Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Summary

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Abstract

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to >10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

Introduction

Serial crystallography (SX) is a technique that was developed initially in the context of X-ray Free Electron Lasers (XFELs)^1,2,3,4. Though fixed target approaches can be used for SX^5,6,7, typically, injector systems are employed to deliver crystals in a continuous stream to the X-ray beam. Because it combines data from a large number of crystals, SX avoids the need for any crystal alignment during the experiment, and enables data to be collected at....

Protocol

1. Preparation of crystals in a high viscous media using glass syringes

1. Centrifuge the crystal solution gently (~1,000 x g, ~10 min at 22 °C) to form a soft crystal pellet and remove the excess buffer. This will result in a high concentration of crystals in the pellet which can be used for data collection.
   NOTE: To prevent dilution of the viscous media increase the crystal concentration at this step. Optimize the ratio of viscous media to crystal volume for each sample to obtain a high concentration of crystals whilst maintaining a high viscosity for the media. Centrifuge the crystal solution to form a pellet and remove excess buffer t....

Results

Lipidico is an HVI built as an alternative delivery system for use on MX2 (Figure 1). It is ideally suited for SX where crystals are either grown in lipidic cubic phase or transferred to a high viscous inert media.

To demonstrate the injector application silicone grease mixed with lysozyme crystals was used to collect SX data at the MX2 beamline at the Australian synchrotron. To mount the injector on the MX2 beamline the cryogenic nozzle is removed and replaced by.......

Discussion

An alternative HVI has been developed, ideal for carrying out SX experiments at synchrotron sources. It has two key advantages over existing HVIs. First, it is easy to install on the beamline allowing rapid switching between conventional crystallography and SX, just ~30 minutes is required for installation and alignment on MX2. Second, the sample syringes used to grow crystals can be directly used as the reservoirs for injection, limiting wastage during sample transfer. The protocol for changing samples has been des.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hen eggwhite lysozyme	Sigma-Aldrich	L6876	Used to grow crystals for testing the injector and the crystals are transferred into silicon grease. https://www.sigmaaldrich.com/
High vacuum silicon grease	Dow Corning	Z273554-1EA	Used for testing of injector. https://www.sigmaaldrich.com/
Injector needle (108 µm ID)	Hamilton	part No: 7803-05	www.hamiltoncompany.com
Glass gas-tight syringes, 100 µl	Hamilton	part no: 7656-01	Syringes used for sample injection. www.hamiltoncompany.com
LCP syringe coupler	Formulatrix	209526	Syringe coupler to mix the samples
Lipidico injector	La Trobe Univerity/ANSTO		This is a specific piece of equipment that can be accessed through La Trobe University / ANSTO Australian Synchrotron Facility