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

Abstract

Macromolecular crystallography is a well established method in the field of structural biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now under development towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand-soaking and cryo-protection. These handling steps can cause significant crystal damage, and hence reduce data quality. Furthermore, in time-resolved experiments based on serial crystallography, which use micrometre-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method that combines protein crystallization and data collection in a novel one-step process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg-white lysozyme and crystallization times of only a few seconds. This method, called JINXED (Just IN time Crystallization for Easy structure Determination), promises high-quality data due to the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

Data reduction in protein serial crystallography

“…To capture these challenges, we selected four representative SX datasets covering a range of detector technologies (both counting and integrating), photon sources (including synchrotron and FELs) and varied sample-delivery methods. These methods include tape drive (Henkel et al, 2023;Zielinski et al, 2022), lipidic cubic phase (LCP) jet (Weierstall et al, 2014) and gas dynamic virtual nozzle (GVDN) jet (DePonte et al, 2008). The dataset specifics are enumerated in Table 1.…”

“…Fig. 2 shows the quality metrics CC* and R split versus resolution as well as R free /R work metrics for the different subsets of lactamase data measured during one of the tape-drive (Beyerlein et al, 2017;Henkel et al, 2023;Zielinski et al, 2022) SX experiments at the P11 beamline of PETRA3. The initial dataset consists of 200 000 diffraction patterns and is processed as smaller subsets equivalent to less measurement time (Table 3).…”

Time-resolved crystallography of boric acid binding to the active site serine of the β-lactamase CTX-M-14 and subsequent 1,2-diol esterification

“…The TapeDrive system 26,27,37 was applied to collect serial diffraction data at the beamline P11, PETRA III/DESY, to explore the kinetics and structural intermediates of ligand binding to the β-lactamase CTX-M-14 in a resolution range of 1.40 -2.04 Å and at delay times of 50 -10000 ms. For this purpose, CTX-M-14 microcrystals were mixed with boric acid to initiate the binding process, and diffraction data were collected after distinct pre-set delay times. To monitor the formation of a diester, microcrystals were pre-soaked with boric acid and subsequently mixed with glycerol and again diffraction data were collected after distinct delay times.…”

Biologia strukturalna: historia, teraźniejszość i perspektywy

“…4 Inny pomysł polega na zmieszaniu roztworów białka i czynnika strącającego tuż przed wstrzyknięciem do wiązki promieniowania synchrotronowego lub laserowego. Ponieważ kryształy nie muszą być duże, wystarczy czas krystalizacji rzędu sekundy [5]. Promieniowania rentgenowskiego do badań dyfrakcyjnych kryształów makromolekularnych dostarczają dziś głównie synchrotrony nowej generacji [6].…”