Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Abstract

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

Fig. 1. Working principle of the ‘spitrobot’ and characterization of the vitrification time.

a Crystals are deposited onto micro-meshes; a reaction is initiated via the LAMA method; after a defined delay time reaction intermediates are vitrified in liquid nitrogen. b, c The spitrobot integrates with high-throughput workflows and enables using macroscopic crystals and microcrystals for canonical rotation as well as cryo-SSX data collections. d (I, II) Vitrification delay determined with 250 and 13 µm temperature sensors, respectively, matching the size range of typical samples; (III) an experimental characterization of the vitrification time demonstrates that the glass-transition temperature (160 K) of a typically-sized sample is reached within ~7.5 ms. The total minimal delay for microcrystalline samples is approximately 50 ms. Plunging time is affected by the air-pressure of the piston (indicated in bar).

Fig. 2. Crystallographic assessment of representative time points.

a, b Structural overview of Xylose isomerase (XI) and CTX-M-14 b-lactamase (c) cryo-SSX: XI 2,3-butanediol complex formation after 50 ms and CTX-M-14:avibactam complex formation after 1 s. d, e Single crystals: CTX-M-14^E166A:ampicillin complexes after 0.5, 1, and 5 s, respectively, and XI:glucose complexes after 50 ms, 250 ms, 500 ms, and 1000 ms after reaction initiation. POLDER omit maps are shown at 3.0 r.m.s.d.

Fig. 3. Time-resolved analysis of Tryptophan synthase (TS) turnover.

a Cartoon representation of Tryptophan synthase AB complex with each subunit represented in a different color. b–e Formation of an external aldimine intermediate (Aex1) at different time points (20 s, 30 s) and serine binding (25 s) after mixing. c–e rearrangement of the residues, Lys87 (green arrow) and Gln114 (orange arrow) during serine binding and Aex1 formation in TrpB, after reaction initiation. Active site residues of TrpB are represented as stick and substrates are represented as ball-and-stick. All electron density maps are represented as composite omit maps contoured at 1.0.