Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

Abstract

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

JINXED: just in time crystallization for easy structure determination of biological macromolecules

“…For the first TapeDrive prototype (Beyerlein et al, 2017;Zielinski et al, 2022), the sample was deposited onto the tape using polished fused silica fibres with inner diameters ranging from 50 to 180 mm and outer diameters of 360 mm. For mixing on the tape, nozzle-in-nozzle assemblies, derived from developments for double flow-focusing nozzles (Oberthuer et al, 2017), were used.…”

“…Sample delivery was performed by the CFEL TapeDrive 2.0 (manuscript in preparation), an updated version of the CFEL TapeDrive (Beyerlein et al, 2017). The general description of sample delivery using a microfluidic controller as described by Zielinski et al (2022) is still valid for TapeDrive 2.0 [see Figs. 1(a) and 2] which was optimized for fast installation at beamlines, ease of use, low sample consumption as well as more accurate tape movement.…”

“…This would solve many challenges such as crystal damage, crystal soaking as well as sample delivery, and open completely new possibilities in time-resolved structural biology and structure-or fragment-based drug discovery. To that end, we developed a method for crystallization on-the-fly using the CFEL TapeDrive (Beyerlein et al, 2017;Zielinski et al, 2022) which yields crystals just in time for easy structure determination of biological macromolecules or JINXED (Just IN time Crystallization for Easy structure Determination). We present here the first structures obtained using JINXED at four different crystallization time points.…”

Introduction to the virtual thematic issue on room-temperature biological crystallography

“…Using X-ray free electron laser (XFEL) radiation they obtained its structure at the 2.25 A ˚resolution that allowed the visualization of the mobile ubiquinone potentially facilitating time-resolved diffraction studies of electron-transfer reactions to this cofactor. Finally, Zielinski and co-workers discussed efficient RT SSX experiments carried out on three different proteins of biological relevance using the CFEL TapeDrive, a conveyor-belt-based sample-delivery system (Zielinski et al, 2022).…”

A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography

“…Like any other experimental technique, X-ray crystallography has evolved and matured, and has re-invented itself over the years. The advent of ultrafast high-brilliance X-ray sources such as free-electron lasers (XFELs) and the latest-generation synchrotrons, in addition to technical developments in continuous sample delivery (Botha et al, 2015;Gru ¨nbein & Kovacs, 2019;Kubo et al, 2017;Lomb et al, 2012;Martin-Garcia et al, 2019;Shimazu et al, 2019;Tono et al, 2015;Weierstall et al, 2012Weierstall et al, , 2014Zielinski et al, 2022), new fastreadout and low-noise detectors (Henrich et al, 2011;Kameshima et al, 2014;Kubo et al, 2017;Leonarski et al, 2018), and new software capable of processing large amounts of data (Grosse-Kunstleve et al, 2002;Kabsch, 2014;Kirian et al, 2011;Nakane et al, 2016;White et al, 2013White et al, , 2016Winter et al, 2018), have enabled the advancement of room-temperature serial crystallography (SX) (Barends et al, 2022;Boutet et al, 2012;Chapman et al, 2011;Dods et al, 2021;Moreno-Chicano et al, 2019;Neutze et al, 2000;Orville, 2020).…”