Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Summary

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Abstract

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Introduction

From its initial application by Perutz, Kendrew and co-workers in determining the structures of hemoglobin and myoglobin, to the modern high throughput automated pipelines of the structural genomics consortia, macromolecular X-ray crystallography has afforded us an unprecedented structural glimpse into the protein world. This technique remains the most widely applicable experimental method that permits the direct visualization of protein structure at atomic, or near atomic resolution (i.e. in the 1-3 Å range). A prerequisite for X-ray diffraction to be applied to a protein is that it must first be crystallized, and it is this stage of the proce....

Protocol

1. Strategic Considerations

1. The choice of seed-crystals used for microseeding experiments will vary depending on the objective of the experiment. At the beginning of a project it is helpful to find several crystallization hits that can provide alternative starting points for crystal optimization. rMMS greatly reduces the need for crystal optimization because good-quality crystals are more likely to grow in the metastable zone of the phase diagram, i.e. in the region where following disturbance or nucleation the system returns to equilibrium. We therefore suggest using rMMS routinely as soon as the first crystals are obtained (or, more accurately, ....

Results

(A) Example of an rMMS experiment

To demonstrate the effectiveness of rMMS screening we applied this method to the crystallization of hen egg white lysozyme (HEWL) and bovine liver catalase (BLC). Both these enzymes are eminently crystallizable and are structurally well characterized targets ^{15, 16}. As such both provide excellent test subjects with which to illustrate the enhanced crystallization success rate achievable with rMMS. Crystallization experiments were established in 96-well.......

Discussion

In this paper we have described a general method for rMMS protein crystallization screening. We have demonstrated using two test proteins a significant enhancement in crystallization success rate using this method. Diffraction analysis using synchrotron radiation of a subset of crystals generated using rMMS and non-rMMS methods revealed little variation in diffraction quality between crystals grown using either method, although previous authors have reported that good quality crystals are more likely to grow in rMM.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MRC 96 well crystallization trays	Molecular Dimensions Ltd	MD11-00-100	Non-UV compatible, for screens established by robot
ClearView sealing sheets	Molecular Dimensions Ltd	MD6-01S
Hen egg white lyzozyme	Sigma-Aldrich	L6876	~95% purity
Bovine liver catylase	Sigma-Aldrich	C9322	>95% purity
Xylanase	Hampton Research	HR7-104
Thaumatin from Thaumatococcus daniellii	Sigma-Aldrich	T7630
Thermolysin from Bacillus thermoproteolyticus rokko	Sigma-Aldrich	P1512
JCSG-plus HT-96 screen	Molecular Dimensions Ltd	MD1-40	For screens established by robot
PACT premier HT-96 screen	Molecular Dimensions Ltd	MD1-36	For screens established by robot
Morpheus HT-96 screen	Molecular Dimensions Ltd	MD1-47	For screens established by robot
Crystal Ph–nix liquid handling system	Art Robbins Instruments	602-0001-10
Seed bead kit	Hampton Research	HR2-320
Binocular stereo microscope	Leica	M165C
Scalpel blades	Sigma-Aldrich	S2646-100EA
ErgoOne 0.1-2.5 μl pipette	Starlab	S7100-0125
ErgoOne 2-20 μl pipette	Starlab	S7100-0221
ErgoOne 100-1000 μl pipette	Starlab	S7100-1000
JCSG-plus screen	Molecular Dimensions Ltd	MD1-37	For screens established by hand
PACT premier screen	Molecular Dimensions Ltd	MD1-29	For screens established by hand
Morpheus screen	Molecular Dimensions Ltd	MD1-46	For screens established by hand
Tweezers	Sigma-Aldrich	T5415-1EA
CrystalClene coverslips 18 mm	Molecular Dimensions Ltd	MD4-17
2 ml glass Pasteur pipettes	Sigma-Aldrich	Z722669
Vortex mixer	Fisher Scientific	02-215-360
24 well XRL crystallization tray	Molecular Dimensions Limited	MD3-11	For screens established by hand
30% (w/v) PEG 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.5
20% (w/v) PEG 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5
20% (w/v) PEG 6000, 100 mM citric acid pH 5.0