We acknowledge financial support for the project from the French grant REPLIPOX ANR-13-BSV8-0014 and by research grants from the Service de Sant&#233; des Arm&#233;es and the D&#233;l&#233;gation G&#233;n&#233;rale pour l'Armement. We are thankful to the ESRF for the SAXS beam time. We thank Andrew McCarthy, Gordon Leonard, Wim Burmeister, and Guy Schoehn for financial and scientific support.

<ol>
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The authors have nothing to disclose.

For many macromolecules, a final purification step using chromatography is required prior to SAXS data collection to obtain a good quality data set. However, not all samples remain stable; they may be prone to aggregation or re-equilibration to a mixture of oligomerization states. Therefore, a final online purification step on the beamline is required to minimize the time between purification and data collection in order to obtain the best-quality SAXS data. Depending on the biophysical properties of the protein of interest, SEC-SAXS or IEC-SAXS might be chosen to obtain optimal sample quality. Here, on a protein construct derived from the helicase/primase D5, both techniques are explained and discussed.
Acquisition of SEC-SAXS data is becoming more and more standardized and is available on many BioSAXS beamlines. Data analysis, especially background subtraction, is relatively straightforward and easy. However, a stable buffer signal and the sufficient separation of the macromolecular species remains essential. Therefore, it is critical to reserve enough time to equilibrate the column thoroughly. Failure of this method can be due to persistent contaminants of similar size to the protein of interest, low concentrations, and radiation-sensitive buffers.
In practice, initially, SEC-SAXS is likely to be used as the method of choice for most macromolecular samples. Still, many purification protocols require a prior IEC step due to the presence of contaminants or aggregation. Given that each concentration and chromatography step is associated with losses of sample (estimated at 30-50%) and time, direct IEC-SAXS is advantageous. For samples that cannot be purified by SEC, be it due to the presence of similarly sized &#34;contaminants&#34; or because they severely aggregate at the necessary concentrations, IEC-SAXS would always be the better-suited approach. Also, the higher flow rates supported by many IEC columns can help to reduce the transit time between purification and measurement. In the example presented here, IEC was used with a step elution, which allows for the separation of the close peaks of D5323-785 from contaminants by carefully choosing the salt concentration steps. In principle, the number of steps is unlimited, but practically, at least 1 step per peak is required, and not too many should be chosen. For the background subtraction method described above, it is crucial to measure a relatively high number of different buffer compositions in order to find the matching one.
A shared downside of both techniques is the lack of precise protein concentration information. Due to this, precise mass determination based on forward scattering is not possible. For globular proteins such as D5323-785, the Porod volume provides an alternative, albeit less precise, mass estimate, but for highly flexible or disordered proteins, this approach would not be valid.
A variation of the step-wise gradient IEC-SAXS method presented here is the use of a linear gradient instead. While it is possible to work with as many steps as desired to isolate sub-peaks using a step-wise elution, in the linear gradient approach, it is required to optimize the gradient conditions carefully in order to separate the peaks entirely before starting the SAXS experiment. Background subtraction in this approach could be done frame-wise and could be verified by the comparison of the individual frames, but it requires more advanced data handling, and a dedicated software does not exist yet.
The choice of a suitable column is critical for both techniques, as it determines the separation of the macromolecular species. Size-exclusion columns differ in loading capacity, the size range of separable macromolecules, and resolution, while ion-exchange columns vary in the kind and the density of their immobilized charges.
While the protocol presented here is specific to the ESRF beamline BM29, adaptation to any other SAXS beamline is, in principle, straightforward. The main requirements are a sufficiently high X-ray flux and a suitable detector (ideally single-photon-counting), to acquire reasonable signal-to-noise data in the range of seconds or less, and an online liquid chromatography system capable of creating gradients. The exact implementation would, of course, depend on the local beamline environment.
The results obtained on D5323-785 using the two methods differ slightly. The radius of gyration is slightly smaller for the IEC data than for the SEC data, and the local minima of the scattering curve are shifted to slightly larger scattering vectors. This means that the D5323-785 measured with IEC-SAXS is slightly more compact than the D5323-785 measured with SEC-SAXS. This might be due to differences in the sample preparation, in the time between purification and measurement (IEC is faster), or, less likely, to a contaminant in the SEC-purified sample. The bead models obtained completely independently with both methods are comparable (Figure 1H). D5323-785 shows the expected hollow, hexameric structure18.
In conclusion, online ion-exchange and online size-exclusion chromatography are important biochemical purification methods that can be coupled directly to SAXS6,7,9-12,25,26. The background subtraction of IEC-SAXS data is slightly more difficult and ambiguous than for SEC-SAXS, but it is nevertheless possible. Depending on the biophysical properties of the protein of interest, both SEC and IEC-SAXS allow for the optimization of species separation with inherent advantages. Providing that the validation steps (as described) are correctly observed, the resulting data can be analyzed with confidence, and models can be determined using the standard tools available within the community. Together, both techniques allow online separation for a broad range of biological macromolecules, yielding data not accessible via standard static measurements.

BioSAXS is a powerful tool to determine the shape of nano-sized objects1-4. The scattering of X-rays by a solution containing macromolecules, sized in the nm range, is recorded at very low angles. This angular range contains information about global parameters: the radius of gyration; the largest intraparticle distance; the particle shape; and the degree of folding, denaturation, or disorder. The technique does not require crystals, and the macromolecule stays in solution and thus can be kept in conditions mimicking certain important parameters of the cell, such as ionic strength, pH, etc. The knowledge of these factors might help to determine, for example, the physiologically relevant oligomeric state of a protein of interest or to validate a proposed model of a complex. The characterization of protein-protein interactions in different buffer conditions, the creation of models of missing domains, the refinement of homology models, and the determination of discrete folded and unfolded states can be performed quickly and easily5.
As with any technique, BioSAXS has intrinsic weaknesses: aggregated or denatured samples, mixtures of particles, heterogeneous samples, radiation damage, and buffer mismatches may result in un-interpretable data. For many analysis methods, it is implicitly assumed that the sample is monodisperse, a requirement that is often difficult to obtain in practice. In many cases, the degradation of the sample is subtle and cannot be detected in the data on its own, and any attempt to interpret the data gives inaccurate or even misleading results. To overcome these obstacles, the combination of size-exclusion chromatography (SEC) and SAXS was implemented on many beamlines to ensure data quality and to make this technique more accessible for increasingly difficult samples6-11. Recently, we added a new method to the repertoire by developing online ion-exchange chromatography (IEC)-coupled SAXS12. Both techniques are opening SAXS to a wide range of biological particles formerly impossible to analyze. The choice of which method to use depends on the biophysical properties of the particles of interest.
SEC separates macromolecules by their size, whereby at least a 10% difference in apparent molecular mass is needed for the separation. Physical limitations of the column and physiological properties of the samples, like hydrophobic surfaces, flexibility, and lack of stability, also complicate data collection, analysis, and interpretation.
Ion-exchange chromatography, which separates molecules based on their charge and, hence, their binding affinity to the IEC column, can be used instead of or in addition to SEC. The total charge can be readily manipulated by changing the pH, or varying the salt concentration of the buffer, providing a relatively simple method for the controlled elution of the molecules from the IEC column. By using the charge, the separation of similar types and sizes of molecules, which would otherwise be difficult to separate, can be performed routinely with IEC. Additionally, IEC has the advantage of being able to deal with diluted samples, allowing one to avoid the concentration steps, which carry the potential risk of denaturing the protein. Unfortunately, as the charge distribution is highly sample-dependent, IEC requires optimization regarding the pH and the salt concentrations13,14.
For many proteins that are difficult to express, purify, or both, only low quantities of sample are available to study. It is important to be efficient and to minimize the number of purification steps and, therefore, the losses. For this reason, the last purification step is online directly prior to SAXS data acquisition, in order to increase the likelihood of collecting a good data set.
Here, we present and compare online SEC-SAXS and IEC-SAXS. Both techniques were implemented on the BioSAXS beamline BM29 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France15. As a test case, we used the D5N and helicase domain of the vaccinia virus protein D5, which was rather difficult to analyze structurally using other methods. The vaccinia virus is a member of the Poxviridae family and is 98% identical to the variola virus, the cause of small pox. Using the vaccinia system, we study the replication machinery, focusing here on the essential helicase-primase D5.
D5 is a 95-kDa protein with an N-terminal archeo-eukaryotic primase (AEP) domain16 followed by a cysteine cluster region (res. 240-345)16. Further towards the C-terminus comes a D5N domain (res. 340-460), which is always associated with D5-type helicases, and finally a superfamily 3 (SF3) helicase domain (res. 460-785) 17 (Figure 1A). The helicase domain of D5 builds a hexameric ring structure that is needed for tight binding to DNA. Thanks to recent SAXS and EM studies, the low-resolution structures of the primase and the helicase domains are now known18.
Here, we show how to use the implemented online chromatography techniques on the BioSAXS beamline BM29 at ESRF to gain insights into the structure of the C-terminal fragment (residue 323-785) of D5.

<table><tbody><tr><td>1,4-dithiothreitol [DTT]</td><td>Euromedex</td><td>EU0006-B</td><td></td></tr><tr><td>10% Mini-PROTEAN TGX Precast Protein Gel</td><td>Biorad</td><td>4561033</td><td></td></tr><tr><td>2x Phusion Flash PCR Master Mix</td><td>ThermoFisher scientific</td><td>F 548</td><td></td></tr><tr><td>acetic acid glacial</td><td>VWR Chemicals (BDH Prolabo)</td><td>20104.298</td><td></td></tr><tr><td>Agarose D-5</td><td>Euromedex</td><td>LF45130653</td><td></td></tr><tr><td>Amicon Ultra -15 Centrifugal filters Ultracel -30K</td><td>Millipore Ltd</td><td>UFC903024</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Euromedex</td><td>EU0400-D</td><td></td></tr><tr><td>Benzonase</td><td>Novagene</td><td>70750-3</td><td></td></tr><tr><td>bromophenol blue&amp;nbsp;</td><td>MERCK</td><td>8122</td><td></td></tr><tr><td>Complete protease inhibitor cocktail&amp;nbsp;</td><td>Roche</td><td>11231400</td><td></td></tr><tr><td>Econo-Pac 10 DG Desalting column</td><td>Bio-rad</td><td>732-2010</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E-5134</td><td></td></tr><tr><td>Glycerol</td><td>VWR Chemicals (BDH Prolabo)</td><td>24388.295</td><td></td></tr><tr><td>Glycine</td><td>Euromedex</td><td>26-128-6405-C</td><td></td></tr><tr><td>HindIII</td><td>Roche</td><td>656313</td><td></td></tr><tr><td>HisSelect HF Nickel Affinity Gel</td><td>Sigma</td><td>H0537</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>VWR Chemicals (BDH Prolabo)</td><td>20252.295</td><td></td></tr><tr><td>Imidazole</td><td>AppliChem Panreac</td><td>A1073,0500</td><td></td></tr><tr><td>InstantBlue</td><td>Expedeon</td><td>ISB1L</td><td></td></tr><tr><td>LB broth Miller</td><td>Fluka Analytical</td><td>L3152</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ICN Biomedicals Inc.</td><td>191421</td><td></td></tr><tr><td>NaCl</td><td>VWR Chemicals (BDH Prolabo)</td><td>27808.297</td><td></td></tr><tr><td>NcoI</td><td>Fermentas</td><td>ER0571</td><td></td></tr><tr><td>One Shot BL21 Star (DE3)&amp;nbsp;</td><td>ThermoFisher scientific</td><td>C6010-03</td><td></td></tr><tr><td>pProEx HTb vector&amp;nbsp;</td><td>Addgene</td><td>10711018</td><td></td></tr><tr><td>Primer</td><td>&amp;nbsp;Eurofins mwg operon</td><td></td><td></td></tr><tr><td>QIAprep Spin Miniprep kit</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>QIAquick Gel extraction kit</td><td>QIAGEN</td><td>28706</td><td></td></tr><tr><td>QIAquick PCR purification kid</td><td>QIAGEN</td><td>28106</td><td></td></tr><tr><td>SDS&amp;nbsp;</td><td>Sigma</td><td>75746</td><td></td></tr><tr><td>Superose 6 10/300 GL</td><td>GE Healthcare</td><td>17-5172-01</td><td></td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Invitrogen life technology</td><td>S33102</td><td></td></tr><tr><td>T4 ligase</td><td>ThermoFisher scientific</td><td>EL0011</td><td></td></tr><tr><td>TEV</td><td></td><td></td><td>home made</td></tr><tr><td>TOP 10</td><td>Invitrogen life technology</td><td>C404003&amp;nbsp;</td><td></td></tr><tr><td>Tris base</td><td>Euromedex</td><td>26-128-3094-B</td><td></td></tr><tr><td>Uno Q 1R column&amp;nbsp;</td><td>Bio-rad</td><td>720 0011</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>MPBiomedicals, LLC</td><td>194834</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beamline control software BsXCuBE</td><td>ESRF</td><td>Pernot &lt;em&gt;et al.&lt;/em&gt; (2013), J. Synchrotron Rad. 20, 660-664</td><td>local development</td></tr><tr><td>Camserver software</td><td>Dectris</td><td>n.a.</td><td>detector control software</td></tr><tr><td>DAMAVER</td><td>ATSAS 2.6.0</td><td>http://www.embl-hamburg.de/biosaxs/download.html</td><td>program to align ab initio models&amp;nbsp;</td></tr><tr><td>DAMMIF</td><td>ATSAS 2.6.0</td><td>http://www.embl-hamburg.de/biosaxs/download.html</td><td>&lt;em&gt;ab initio&lt;/em&gt; model reconstruction program</td></tr><tr><td>HPLC program Biologic Duo Flow</td><td>Bio-rad</td><td></td><td></td></tr><tr><td>HPLC program LabSolutions</td><td>Shimadzu</td><td>n.a.</td><td></td></tr><tr><td>ISPyB</td><td>ESRF</td><td>De Maria Antolinos &lt;em&gt;et al.&lt;/em&gt; (2015). Acta Cryst. D71, 76-85.</td><td>local development</td></tr><tr><td>PRIMUS</td><td>ATSAS 2.6.0</td><td>http://www.embl-hamburg.de/biosaxs/download.html</td><td>program, which performs the manipulation with experimental SAXS</td></tr><tr><td>PyMOL</td><td>DeLano Scientific LLC</td><td>https://www.pymol.org/</td><td>software for visualization</td></tr><tr><td>SUPCOMB</td><td>ATSAS 2.6.0</td><td>http://www.embl-hamburg.de/biosaxs/download.html</td><td>program to superimpose 3D structures</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioLogic Duo Flow</td><td>Biorad</td><td></td><td></td></tr><tr><td>BioLogic Biofrac Fraction collector</td><td>Biorad</td><td></td><td></td></tr><tr><td>HPLC system</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Labsonic P</td><td>Sartorius Stedim biotech</td><td>BBI8535108</td><td></td></tr></tbody></table>

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 results of the model-free invariant analysis are listed in Table 1. The analysis of the D5323-785 SEC-SAXS data showed a molecular mass estimate (from a Porod analysis) of 345 kDa versus 338 kDa, observed using IEC-SAXS. Both are in agreement with the expected mass of 6 times 53.5 kDa (321 kDa) for a hexamer. The ab initio modeling of both data sets was undertaken with no imposed symmetry (SEC-SAXS: &#967;2 = 0.88, IEC-SAXS: &#967;2 =3.1) and using C6 symmetry (SEC-SAXS: &#967;2 = 1.0, IEC-SAXS: &#967;2 = 4). As the overall fits to the scattering data for both reconstructions are comparable in both cases (Figure 1D and E), C6 symmetry can be assumed. Thus, the model corresponds to a hexagonal cone-like structure with a central channel, which appears partially obstructed (SEC: Figure 1F; IEC: Figure 1G, overlay Figure 1H). An examination of individual models before averaging shows that this obstruction is likely to be an artifact of the averaging process.
<img alt="Figure 1" src="/files/ftp_upload/54861/54861fig1x.jpg" /> 
Figure 1: SAXS data analysis and comparison of D5323-785 (figure adapted from References 12 and 18). A) Schematic domain structure of D5 protein and D5323-785. B) Offline ion-exchange chromatogram with the indication of the three peaks. Orange curve: Absorbance at 280 nm (au), blue curve: percentage of buffer B. Percentages of buffer B at the peaks are indicated. C) Online ion-exchange chromatogram. Color key as in B. Additionally, the measured intensity is indicated in green. The experimental scattering curve and calculated curve of the model obtained by SEC-SAXS D) and IEC E) data analysis of D5323-785. F) Bead model of D5323-785 based on SEC data and G) IEC data. H) Overlay of the SEC and IEC models. The panels in F) to H) were created using a molecular visualization software24. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54861/54861fig1large.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>Data-collection parameters</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument:</td>
			<td>ESRF BM29</td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>0.99</td>
		</tr>
		<tr>
			<td>q-range (&#197;-1)</td>
			<td>0.0032 &#8211; 0.49</td>
		</tr>
		<tr>
			<td>Sample-to-detector distance</td>
			<td>2.864 m</td>
		</tr>
		<tr>
			<td>Exposure time (sec)</td>
			<td>1 per frame</td>
		</tr>
		<tr>
			<td>Concentration range</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>293</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Pilatus 1M (Dectris)</td>
		</tr>
		<tr>
			<td>Flux (photons/s)</td>
			<td>1 &#215; 1012</td>
		</tr>
		<tr>
			<td>Beam size (&#181;m2)</td>
			<td>700 &#215; 700</td>
		</tr>
		<tr>
			<td>Structural parameters for D5323-785, SEC</td>
			<td></td>
		</tr>
		<tr>
			<td>I0 (cm-1) [from Guinier]</td>
			<td>0.0237</td>
		</tr>
		<tr>
			<td>Rg (&#197;) [from Guinier]</td>
			<td>48</td>
		</tr>
		<tr>
			<td>qminRg &#8211; qmaxRg used for Guinier</td>
			<td>0.77 - 1.24</td>
		</tr>
		<tr>
			<td>Dmax (&#197;) [from p(r) ]</td>
			<td>145</td>
		</tr>
		<tr>
			<td>q-range used for p(r) (&#197;-1)</td>
			<td>0.02 - 0.17</td>
		</tr>
		<tr>
			<td>Porod volume Vp (&#197;3) [from Scatter]</td>
			<td>(570 &#177; 5) &#215; 103</td>
		</tr>
		<tr>
			<td>Molecular mass Mr (kDa) [from Vp]</td>
			<td>345</td>
		</tr>
		<tr>
			<td>Calculated monomeric Mr from sequence (kDa)</td>
			<td>321</td>
		</tr>
		<tr>
			<td>Structural parameters for D5323-785, IEC</td>
			<td></td>
		</tr>
		<tr>
			<td>I0 (cm-1) [from Guinier]</td>
			<td>0.386</td>
		</tr>
		<tr>
			<td>Rg (&#197;) [from Guinier]</td>
			<td>46.5 &#177; 0.1</td>
		</tr>
		<tr>
			<td>qminRg &#8211; qmaxRg used for Guinier</td>
			<td>0.44 - 1.29</td>
		</tr>
		<tr>
			<td>Dmax (&#197;) [from p(r) ]</td>
			<td>120</td>
		</tr>
		<tr>
			<td>q-range used for p(r) (&#197;-1)</td>
			<td>0.03 - 0.18</td>
		</tr>
		<tr>
			<td>Porod volume Vp (&#197;3) [from Scatter]</td>
			<td>(577 &#177; 5) &#215; 103</td>
		</tr>
		<tr>
			<td>Molecular mass Mr (kDa) [from Vp]</td>
			<td>339</td>
		</tr>
		<tr>
			<td>Calculated hexameric Mr from sequence (kDa)</td>
			<td>321</td>
		</tr>
	</tbody>
</table>
Table 1: SAXS data parameters. The table summarizes the parameters of SAXS data acquisition and analysis.

Watch this Scientific Journal Video about Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline at JoVE.com

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

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, ...

online-size-exclusion-ion-exchange-chromatography-on-saxs

Nanyang Technological University

Research

JoVE Journal

Biochemistry

17.2K Views.  European Synchrotron Radiation Facility.  De bepaling van de oplossing structuur van een eiwit door kleine hoek röntgen verstrooiing (SAXS) vereist monodisperse monsters. Hier presenteren we twee mogelijkheden te verzekeren minimale vertragingen tussen monstervoorbereiding en data acquisitie: online size-exclusion chromatography (SEC) en online ionenuitwisselingschromatografie (IEC). 

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

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 ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

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 ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.

This paper describes the complete XChem process for crystal-based fragment screening, starting from applying for access and all subsequent steps to data dissemination.

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify ...

Chemistry

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

Neuroscience

Ion-Exchange Chromatography

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on charge. A column is used that is filled with a charged stationary phase on a solid support, called an ion-exchange resin. Strong cation-exchange chromatography preferentially separates out cations by using a negatively-charged resin while strong anion-exchange chromatography preferentially selects out anions by using a positively-charged resin. This type of chromatography is popular for sample preparation, for example in the cleanup of proteins or nucleic acid samples.
Ion-exchange chromatography is a two-step process. In the first step, the sample is loaded onto the column in a loading buffer. The binding of the charged sample to the column resin is based on ionic interactions of the resin to attract the sample of the opposite charge. Thus, charged samples of opposite polarity to the resin are strongly bound. Other molecules that are not charged or are of the opposite charge are not bound and are washed through the column. The second step is to elute the analyte that is bound to the resin. This is accomplished with a salt gradient, where the amount of salt in the buffer is slowly increased. Fractions are collected at the end of the column as the elution occurs and the purified sample of interest can be recovered in one of these fractions. Another technique, such as spectroscopy, may be needed to identify which fraction contains the sample. Ion-exchange chromatography is especially useful in protein studies, to isolate proteins of interest that have a specific charge or size, as size can determine the number of interactions with the resin.
Ion-exchange chromatography is a more general separation technique than affinity chromatography, which is also often used in preparing protein samples, where an antibody is attached to a column to bind one specific analyte. A new affinity column must be purchased for each analyte, while the same type of ion-exchange column, often with different eluting conditions, can be used to clean up many proteins of the same charge. Ion-exchange chromatography can also be used in conjunction with other types of chromatography that separate based on other properties. For example, size-exclusion chromatography separates based on size and could be used before ion-exchange chromatography to choose compounds of only a given size.

Ion-Exchange Chromatography: Principle and Protein Separation

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on ...

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Analytical Chemistry

Size-Exclusion Chromatography with Multi-Angle Light Scattering for Protein Oligomers

Source: Some, D. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59615">Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS).</a> J. Vis. Exp. (2019).
This video describes SEC-MALS, which leverages the abilities of size-exclusion chromatography (SEC) and multi-angle light scattering (MALS). In SEC-MALS, the SEC separates proteins or protein complexes based on their size, and the MALS detector determines the average molecular weight by measuring the intensity of the light scattered by the molecule at different angles.

Source: Some, D. et al., Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS). J. Vis. Exp. ...

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

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