Name: study of protein dynamics via neutron spin echo spectroscopy
Uploaded: 2022-04-13T12:30:00
Description: Watch this Scientific Journal Video about Study of Protein Dynamics via Neutron Spin Echo Spectroscopy at JoVE.com

This research used resources at the Spallation Neutron Source (BL-15, BL-6, Biology and Chemistry labs), a DOE Office of the Science User Facility operated by the Oak Ridge National Laboratory. This research also used resources at the MLZ-FRM2 reactor Garching (KWS-2, Phoenix-J-NSE) and the JCNS1 at Forschungszentrum J&#252;lich GmbH, Germany. The author acknowledges Dr. Ralf Biehl and Dr. Andreas Stadler for their help with modeling and their contribution to both IgG and MBP proteins research, Dr. Piotr A. &#379;o&#322;nierczuk for NSE data reduction support, Dr. Changwoo Do for support with SANS measurements, and Rhonda Moody and Dr. Kevin Weiss for SNS biochemistry lab support.

The present work characterizes two proteins: a regular human antibody protein IgG, and the intrinsically disordered MBP. The lyophilized form of the proteins was obtained from commercial sources (see Table of Materials).
1. Protein sample preparation
<ol>
	<li>Prepare a 50 mM sodium phosphate + 0.1 M NaCl buffer by weighing and dissolving the respective solid reagents in heavy water (D2O) (see Table of Materials). This is the deut...

NSE spectroscopy delivers a unique and detailed view of the dynamics of proteins, which other spectroscopic techniques cannot produce. Measurements over an extended time scale provide observations of both the proteins&#39; translational and rotational diffusion, as presented here. The segmental dynamics and other internal oscillations reveal themselves as a strong decay of the coherent scattering function S(Q, t) at a short time scale and are well separated from the overall diffusional relaxation processes. The ...

Probing dynamics of soft matter with neutrons 
Investigating the dynamical properties of proteins and peptides is a major part of biophysical research, and many well-developed methods exist today to access a wide range of energy landscapes1. Relating the experimentally revealed dynamics of the proteins to their biological function is a far more difficult task, requiring complex mathematical models and computer-aided dynamics simulations. The importance of neutron spectroscopy for the analysis of protein motions has been emphasized in several well-received and widely recognized studies1,2,3,4,5. Before exploring the diverse energy landscape of internal protein dynamics, a short overview of the dynamical processes in soft matter and how neutrons can access them is required.
The sensitivity of neutrons to isotopic configuration and the type of interactions they display with soft matter makes neutron scattering one of the most versatile investigation techniques6. There is a broad spectrum of correlation length scales and correlation times that neutrons can access, from nuclear excitations and atomic vibrations to collective motions and slow relaxation processes like isotropic rotations and diffusive motions. When investigating the scattered neutrons for their energy transfer, three main interactions can be distinguished: the elastic scattering, in which there is no energy exchange between incoming neutron and particle in the sample; the inelastic scattering, with a large, quantifiable energy exchange between neutron and particle; and the peculiar case of quasi-elastic scattering that designates a very small energy transfer compared to the incident neutron energy1,7. These interactions give precise information about the material investigated and form the theoretical basis of a wide variety of neutron scattering techniques.
In elastic scattering, the detector records the directions of the neutrons as a diffraction pattern, which shows the position of the sample atoms relative to one another. Information about the correlations of atomic positions is acquired (i.e., integrated intensity S(Q) concerning the momentum transfer Q, which pertains to structural information alone). This principle forms the basis of neutron diffraction8.
Complexity arises when the energy transfer is no longer zero due to excitations and internal fluctuations in the sample material. This forms the basis of neutron spectroscopy, in which the scattered neutrons are investigated as a function of both the energy transfer E and the momentum transfer Q. Dynamical and structural information is obtained. Neutron spectroscopy measures the same integrated intensity S(Q) for energy transfer (i.e., velocity change of the neutrons due to sample scattering, S(Q,&#969;) = S(Q, E), which is also referred to as the dynamic structure factor)9.
For calculating the scattering from a material, it is more adequate to use the pair correlation function7,10. In the diffraction case, the static pair correlation function G(r) gives the probability of finding the center of a particle at a given distance r from the center of another particle. The spectroscopy generalizes the static pair correlation function and includes energy/frequency/time in the scattering equation. The pair correlation function G(r) becomes a function of time G(r, t), which may be decomposed into a distinct atom pair correlation function GD(r, t), and a self-correlation function GS(r, t). These describe two types of correlations: pair-correlated motions of atoms that govern the coherent scattering, and self-correlation that governs the incoherent scattering10.
Coherent scattering is the scattering from &#34;the average&#34; and depends on the relative phase of the scattered waves. In the small-angle scattering regime, the scattered neutron waves from different scattering centers (different atoms) interfere constructively (have similar phases), and the collective motion of the atoms is observed with strong intensity enhancement. Coherent scattering essentially describes the scattering of a single neutron from all the nuclei in the sample10.
When no constructive interference occurs between the scattered neutron waves from different centers, a single atom is followed in time, and the self-correlation between the position of the atom at time t = 0 and the same atom at time t is observed. Thus, the information on the relative positions of atoms is lost, and the focus is only on local fluctuations. Scattering from local fluctuations governs incoherent scattering. Incoherent scattering is isotropic, contributes to the background signal, and degrades the signal-to-noise10,11.
Combining all of the above, we distinguish four major neutron scattering processes10: (1) elastic coherent (measures the correlations of atomic positions), (2) inelastic coherent (measures collective motions of atoms), (3) elastic incoherent (contributes to the background, reduces scattering intensity by Debye-Waller factor (DWF) and measures elastic incoherent structure factor (EISF), describing the geometry of diffusive motions in confined geometry, and (4) inelastic incoherent (measures single atom dynamics and self-correlation).
Dynamics processes that neutrons can access in biology range from the damping of low frequency atomic and molecular vibrations, the interaction of solvent molecules with bio-surfaces, and diffusion processes in the hydration layer of macromolecules and confined geometry, to short-range translational, rotational, and tumbling diffusive motions, and protein domains and allosteric motions1. The wide diversity of neutron methods and instruments for measuring protein dynamics is based on how the achromatization of the incident or outgoing neutron beam is achieved and how the energy analysis of the scattered neutrons is performed. From triple-axis to time-of-flight, backscattering, and spin-echo spectrometers, one can explore dynamical processes with characteristic times between 1 x 10-14 s and 1 x 10-6 s (femtoseconds to microseconds)12.
Oak Ridge National Laboratory, with its two renowned neutron sources, the Spallation Neutron Source - SNS13 and the High Isotope Flux Reactor - HFIR14, has one of the best suites of spectrometers for investigating dynamics in bio-materials. Some of the most eloquent examples include the use of the cold neutron chopper spectrometer (CNCS) at SNS15&#160;to investigate the dynamical perturbation of hydration water around green fluorescent protein in solution16 or the sub-picosecond collective vibrations of several proteins17. A recurring problem of inelastic neutron scattering investigations is that some biological processes are too slow to be observed. Without extreme setups that lead to a huge loss of neutron intensity, time-of-flight spectrometers are limited to 10 &#181;eV energy resolution, corresponding to a maximum time scale of ~200 ps10,11. This is not sufficient to observe large-scale motions in proteins. Therefore, instruments with higher energy resolution like the backscattering spectrometers are often needed. Combining the time-of-flight and backscattering techniques has proven powerful for investigating the change in internal dynamics of Cytochrome P450cam (CYP101), an enzyme that catalyzes the hydroxylation camphor18.
Microscopic diffusivity measured by the backscattering spectrometer at SNS-BASIS19 was surprisingly well defined and could be separated into the diffusivity of water (hydration, cytoplasmic, and bulk-like water) and the diffusivity of cell constituents in planarian flatworms, the first living animal to be studied by neutron scattering20. Backscattering is a high-resolution spectroscopic technique, but it is also limited to several &#181;eV = several nanoseconds, while the slow dynamics in biomaterials also manifest as the survival time of correlation between atomic position or spin orientations (e.g., relaxation processes, which regularly happen in the time range of ten to hundreds of nanoseconds).
Neutron spin echo spectroscopy (NSE) is the only neutron scattering technique to reach such high resolution. Unlike other neutron techniques, NSE does not require achromatization of the beam since it uses the quantum mechanical phase of the neutrons, which is their magnetic moments. The manipulation of magnetic moments allows the use of a broad neutron beam wavelength distribution, while the technique is sensitive to very small neutrons velocity changes in the order of 1 x 10-4. NSE has been successfully used to investigate the slow dynamics of proteins in solution for many proteins. Among these many pioneer studies, we acknowledge the study of the segmental flexibility of pig immunoglobulin21; the coupled domain motions in Taq polymerase22; the domain motions in the tetramer of yeast alcohol dehydrogenase23; the change of conformation in phosphoglycerate kinase upon substrate binding3; the activation of domain motions and the dynamic propagation of allosteric signals in the Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein4,24,25; the dynamics of a compact state of mercuric ion reductase26; and the diffusion of hemoglobin in red blood cells27. Two more recent studies in protein dynamics have exposed the flexibility of human antibody Immunoglobulin G (IgG) as an entropic spring28 and the characteristics of solvent contribution to the dynamics of intrinsically disordered myelin basic protein (MBP)5.
The present article explains the basic principles of NSE, the multiple preparatory methods recommended for a thorough protein dynamics investigation, as well as the methodology and the experimental protocol for NSE data acquisition at the NSE spectrometer at SNS, SNS-NSE. The protocol characterizes two proteins: IgG, a regular human antibody protein, and the intrinsically disordered protein MBP. The biophysical implications, the research relevance of the examples, and the limitations of the technique are discussed briefly.
NSE spectroscopy, the method for slow dynamics measurements 
NSE is a polarized technique that uses neutron&#160;time-of-flight to measure the exchange of energy (loss of polarization) due to the quasi-elastic interaction between neutrons and atoms in a sample. At the core of NSE spectroscopy lie two basic principles: (1) the ability of the neutron spin to precess in the magnetic field with a frequency proportional to magnetic strength <img alt="Equation 1" src="/files/ftp_upload/61862/61862eq01.jpg" style="margin: auto;" />, namely the Larmor frequency29, and (b) the spin-echo or Hann echo, representing the manipulation and refocusing of the polarization signal when applying a series of radiofrequency pulses30.
The basics of the NSE process can be summarized in a few simple steps6,11 using Figure 1. (1) The neutron beam produced by the source (position 1) is polarized (position 2), guided, and transported (position 3), and arrives at the entrance of the NSE spectrometer, where it gets rotated by 90&#176; by the first pi-half flipper (position 4). (2) The polarized beam (e.g., neutron magnetic moments) becomes perpendicular to the first magnet&#39;s magnetic field lines (first precession zone, position 5) and starts to precess. (3) At the end of the magnet, neutron spins accumulate a certain precession angle proportional to the magnetic field strength and the time-of-flight spent inside (basically inversely proportional to the neutron velocity). The individual neutron velocities are encoded within their precession angle at the end of the first precession zone. (4) Close to the sample position, the pi-flipper (position 6) reverses the orientation of the spin by 180&#176;, changing the sign of the precession angle. (5) The neutrons interact with the sample&#39;s molecules (position 7) and get scattered. (6) The scattered neutrons enter and precess in the second precession zone (position 8) but become reversed-oriented. (7) Another pi-half flipper (position 9) is used to rotate the orientation of the spin from perpendicular to the horizontal direction. This will stop the precession, translating the precession angle &#966; into polarization proportional to cos(&#966;). (8) The analyzer (position 10) selects the neutrons based on one orientation. If the interaction with the sample is elastic, the neutron&#39;s velocity will not change. The neutrons will spend an identical amount of time flying in the first and second precession zones, and the accumulated precession angles are fully recovered. The full polarization is restored on the detector (position 11) as an echo of the original polarization (i.e., spin-echo). (9) However, in NSE, the scattering is quasi-elastic, so a small energy exchange between neutrons and sample molecules leads to different neutron velocities after scattering by the sample. Due to the different velocities, the neutrons will spend an additional time flying through the second precession zone and will not have properly recovered their precession angle. A partial polarization is retrieved on the detector, and the loss of polarization due to spin relaxation is proportional to the cos-Fourier-transform of the spectral function S(Q, &#969;), the intermediate scattering function F(Q, t). (10) The time parameter of the function F(Q, t) is proportional to the precession magnetic field strength. Scanning the loss of polarization as a function of magnetic field strength yields, therefore, a relaxation function that depends on the dynamical processes within the sample.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61862/61862fig01.jpg" /> 
Figure 1: Photograph of the NSE spectrometer at SNS (SNS-NSE) and neutron fly path schematic with the most important functional components. From right to left: 1 = neutron source; 2 = choppers-bender-polarizer-secondary shutter system; 3 = beam transport guides; 4 = pi/2 flipper for first 90&#176; spin-turn; 5 = first precession zone; 6 = pi flipper for 180&#176; spin-turn; 7 = sample area and sample environment (here, the cryo-furnace is shown); 8 = second precession zone; 9 = pi/2 flipper for second 90&#176; spin-turn; 10 = analyzer; 11 = detector. (Note that portions of 3, as well as 2 and 1 ,are situated behind the blue wall inside shielding; the choppers are replaced by a velocity selector for reactor-based NSE). <a href="https://www.jove.com/files/ftp_upload/61862/61862fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine MBP protein solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1891</td>
			<td>lyophilized powder reconstituted in&#160; D2O</td>
		</tr>
		<tr>
			<td>D2O - heavy water</td>
			<td>Sigma-Aldrich</td>
			<td>Product No. 151882</td>
			<td>liquid</td>
		</tr>
		<tr>
			<td>Dionized water</td>
			<td>in house</td>
			<td>-</td>
			<td>for washing / cleanning cells</td>
		</tr>
		<tr>
			<td>DLS instrument</td>
			<td>Zetasizer Nano ZS, FZ-J&#252;lich</td>
			<td>-</td>
			<td>dynamic light scattering instrument</td>
		</tr>
		<tr>
			<td>Elastic scattering standards</td>
			<td>SNS-NSE, ORNL</td>
			<td>-</td>
			<td>Al2O3&#160; and Graphite powders</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>65350-M</td>
			<td>70% ethanol for cleaning cells</td>
		</tr>
		<tr>
			<td>IgG protein solution</td>
			<td>Sigma-Aldrich</td>
			<td>I4506</td>
			<td>lyophilized powder reconstituted in&#160; D2O</td>
		</tr>
		<tr>
			<td>KWS-2 instrument</td>
			<td>JCNS outstation at the MLZ,&#160; Garching, Germany</td>
			<td>-</td>
			<td>small angle neutron instrument</td>
		</tr>
		<tr>
			<td>Liquinox dish detergent</td>
			<td>Alconox</td>
			<td>-</td>
			<td>Phosphate-free liquid lab glassware cleaner</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Product No.S9390</td>
			<td>disodium phosphate heptahydrate salt</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>Product No.S9888</td>
			<td>sodium chloride salt</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Product No. S9638</td>
			<td>monosodium phosphate monohydrate salt</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Catalog number:&#160;ND-2000</td>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
		</tr>
		<tr>
			<td>Neutron alignment camera</td>
			<td>NeutronOptics, Grenoble</td>
			<td>NOG210222</td>
			<td>100 x 100 mm camera with Sony IMX249 CMOS sensor</td>
		</tr>
		<tr>
			<td>Parafilm M - wax parafilm</td>
			<td>Bemis</td>
			<td>Parafilm M - 5259-04LC PM996</td>
			<td>all-purpose laboratory film in cardboard dispenser</td>
		</tr>
		<tr>
			<td>Phoenix-J-NSE Spectrometer</td>
			<td>JCNS outstation at the MLZ,&#160; Garching, Germany</td>
			<td>-</td>
			<td>neutron spectrometer</td>
		</tr>
		<tr>
			<td>SasView</td>
			<td>https://www.sasview.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SAXSpace, Anton Paar instrument</td>
			<td>FZ-J&#252;lich</td>
			<td>-</td>
			<td>small angle x-ray instrument</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer dialysis membranes</td>
			<td>Thermo Scientific</td>
			<td>88400-88405</td>
			<td>Slide-A-Lyzer mini dialysis devices tubes of 3.5 K MWCO</td>
		</tr>
		<tr>
			<td>SNS Remote Analysis Cluster</td>
			<td>Neutron Science Remote Analysis (sns.gov)</td>
			<td>https://analysis.sns.gov</td>
			<td></td>
		</tr>
		<tr>
			<td>SNS-NSE spectrometer</td>
			<td>ORNL, Oak Ridge, TN, USA</td>
			<td>-</td>
			<td>neutron spectrometer</td>
		</tr>
		<tr>
			<td>Sterile syringe filters</td>
			<td>VWR</td>
			<td>N.A. PN:28145-501</td>
			<td>0.2 &#181;m pore size filters</td>
		</tr>
		<tr>
			<td>Temperature Forcing System (TFS)</td>
			<td>SP Scientific</td>
			<td>Part Number 100004055</td>
			<td>sample environment equipment</td>
		</tr>
		<tr>
			<td>Urea -d4</td>
			<td>Sigma-Aldrich</td>
			<td>Product No. 176087</td>
			<td>deuterated Urea salt</td>
		</tr>
		<tr>
			<td>Viscometer</td>
			<td>FZ-J&#252;lich</td>
			<td>-</td>
			<td>falling ball viscometer</td>
		</tr>
	</tbody>
</table>

study of protein dynamics via neutron spin echo spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

IgG protein from human serum and bovine MBP proteins were reconstituted at high concentrations (~50 mg/mL) in D2O-base buffers. Since the proteins were dissolved in high concentrations, the solutions obtained were crowded proteins solutions. The dynamics investigated using NSE suffer from the crowded environment that the proteins reside in (structure factor interactions and hydrodynamics effects)5,28,39. DLS was perfo...

Watch this Scientific Journal Video about Study of Protein Dynamics via Neutron Spin Echo Spectroscopy at JoVE.com

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

The following protocol presents how to set up a Neutron Spin Echo experiment to measure the intermediate scattering function and investigate the dynamics of proteins in solution. The main advantage of Neutron Spin Echo Spectroscopy is its ability to look at rearrangement of protein domains and sub domains on the pico to nanosecond time scale;which is the range of slow motions in proteins in their nearly natural environment, and in the crowded protein solution. Neutron Spin Echo is also sensitive to isotopic configuration, which allows very specific and targeted studies using contrast matching.Insights into protein domain dynamics is a major part of biophysical research in the ongoing difficult task to relay the proteins'domain motions with their biological functionality. The protocol presented here can be generally applied to any Neutron Spin Echo measurement performed at the SNS-NSE spectrometer, regardless of your choice of samples, if one stays within the realm of soft matter materials. To set up the experiment begin by selecting the thickness of the cell sample loading, based on the concentration of the protein sample, the temperature needed for measurement, and the amount of solution available.Clean the cell with phosphate-free dish detergent, deionized water, and 70%diethanol. Dry the cell in the convection oven. Load four milliliters of protein solution into the cell, and close with caps.Use wax film or any sealant to seal the sample cells. Load four milliliters of dialysis buffer into an identical container as the protein sample and seal. Transport samples to the beam line.Close the shutter, and enter the spectrometer enclosure cave area. Mount the sample cell on the aluminum sample holder by tightening the screws and the holding plates. Mount the graphite sample and aluminum oxide powder sample loaded in an identical container as the protein sample.Place the same holder by gently sliding it into the can of the temperature forcing system. Close the temperature forcing system lid, and set the temperature to the desired value by accessing the interactive screen of the temperature forcing system. Mount the neutron camera for the alignment of the samples to the beam.To collect the Neutron Spin Echo Spectroscopy data, align the sample in the neutron beam using the neutron camera, and the four independent sample apertures. Open the Spallation Neutron Source, Neutron Spin Echo Spectroscopy data collection software, and collect sample statistics by running defraction scans for the desired scattering angles and wavelength. Set up the measurement parameters based on the statistics collected for each sample by editing the measurement macros provided by the Assisting Instrument Scientist.Start scanning by typing the protocol name at the command prompter and acquiring echoes for the sample. Log in to Neutron Sciences Remote Analysis cluster with the ORNL user credentials and press the launch session button. In the user directory, open a terminal window and type the software setup command.Next, type the environment command. Create a folder for the data reduction in the home directory and copy the provided scripts and macros from the shared directory. Edit, rename and save the reduction macro provided accordingly.Type drspine"at the command prompter and press Enter to start the software reduction environment. Type the name of the reduction macro in the command prompter within the software environment, and press Enter. Edit the Python script provided by the Assisting Instrument Scientist with the names of the reduced file data.Edit the function to fit from the library provided. Type the name of the edited script at the command prompter, and press Enter to read, fit, and plot reduced NSE data. The intermediate scattering function measured by NSE for IgG and MBP proteins shows a clear deviation from a simple diffusion-like relaxation process on the short four year time scale, indicating the accessibility of protein internal dynamics by NSE, and the need for a more complex model to describe the observed dynamical processes.The results of intermediate scattering function calculations fitted by atomic modeling of both proteins using models developed by were in excellent agreement with the experimental NSE data. The results proved that slower dynamics observed at longer furrier times can be attributed to the overall translational and rotational diffusion processes, while the dynamics observed at the short time scales can be attributed to the dynamics of protein domains. One important thing to remember is the need of well prepared and clean samples for NSE measurements.A good sample for a Spin Echo has a flip ratio higher than three, which is the ratio between coherent and incoherent scattering of the sample. Also during sample preparation and sample loading into the NSE cells, the sample solution should be kept safe and free from any chemical or biological cross contamination. For safety reasons the entrance in the spectrometer enclosure should only be attempted after the shutter is closed, and an additional half an hour time has been allowed for radiation cooling of the enclosure.Small-Angle Neutron Scattering is recommended to assess the shape and the structure of proteins in a concentrated solution. It can be done before or in parallel with the NSE experiment. Additional methods like dynamic light scattering and viscosity measurements provide information about the translational diffusion and the hydrodynamics interaction within the concentrated protein solution, and are also recommended.

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