editing Deliverable no. D5.4 2 5 14/01/2025 D. de Sanctis editing Table of Contents Project information ................................................................................................................................. 1 Deliverable information .......................................................................................................................... 1 Document information ............................................................................................................................ 1 Introduction ............................................................................................................................................. 2 Fixed target - Scope of the description ................................................................................................... 3 Fixed target parameters and descriptions .............................................................................................. 4 Conclusion ............................................................................................................................................... 5 Introduction Fixed targets have become the most widely used method for sample delivery in serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFEL) and serial synchrotron crystallography (SSX) at synchrotron light sources. Their popularity is due to their ease of design, adaptation, and adoption, leading to the creation and successful use of a diverse range of fixed-target devices across numerous facilities. However, due to the variety of solutions and ongoing developments, it can be unclear which targets are compatible with specific beamlines or whether a new fixed target can be accommodated. Therefore, some level of standardization was considered to be advantageous . This deliverable aimed to identify potential areas for standardization among fixed targets and to eventually promote best practices for manufacturing and data collection. The primary goals were to facilitate the exchange of fixed targets between different facilities without hindering innovation or imposing restrictions on the specialized hardware currently used at various beamlines or instruments. Achieving consensus on a single, or even a few, cost-effective fixed-target designs that would suit all existing serial experiments would be highly challenging, and most likely detrimental. Instead, here we propose a simple standard descriptor that can detail all aspects of any fixed target relevant to motion during data collection. The goal is to ensure that, with this descriptor provided to beamline software, data collection can be easily implemented. The description was presented and discussed at the “High Rep Rate Fixed Target Delivery: Chip standardisation and workflow” workshop held at European XFEL on 23 January 2023 and included contributions from representative scientists and engineers from European XFEL, European Synchrotron Radiation Facility, European Molecular Biology Laboratory, Paul Scherrer Institute, Diamond Light Source, University of Hamburg, SOLEIL Synchrotron, MAXIV, Deutsches Elektronen-Synchrotron, Helmholtz-Zentrum Dresden-Rossendorf, Australian Nuclear Science and Technology Organisation, Brookhaven National Laboratory, SLAC National Accelerator Laboratory and Lawrence Livermore National Laboratory. Deliverable no. D5.4 3 Fixed target - Scope of the description Figure 1. Outline of the main components of a fixed target support Serial crystallography fixed targets typically consist of four main components: a mounting base, a connecting link, a frame, and an active area (Figure 1). The active area is where the crystals are placed, with the frame holding this area in position. The mounting base serves as the interface with the beamline scanning stages, and the connecting link joins the frame to the base. The link is specified as a separate component because the distance between the mounting base and the active area is crucial for positioning the chip's active area, which can vary across different fixed-target solutions and scanning stage geometries. This aspect is identified as a potential area for future standardization. In most cases, a single mounting base, connecting link, and frame can accommodate various fixed targets (active-area configurations) at a given endstation. An alignment strategy is essential for fixed-target serial crystallography to ensure the accurate positioning of the active area in the x, y, and z axes relative to the X-ray beam during data collection. The combined movement of the translation stages holding the fixed target allows predefined positions of the active area to be moved into the X-ray beam path, ensuring a constant crystal-to-detector distance. Consistent z-axis placement also ensures that the spatial overlap of any laser or droplet ejection used to initiate reactions in time-resolved experiments remains uniform across the entire active area. Fixed-target data collection can be broadly categorized into two modes: 1. Directed Raster: A raster grid is created over a chip where crystals are randomly distributed on the active area. The stages then move sequentially through this grid, with X-rays potentially striking any part of the target. 2. Aperture Aligned: Crystals are placed in defined cavities on the chip's active area, and the stages move sequentially through these defined positions. Deliverable no. D5.4 4 The apertures are aligned with the X-ray beam, ensuring that X-rays will only pass through the apertures. We propose a standardized dictionary to describe both types of fixed-target active areas. Each chip type will have its own unique set of definitions, but these will be formatted in a common file that can be read by either generic or facility-specific software. The descriptor provided aims to fully detail regular arrays, enabling alignment and data collection in a concise, human-readable format. YAML (https://yaml.org) offers a straightforward way to accomplish this, as it can be easily generated from a text editor or command line and interpreted by beamline data-acquisition software. While the descriptor is designed to include all necessary parameters for motion control, it does not provide the details required to design a suitable frame since the beamline software and motion control are independent of this. Fixed target parameters and descriptions The set of geometrical descriptors is represented in Figure 2. Three Fiducials (0,1,2) are needed to pre-orient the fixed target relatively to the beamline coordinate system. The fixed target is organized in City blocks that define the number of compartments. Each City block is structured in Apertures which define the number of features where the crystals are located (Figure2). Table 1 lists all and defines parameters required for aligning and moving a fixed target through the X-ray beam. Figure 2. Schematic of a fixed target with the parameters labelled. Left image shows the entire fixed target, which comprises 64 city blocks (such as A1, A2, …) with outer dimensions of approximately 30 x 30 mm. Each city block contains 400 apertures. There is a fiducial marker for alignment at each corner of the fixed target. Right image highlights a subregion of the fixed target showing a single city block Table 1 Parameters included in the standard description and their definitions. Deliverable no. D5.4 5 Name Key Description Geometry type fixedtarget_geom Geometry type. If absent, square is assumed. Fiducials Fiducial 0 x position Fiducial 0 y position Fiducial 0 z position Fiducial 1 x position Fiducial 1 y position Fiducial 1 z position Fiducial 2 x position Fiducial 2 y position Fiducial 2 z position fixedtarget_F0_x fixedtarget_F0_y fixedtarget_F0_z fixedtarget_F1_x fixedtarget_F1_y fixedtarget_F1_z fixedtarget_F2_x fixedtarget_F2_y fixedtarget_F2_z Distance from fiducial 0 to aperture 0 in x Distance from fiducial 0 to aperture 0 in y Distance from fiducial 0 to aperture 0 in z Distance from fiducial 0 to fiducial 1 in x Distance from fiducial 0 to fiducial 1 in y Distance from fiducial 0 to fiducial 1 in z Distance from fiducial 0 to fiducial 2 in x Distance from fiducial 0 to fiducial 2 in y Distance from fiducial 0 to fiducial 2 in z City blocks Number in x Number in y Spacing in x Spacing in y fixedtarget_BLnum_x fixedtarget_BLnum_y fixedtarget_BLgap_x fixedtarget_BLgap_y Number of city blocks in x Number of city blocks in y Gap between city blocks in x Gap between city blocks in y Apertures Number in x Number in y Spacing in x Spacing in y fixedtarget_APnum_x fixedtarget_APnum_y fixedtarget_APgap_x fixedtarget_APgap_y Number of apertures in x in each block Number of apertures in y in each block Distance between adjacent apertures in x Distance between adjacent apertures in y Conclusion The SPINE collaboration introduced about 20 years ago the definition of the standards for sample supports for macromolecular crystallography. This led to a unification of the data collection protocols, enabling more efficient sample handling across different facilities. This harmonization improved reproducibility, data sharing, and collaboration, leading to more streamlined and accelerated structural biology research. The Definition of a European-wide standard for fixed target support sample handling aimed at a similar goal for the emerging field of serial macromolecular crystallography. Today, fixed target supports are used at many European light sources, most of which are part of the LEAPS collaboration, (https://www.leaps-initiative.eu/) such as European Synchrotron Radiation Facility (ID29), Deutsches Elektronen-Synchrotron (P14-2 and P11), Paul Scherrer Institute (Cristallina at SwissFEL and PXI at SLS), Diamond Light Source (I24), and MAXIV (MicroMAX). Additionally, they can also be deployed at the National Synchrotron Light Source II, Stanford Synchrotron Radiation Lightsource, Linac Coherent Light Source, Advanced Photon Factory in the US, the Laboratório Nacional de Luz Síncrotron in Brazil, ANSTO in Australia and SPring8 in Japan. Most of these facilities share the same support designs. The definition is published in Owen, R. L., de Sanctis, D., Pearson, A. R., & Beale, J. H., “A Deliverable no. D5.4 6 standard descriptor for fixed-target serial crystallography”, Acta Crystallographica. Section D, Structural Biology (2023), https://doi.org/10.1107/S2059798323005429 The description library for Deliverable 5.4 ensures compatibility across light sources, facilitating the sharing and adoption of new designs while laying the foundation for manufacturing with industrial partners. Additionally, a standardized approach to these experiments establishes the basis for a highthroughput strategy in large-scale screening campaigns aimed at developing novel drugs. By studying dynamic molecular behavior at room temperature and capturing these interactions in real time, fixed-target serial crystallography accelerates the drug discovery process and aids in the design of more effective and precise therapeutic compounds. Based on the description library developed in task 5.4 of LEAPS-INNOV, already examples of its application are presented in Orlans et al. “Advancing macromolecular structure determination with microsecond X-ray pulses at a 4th generation synchrotron”, Communications Chemistry, 8(1), 1–12 (2025), https://doi.org/10.1038/s42004-024-01404-y.