On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Summary

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Abstract

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

Introduction

Due to the ionizing effects of X-ray radiation, protein crystallography, to a great extent, has been limited to cryogenic conditions in the last three decades. Therefore, the current knowledge of protein motions during its function largely arises from comparisons between static structures observed in different states under cryogenic conditions. However, cryogenic temperatures inevitably hinder the progression of a biochemical reaction or interconversion between different conformational states while protein molecules are at work. To directly observe protein structural dynamics at atomic resolution by crystallography, robust and routine methods are needed for conducting....

Protocol

1. Device pre-assembly

1. Label the outer ring (30 mm diameter) for sample identification. If necessary, include the project name, device number, crystallization condition, and date (Figure 1A). Place the outer ring upside down on a clean surface (Figure 1B), and carefully place one quartz wafer inside the ring (Figure 1C). This first quartz wafer serves as an entrance window for the incident X-rays.
2. Pour a small amount of microscope immersion oil (viscosity of 150 cSt) into a Petri dish. Dip a shim in the oil, and make sure that both sides of ....

Results

Several representative datasets have been published In the past few years^10,12 along with the crystallographic results and scientific findings from a diverse range of protein samples, including photoreceptor proteins and enzymes, for example, a plant UV-B photoreceptor UVR8, a light-driven DNA repair photolyase PhrB¹⁰, a novel far-red-light sensing protein from a multi-domain sensory histidine kinase¹⁴, ligand/light.......

Discussion

Protein crystallography in the early years conducted at room temperature experienced tremendous difficulty in battling X-ray radiation damage. Thus, it has been superseded by the more robust cryocrystallography method as synchrotron X-ray sources became readily available²⁰. With the advent of X-ray free-electron lasers, room-temperature protein crystallography has been revived in recent years, with many new developments driven by a desire to observe protein structural dynamics at a physiologically.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Analysis software	In-house developed
Cerium doped yttrium aluminum garnet	MSE Supplies		Ce:Y3Al5O12, YAG single crystal substrates
Chip holder	In-house developed
Control software	In-house developed
Immersion oil	Cargille Laboratories	16482	Type A low viscosity 150 cSt
inSituX platform	In-house developed
IR light source	Thorlabs Incorporated	LED1085L	LED with a Glass Lens, 1085 nm, 5 mW, TO-18
Microscope	Zeiss		SteREO Discovery V8
Outer ring	In-house developed
Petri dish	Fisher Scietific	FB0875713
Pipette	Pipetman	F167380	P10
Pump lasers	Thorlabs Incorporated	LD785-SE400	785 nm, 400 mW, Ø9 mm, E Pin Code, Laser Diode
Raspberry Pi	Raspberry Pi Fundation
Retaining ring	Thorlabs Incorporated	SM1RR	SM1 retaining ring for Ø1" lens tubes and mounts
Seedless quartz crystal	University Wafers, Inc.	U01-W2-L-190514	25.4 mm diameter Z-cut 0.05 mm thickness double side polish 8 mm on -X
Shim	In-house developed
X-ray beam stop	In-house developed