Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

Summary

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

Abstract

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

Introduction

Serial femtosecond crystallography (SFX) is a structural biology technique that exploits the unique properties of X-ray free electron lasers (XFEL) to determine room temperature structures from thousands of micrometer-sized crystals while outrunning most of the radiation damage by the "diffraction before destruction" principle^1,2,3.

In a time-resolved extension of SFX (TR-SFX), the femtosecond pulses from the XFEL are used to study structural changes in proteins^4,5. The protein of i....

Protocol

1. Protein Crystal Sample Preparation

1. About 30 min before the sample is to be injected, load 50 µL of crystal-laden monoolein based LCP in a 100 µL syringe.
2. For injection at atmospheric pressure: Load 10 µL of liquid paraffin into the back of a second syringe. Holding the syringe vertically, expel the air bubbles from the syringe.
   1. For injection into vacuum environment: Load 5 µL of MAG 7.9 and 5 µL of liquid paraffin into the back of a second syringe. Holding the.......

Discussion

The TR-SFX method with the viscous extrusion injector has proven to be a viable technique for structural dynamics studies of bacteriorhodopsin^9,10 and photosystem II¹³ and now seems ready to study proteins driving other photo biological processes such as light-driven ion transport or sensory perception^5,50. The protocols described above were designed to maximize the success of TR-S.......

Materials

Name	Company	Catalog Number	Comments
Mosquito LCP Syringe Coupling	TTP labtech store	3072-01050
Hamilton Syringe 1710 RNR, 100 µl	Hamilton	HA-81065
Hamilton Syringe 1750 RNR, 500 µl	Hamilton	HA-81265
Monoolein	Nu-Chek Prep, Inc.	M-239
7.9 MAG	Avanti Polar Lipids Inc.	850534O
50% w/v PEG 2000	Molecular Dimensions	MD2-250-7
Paraffin (liquid)	Sigma-Aldrich	1.07162
High speed camera	Photron		Photron Mini AX
High magnification lens	Navitar		12X Zoom Lens System
Three axis stage	ThorLabs	PT3/M
Fiber light	Thorlabs	OSL2
Fused silica fiber	Molex/Polymicro	TSP-505375
Lite touch ferrule	IDEX	LT-100
ASU high viscosity injector	Arizona State University		Purchasable from Uwe Weierstall (weier@asu.edu)
HPLC pump	Shimadzu	LC-20AD
Electronic gas regulator	Proportion Air	GP1