Name: small and wide angle x-ray scattering studies of biological macromolecules in solution
Uploaded: 2013-01-08T12:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

We would like to thank Dr. Manfred Kriechbaum of Hecus XRS and the Institute of Biophysics and Nanosystems Research at the Austrian Academy of Sciences in Graz, Austria. LL and XW were supported in part by U.S. Department of Energy, under NERI-C Award No. DE-FG07-07ID14889, and U.S. Nuclear Regulatory Commission, under Award No. NRC-38-08-950. The SWAXS instrument is also supported in part by U.S. Department of Energy, under Award No. DE-NE0000325.

1. Preparation of the Sample
<ol>
 <li>Use a needle to remove some of the sample from the sample container.*</li>
 <li>Use the needle to fill the capillary (maximum diameter of 2.2 mm) with sample. The capillary must be filled between 2 and 3 cm from the bottom.</li>
 <li>Close the capillary by melting wax on its tip.</li>
 <li>Unscrew the vacuum sample holder from the system.</li>
 <li>Take the capillary by the fused end (wax end) and insert the un-fused end into the sample holder.</li>
 <li>Place the holder back into the system and screw in place.</li>
</ol>
* Solid samples (including powder samples) can be directly placed in the sample holder (no capillary necessary), whereas liquid samples must be placed in a capillary.
Start-Up of the SWAXS Machine
2. Source Cold Startup Procedure
<ol>
 <li>Close the shutter by pushing the safety shutter button.</li>
 <li>Switch the main power "ON" by pushing the green on button.</li>
 <li>Verify the Emergency "Shut-Off" button is in the extended position.</li>
 <li>Switch the main power on by pushing the green button.</li>
 <li>Verify the interlock LED light is green, indicating all interlocks are ok.</li>
 <li>Wait for the touch screen to load. Once it has loaded, press "R6" on the menu.</li>
 <li>Turn the Standby key to the "ON" position.</li>
 <li>Turn the X-ray Key to the "ON" position. The red X-ray light should light up.</li>
 <li>Wait for the touch screen to read standby levels (30 kV and 0.4 mA).</li>
 <li>Turn the Standby Safety Key to the "OFF" position. Press the "Start Cycle" button on bottom of the touch screen.</li>
 <li>Wait for the touch screen to read nominal power (50 kV and 1 mA). If a different power level is desired, enter the configuration screen by pressing "R4" on the touch screen and entering the desired settings.</li>
</ol>
3. Chiller Procedure
<ol>
 <li>Turn the power switch to the "ON" position on the temperature control panel. A control light and the LED temperature display will light up.</li>
 <li>Turn the power switch to the "ON" position on the chiller.</li>
 <li>Wait until the temperature display reads "OFF." This is standby mode.</li>
 <li>Press and hold the enter button (return symbol) for about 4 sec or until the temperature display indicates an actual temperature.</li>
 <li>To change the temperature, press the up or down arrows. The set value will be shown on the temperature display for approximately 8 sec before it will return to the actual value.</li>
 <li>Press the enter button once the desired set value is displayed. This will store that value.</li>
 <li>Wait until the actual temperature reaches the desired set value.</li>
</ol>
NOTE: If at any time the error "E 01" shows up on the temperature display, follow the Chiller Shutdown instructions, refill the bath unit by pouring purified water into the tank fill, and then perform the chiller Startup procedure again.
4. Shutdown of Chiller
<ol>
 <li>Press "enter" for approximately 4 sec.</li>
 <li>Turn the power switch to the "OFF" position.</li>
</ol>
5. Turning on the Vacuum
<ol>
 <li>Ensure the vacuum door is secured in place.</li>
 <li>Turn the Vacuum 1 and 2 knobs to the "ON" positions.</li>
 <li>Wait until the VAP5 vacuum gauge reads a vacuum level less than 1.5 mbar.</li>
</ol>
6. Detector System Setup
<ol>
 <li>To set up the gas pressure, verify the Main Pressure Gauge of the reduction valve is at least 10 bar.</li>
 <li>Open the Main Valve.</li>
 <li>Slowly open the Operating Pressure Valve until the pressure reaches 8 bar at the Operating Pressure Gauge.</li>
 <li>Open the Second Main Valve</li>
 <li>To adjust gas flow, open the Main Pressure valve by turning the knob to the vertical position on the Gas Control panel.</li>
 <li>Adjust the flow rate with the needle valve until the gauge reads approximately 8 bar.</li>
 <li>Turn the Main Power switch to the "ON" position. The LED light should light up.</li>
 <li>To adjust the high voltage, activate the high voltage by turning the switch to the "ON" position. The LED light should light up.</li>
 <li>Slowly turn the voltage control knob until approximately 3.5kV is reached on the gauge. DO NOT EXCEED 4kV.</li>
</ol>
7. Calibration
<ol>
 <li>The experimental steps above are performed with a sample of known peaks.</li>
 <li>ASA 3.3 is used to obtain graph of intensity vs. channel with 5 peaks.</li>
 <li>Go to Menu &rarr;Options&rarr;Enter channels and corresponding intensities.</li>
 <li>Go to ASA3, "TPF" tab first.</li>
 <li>Change filter, top left 1 mm Nickel (beam filter).</li>
 <li>Set position 32,000 (range 28,000 to 32,000, critical region around 28,000) &rarr; go to position, make sure vacuum is below 5 mbar, make sure the detector is reading between 1k -10K.</li>
 <li>Run for 10 sec for approximately 52 counts per sec.</li>
 <li>Change the energy windows, (or measure the energy window with the sample only, not including the filter case). Set the final run for 10 sec.</li>
 <li>Find the location of the primary beam (channel 233 for example).</li>
 <li>For other runs, change filter again to 2 mm Tungsten (W, beam stopper) and start from 30,000.</li>
 <li>Find the location of all other peaks of the samples.</li>
 <li>Use ImageJ-macro software to calibrate SAXS - making transitions from intensity vs. channel to intensity vs. q (or d spacing) (Figure 1).</li>
 <li>Use ImageJ-macro software to calibrate WAXS - making transitions from intensity vs. channel to intensity vs. q (or d spacing) (Figure 2).</li>
</ol>
8. Software Procedure
<ol>
 <li>Use the software ASA 3.3, to collect the live data: Live Data&rarr; TPF tab&rarr; Save file.</li>
 <li>For EasySWAXS, click on the Device Settings tab.</li>
 <li>Enter the "a", lambda, and d values, as well as the center of gravity.</li>
 <li>Select Point Collimation.</li>
 <li>Select Globular particle type (unless it is a known to be a rod of flat shaped particle).</li>
 <li>Click on the Guinier-Plot tab. An I vs. q2 graph will be displayed.</li>
 <li>Drag the vertical lines to surround a section of approximately linear slope.</li>
 <li>At the bottom of the screen, an R value will be displayed. Next to this there is a validation requirement.</li>
 <li>Continue to drag the vertical lines closer together until the validation value is between 1 and 2. This is subjective. The R value, or Radius of Gyration, obtained is an estimate.</li>
</ol>
9. Source Shutdown Procedure
<ol>
 <li>Verify the safety and fast shutters are closed.</li>
 <li>Turn the Standby key to the "ON" position.</li>
 <li>Wait for the touchscreen to read standby levels.</li>
 <li>Press R5 on the touchscreen.</li>
 <li>Reduce the current to 0 A.</li>
 <li>Reduce the voltage to 0 A.</li>
 <li>Turn the X-ray key to the "OFF" position.</li>
 <li>Switch the main power "OFF" by pushing the red off button.</li>
</ol>

<ol>
	<li>Bernad&#243;, P., Blackledge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology:+Proteins+in+dynamic+equilibrium.">Structural biology: Proteins in dynamic equilibrium.</a> Nature. 468, 1046-1048 (2010).</li><li>Zhang, F., Skoda, M. W. A., Jacobs, R. M. J., Martin, R. A., Martin, C. M., Schreiber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Interactions+Studied+by+SAXS:+Effect+of+Ionic+Strength+and+Protein+Concentration+for+BSA+in+Aqueous+Solutions.">Protein Interactions Studied by SAXS: Effect of Ionic Strength and Protein Concentration for BSA in Aqueous Solutions.</a> J. Phys. Chem. B. 111, 251-259 (2007).</li><li>Maranas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+environment+on+local+dynamics+of+macromolecules.">The effect of environment on local dynamics of macromolecules.</a> Current Opinion in Colloid &amp; Interface Science. 12, 29-42 (2007).</li><li>Stribeck, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-Ray Scattering of Soft Matter. , (2007).</li><li>Mertens, H. D., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+proteins+and+complexes+using+small-angle+X-ray+solution+scattering.">Structural characterization of proteins and complexes using small-angle X-ray solution scattering.</a> J. Struct. Biol. 172 (1), 128 (2010).</li><li>Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+X-ray+and+neutron+scattering+as+a+tool+for+structural+systems+biology.">Small-angle X-ray and neutron scattering as a tool for structural systems biology.</a> Biol. Chem. 391 (7), 737 (2010).</li><li>Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+solution+scattering+(SAXS)+combined+with+crystallography+and+computation:+defining+accurate+macromolecular+structures,+conformations+and+assemblies+in+solution.">X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.</a> Quat. Rev. Biophys. 40, 191-285 (2007).</li><li>Bonini, M., Fratini, E., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAXS+study+of+chain-like+structures+formed+by+magnetic+nanoparticles.">SAXS study of chain-like structures formed by magnetic nanoparticles.</a> Materials Science &amp; Engineering C-Biomimetic and Supramolecular Systems. 27 (5-8), 1377-1381 (2007).</li><li>Falletta, E., Ridi, F., Fratini, E., Vannucci, C., Canton, P., Bianchi, S., Castelvetro, V., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tri-block+copolymer+templated+synthesis+of+gold+nanostructures.">A tri-block copolymer templated synthesis of gold nanostructures.</a> Journal of Colloid and Interface Science. 357 (1), 88-94 (2011).</li><li>Glatter, O., Scherf, G., Schillen, K., Brown, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Poly(ethylene+oxide)+Poly(propylene+oxide)+Triblock+Copolymer+(EO(27)-PO39-EO(27))+in+Aqueous-Solution.">Characterization of a Poly(ethylene oxide) Poly(propylene oxide) Triblock Copolymer (EO(27)-PO39-EO(27)) in Aqueous-Solution.</a> Macromolecules. 27 (21), 6046-6054 (1994).</li><li>Mittelbach, R., Glatter, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+structure+analysis+of+small-angle+scattering+data+from+polydisperse+colloidal+particles.">Direct structure analysis of small-angle scattering data from polydisperse colloidal particles.</a> Journal of Applied Crystallography. 31, 600-608 (1998).</li></ol>

No conflicts of interest declared.

The comparative procedure of the SWAXS system allows for numerous variables to be determined from experimental analysis. The parameters that are attained from the analysis can be used for different purposes according to the sample and experimental setup. SAXS provides information about nano-scale size and shape of the object, whereas WAXS focuses on the atomic and micro-scale structure (e.g. molecular lattice, unit cell dimension symmetry). More specifically, for particles in dilute solutions, SAXS can st...

<table><tbody><tr><td>The System3 Small- and Wide-Angle X-Ray Scattering (SWAXS) Camera</td><td>Hecus XRS and IBN, &lt;br/&gt; Graz, Austria</td><td></td><td></td></tr><tr><td>GNOM </td><td>ATSAS 2.3 package by D. Svergun EMBL-Hamburg</td><td></td><td></td></tr></tbody></table>

small and wide angle x-ray scattering studies of biological macromolecules in solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

SAXS and WAXS altogether can provide structural information of the sample through the following parameters: the radius of gyration, particle size and shape, solution structure factor, specific inner surface and pore size, lattice type and dimension, and electron density. SAXS and WAXS can also be applied to the study of protein dynamics 1.
The structural information of SWAXS experiments is obtained by comparing the experimentally detected spectra and the computational results of the...

Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution | Bioengineering | Video

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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18.1K Views.  Rensselaer Polytechnic Institute. De demonstratie van de kleine en grote hoek X-ray scattering (SWAXS) procedure is van fundamenteel belang in de studie van biologische macromoleculen. Door het gebruik van de instrumenten en procedures van specifieke hoek methoden en voorbereiding, de experimentele gegevens van de SWAXS toont de atomaire en nanoschaal karakterisering van macromoleculen.

Video: Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Biochemistry

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

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.

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.

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

Engineering

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Chemistry

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

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