A prototype system for dynamically polarized neutron protein crystallography

doi:10.1016/j.nima.2019.06.023

Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment

Volume 940, 1 October 2019, Pages 430-434

https://doi.org/10.1016/j.nima.2019.06.023 Get rights and content

Abstract

The sensitivity of Neutron Macromolecular Crystallography to the presence of hydrogen makes it a powerful tool to complement X-ray crystallographic studies using protein crystals. The power of this technique is currently limited by the relative low neutron flux provided by even the most powerful neutron sources. The strong polarization dependence of the neutron scattering cross section of hydrogen will allow us to use Dynamic Nuclear Polarization to dramatically improve the signal to noise ratio of neutron diffraction data, delivering order of magnitude gains in performance, and enabling measurements of radically smaller crystals of larger protein systems than are possible today. We present a prototype frozen spin system, built at Oak Ridge National Laboratory to polarize single protein crystals on the IMAGINE beamline at the High Flux Isotope Reactor (HFIR). Details of the design and construction will be described, as will the performance of the system offline and during preliminary tests at HFIR.

Section snippets

Introduction and motivation

Macromolecular crystallography is a powerful tool to measure the structure of proteins. This is usually done by measuring the diffraction from a protein crystal in a high intensity X-ray beam. The incredibly high flux of these beams has resulted in the determination of tens of thousands of protein structures. Unfortunately, since X-rays scatter primarily from the high Z atoms in a sample, these structures often lack direct information about the location of the lighter elements, especially

Requirements

The DNP system used for NMC measurements shares aspects from the more commonly seen systems used in medium energy nuclear physics [11], [12], [13] or NMR [14]. In order to not block the acceptance required for diffraction measurements, the system has to run in frozen spin mode. The refrigerator, magnet, microwave, and NMR systems will be described in the following sections, as will the custom sample interface which was required for compatibility with the protein crystal samples to be polarized.

Refrigertator

Operation

Crystals were soaked for 30 to 60 min in a solution containing 100 mM hydroxy-TEMPO and flash frozen (with some of samples first being passed through a glycerol cryo-protectant solution) in liquid nitrogen before being placed in the capillary and stored under liquid nitrogen. In order to ensure that the crystals did not warm during loading, samples were loaded into the refrigerator while it was in standby mode (no helium circulating in the dilution unit, pulse tube at base temperature). After

Performance and results

Over a dozen protein crystal samples were tested at the IMAGINE beam-line over two separate HFIR operating cycles under different conditions (including room temperature tests, cold tests, and polarized tests) in order to first commission the apparatus and the detector, and then measure the effect of the polarization on the diffraction. The samples were hen-egg-white lysozyme and T4-lysozyme, well characterized standard proteins that are typical of the medium unit cell (around $100$ Å) systems

Conclusion and outlook

Tests using the system described in this paper have shown that it is possible to increase the intensity of diffraction from a protein crystal by manipulating the nuclear polarization. The test apparatus built is sufficient to polarize these samples and allow neutron scattering data to be taken, although the data collection time is limited by the relatively high sample temperature in frozen spin mode. Since the tests described were completed, the sample interface has been redesigned to lower the

Acknowledgments

The authors would like to acknowledge the support provided by the HFIR in the installation of the DNP system on the IMAGINE beam-line. The Neutron Sciences Directorate Detector group provided the prototype detector and assisted with the data acquisition. The Jefferson Lab Target Group offered insight into the design of the sample space. The authors also would like to acknowledge the help of all of the IMAGINE instrument team.

Research sponsored by the Laboratory Directed Research and Development

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Exploiting the spin property of neutrons is also envisaged as a means to improve biological neutron crystallography using dynamic nuclear polarization methods. In this approach, described in the chapter by Pierce et al. (2019), a spin labeled protein crystal is cooled below 1 K and subjected to a magnetic field of > 2.5 T, to polarize electrons in the sample, and then irradiated with microwaves (at the Larmor frequency) to transfer this spin polarization to protons. With the neutron beam spin polarized and the protons in the sample polarized the scattering varies significantly when the spins are aligned parallel or antiparallel.
This chapter introduces this topic for the whole volume. It is not a review, rather it presents the basics, the key considerations and forward references to the other chapters. This starts by setting the scene of principles and overall strategy, moves onto planning an experiment including its feasibility and then outlines practicalities with options for the experiment. The crystal structure that results will lead to publication and associated with it, Protein Data Bank deposition.
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Fig. 4A shows a schematic of the system and mode of operation. Detailed technical design specifications and descriptions of the sample space configuration, the sample loading scheme and polarization protocols are described elsewhere (Pierce et al., 2019). Fig. 4B shows the apparatus assembled and installed on the IMAGINE beamline at HFIR.
Dynamic nuclear polarization (DNP) can provide a powerful means to amplify neutron diffraction from biological crystals by 10–100-fold, while simultaneously enhancing the visibility of hydrogen by an order of magnitude. Polarizing the neutron beam and aligning the proton spins in a polarized sample modulates the coherent and incoherent neutron scattering cross-sections of hydrogen, in ideal cases amplifying the coherent scattering by almost an order of magnitude and suppressing the incoherent background to zero. This chapter describes current efforts to develop and apply DNP techniques for spin polarized neutron protein crystallography, highlighting concepts, experimental design, labeling strategies and recent results, as well as considering new strategies for data collection and analysis that these techniques could enable.
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^☆: This manuscript has been authored in part by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

¹: Present address: Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA..

²: Present address: Abilita Bio, Inc, 3210 Merryfield Row, San Diego, CA 92121, USA..

³: Present address: Department of Chemistry, East Tennessee State University, Johnson City, TN 37614, USA..

⁴: Present address: Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China and Songshan Lake Materials Laboratory, Dongguan 523808, China..

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A prototype system for dynamically polarized neutron protein crystallography☆

Abstract

Section snippets

Introduction and motivation

Requirements

Refrigertator

Operation

Performance and results

Conclusion and outlook

Acknowledgments

Arch. Biochem. Biophys.

Nucl. Instrum. Methods Phys. Res. A

Nucl. Instrum. Methods Phys. Res. A

Nucl. Instrum. Methods Phys. Res. A

Nucl. Instrum. Methods Phys. Res. A

Nucl. Instrum. Methods Phys. Res. A

Acta Crystallogr. D Struct. Biol.

Crystallogr. Rev.

Rev. Sci. Instrum.