Emerging applications of small angle
solution scattering in structural biology

Barnali N. Chaudhuri*
Faculty of Life Sciences and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi, India

Received 22 August 2014; Accepted 5 December 2014
DOI: 10.1002/pro.2624
Published online 16 December 2014

Abstract: Small angle solution X-ray and neutron scattering recently resurfaced as powerful tools to
address an array of biological problems including folding, intrinsic disorder, conformational transitions,
macromolecular crowding, and self or hetero-assembling of biomacromolecules. In addition, small
angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy,
and other structural methods to aid in the structure determinations of multidomain or multicomponent
proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation,
or X-ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for charac-
terizing the organizations of two-component systems such as a nucleoprotein or a lipid-protein assem-
bly. Time-resolved small and wide-angle solution scattering to study biological processes in real time,
and the use of localized heavy-atom labeling and anomalous solution scattering for applications as
FRET-like molecular rulers, are amongst promising newer developments. Despite the challenges in
data analysis and interpretation, these X-ray/neutron solution scattering based approaches hold great
promise for understanding a wide variety of complex processes prevalent in the biological milieu.
Keywords: small angle X-ray scattering; anomalous solution scattering; solution neutron scattering;
solution structure determination

Introduction acterizations of protein/RNA folding, intrinsic disor-
Modern biological applications of small angle X-ray der, conformational transitions, and protein-protein
and neutron solution scattering (SAXS/SANS) or protein-nucleic acid assembling processes (Fig.
include low-resolution shape modeling, and the char- 1).1–5 Even though small angle solution scattering
(SAS) typically requires very pure and mono-
Abbreviations: ASAX, anomalous solution X-ray scattering; disperse sample, no specialized sample treatments
FRET, Fo €rster resonance energy transfer; IDP, Intrinsically like chemical fixation or crystallization are neces-
disordered protein; NMR, Nuclear magnetic resonance; sary, making the use of a range of physiologically
SANS, small angle neutron scattering; SAS, small angle
relevant buffer conditions possible. Furthermore,
scattering; SAXS, small angle X-ray scattering; SAXS-SEC,
small angle X-ray scattering–size exclusion chromatography; small sample volume at 1 to 10 mg/mL concentra-
WAXS, wide angle X-ray scattering. tion, rapid data collection at the synchrotron sites
*Correspondence to: Barnali Chaudhuri, Faculty of Life Sciences (0.5–5 s), practically no size-limitation (depending
and Biotechnology, South Asian University, Akbar Bhawan, upon the experimental setup), several dedicated
Chanakyapuri, New Delhi, India 110021. E-mail: barnalichaud- beam-lines at the synchrotron facilities including those with options for automated high-throughput
Barnali Chaudhuri current address is GN Ramachandran protein
center, CSIR Institute of Microbial Technology, Chandigarh-
data collection and the availability of user-friendly,
160036, India. automated software for data analysis makes SAXS a

Published by Wiley-Blackwell. V
C 2014 The Protein Society PROTEIN SCIENCE 2015 VOL 24:267—276 267

25. Despite the tremendous methodological advan- age molecular shape of a mono-disperse. SAXS ing component within the structure.22. While X-ray crystallography is a routine tool for the wise distance distribution profile computed from atomic resolution structure determinations of bio- SAS data are useful for testing various structural molecules held in crystal lattices.ORG Biological Applications of Small-Angle Solution Scattering . low-resolution shape model. aid in small angle scattering. globular mac.40 provides a quick way to compute a low-resolution. SAXS at ultra-low scattering angles can aid in ary or quaternary organizations. very popular technique.16 On the other the existing substructures to model their overall terti- hand.24 Recently.12 Comparisons of the SAS-derived shape models with corresponding known structures indicated Size and shape of a macromolecule excellent agreements for a number of cases. higher-order struc.13–15 These quantities and the pair. endorsed.27–32 In addition to ab initio at higher scattering angles contains information shape modeling. SAS can be used in conjunction with about its finer structural features. such as the nucleosome fiber. con- actively discussed in the research community.1.13 In ture prediction algorithms.2. cross-sectional size and linear mass density (mass per unit length) of a rod-shaped filament and SAS as a complementary technique in structural thickness of a disk-shaped particle can be estimated biology from SAS data. and multiple ab For the theoretical basis and technical details of initio shape computations with consistent results. guidelines for publications are Impositions of anticipated point-group symmetry. A schematic diagram showing various biological applications of small angle solution scattering technique. radius of gyration. Analysis of SAS data allows ready estimations of methods were developed to model RNA structures using mass. While small angle X-ray/neutron tions in solution.17 structures can help to predict the location of a miss- When a crystal structure is not available.18–21 ularity of SAXS. SAS can readily hypotheses and can be used as restraints for struc. recently published text-book.10.2 Solution scattering profiles are scattering1–4 provides information about the global computed from the atomic co-ordinates of available size and shape of the macromolecule in solution. the elucidation of large-scale.12 In this Due to the inherent ambiguity of SAS-based shape mod- review.26 addition. aver.11 nectivity and compactness conditions. crystal structures for comparisons with the experi- wide-angle X-ray scattering (WAXS) data obtained mental SAXS/WAXS data.2.1. and maximum diameter of experimental SAXS data in conjunction with the struc- a monodisperse macromolecule in solution. mental data that are “not seen by the model” are lems in structural biology. ces in structure determination techniques. reveal their oligomeric states and domain organiza- tural modeling. validations of the model using additional experi- applications of SAXS and SANS in a variety of prob. we refer the readers to an obtaining an average. we will focus our attention on the modern eling.23 excellent.Figure 1. romolecule from its scattering profile.1.5–9 Due to the immense pop.33–39 Furthermore. many 268 PROTEINSCIENCE. SAS-based shape computations with known partial tures.

49–52 Thus.57 In addition.72 High salt conditions are scattering macromolecule. to learn about the structural organizations membrane proteins. q. component.12 ful approach to elucidate the structural organiza. from which individ. The “matching with the changes in sizes and pair-distribution func- out” conditions for lipids. are used to track folding-unfolding DNA/RNA are at about 10 to 14%. unaddressed issues such as bly.multicomponent or multidomain biological entities Scattering with contrast variation aided in elu- continue to pose considerable challenge to structure cidating the organizations of many two-component determination. acids.73 However. which can be changed by tal conditions.57 Neutron scattering with contrast pair-distributions functions are model-free indicators matching is an exceptional tool for observing a selected of large-scale conformational changes in folded. a large amount of sample requirement particular.13. possible to achieve solely based on SAXS.68–72 Moreover.q2 neutron scattering contribution from one of the compo.74 Furthermore. and q is nents can be selectively “matched out” or abolished by the momentum transfer).46–49 In facilities.1. a renewed assembly as well as the cross-term describing their search for additional contrast variation agents for relative orientations in a two-component assembly SAXS that are suitable for biological samples will be can be retrieved. ods are developed to integrate SAS data with other Due to a limited number of neutron scattering experimental data for structural modeling.74 Kratky plots. Thus. and tion profiles.72 Due to the relative ease of obtaining ual scattering profile for each component in the SAXS data in comparison to SANS. and behaviors of biomolecules under varying experimen- 65 to 72% D2O. conformational flexibility. respectively. 47%. Folding.53–57 A typical contrast var.66 Recently. shape or structural modeling. A global indication of “folded-ness” of the bio- In an alternative contrast matching experiment.13. problems. noisier data due to in-coherent scattering and long tions of various biological entities. SANS is useful for studying and SAS. and intrinsic SANS with H/D contrast variation is very well suited disorder for analyzing relative positioning of the components SAS is a powerful global sensor of the folding states within an assembly and an induced-fit or a mutual and conformational changes in proteins and nucleic induced fit mode of molecular recognition. such as crystallography. An effective way to tackle this chal. ing densities between proteins and lipids as well as transmission electron microscopy. SAXS ques can be an effective tool to address structural typically requires only 20 to 30 mL of sample and a problems that are not easily amenable to a single few seconds of exposure at a powerful synchrotron technique.. where I is the scattering intensity. but have limited applicability in biological a number of different H/D ratio. carbohydrates. together adjusting the H/D ratio of the buffer.44 Hybrid meth. NMR with SAS has emerged as a power.4. SAS data collection times (a few hours). which can be combined with ponent in a heteromeric assembly. Although less potent than SANS with H/D contrast variation.67 information about overall shape. 40 to 45%. protein-protein assemblies with a deuterated protein plementary methods. changes in sizes and deuteration.58–64 Owing to the differences in scatter- nuclear magnetic resonance spectroscopy (NMR).4.44. resurgence of in combination with other complementary techni. ces. proteins. sucrose can reduce the effect of iation experiments reveal the relative dispositions of radiation damage at the powerful synchrotron sour- components within a multicomponent assem. a range of two-component assemblies. centers of mass of the two components can be esti- mated from SANS contrast variation dataset. Although the Chaudhuri PROTEIN SCIENCE VOL 24:267—276 269 . distance between the rewarding to the SAS research community. (150 mL or more at 5–10 mg/mL concentration). In comparison. source.57 Thus. mass spectrometry proteins and detergents.53–56 A difference in the neutron scattering enhanced viscosity at higher sucrose concentration length density of hydrogen (H) and deuterium (D) is may limit the utility of these SAXS-based contrast exploited in a SANS contrast variation experiment to variation experiments and care should be taken to vary the excess scattering density or contrast of the account for these effects. relative orienta. romolecules for changing contrasts and the power of interface residues obtained from SAXS. NMR and H/D contrast variation allows SANS to characterize mass spectrometric data to learn about the oligo. such as nucleoprotein assemblies and lenge is to combine information obtained from com. non- component in the presence of another “invisible” com. tion. used by combining SAXS and SANS data was described. effective for SAXS-based contrast variation experi- iation dataset is obtained by collecting SANS data at ments. a hybrid strategy of proteins and their assemblies at data-dependent to model membrane proteins in lipidic environment levels of details. Wang et al. neutron scattering is gradual.41–45 For example. vs. molecule can be obtained from the Kratky plot (I. assemblies.65. aggregated proteins.1. the feasibility of deuterium-labeling the mac- tions of the components and the footprints of buried. which is not meric states of a chemokine CCL5. SAXS with varying amounts of Neutron and X-ray scattering with contrast sucrose provides another avenue to protein contrast variation matching and a limited range of contrast varia- Small angle neutron scattering or SANS contrast var.

and their complexes.96 Furthermore.105 Grishaev et al.81 proteins. SANS study on the intrinsically disordered. both folded tion function or a Porod-Debye plot. other hydrodynamic data analyses. when possible. concentrated conditions inhibit breathing motions in tude structural changes in proteins.99. the SAS data. quaternary structures of certain modular enzymes ordered regions often fold in the presence of a bind. esses and multimeric organizations. particularly in combi. a number of SANS in the presence of these “invisible” contrast- different computational methods were developed to matched.91 Synergy determined the structural organization of lead- between NMR and SAS were exploited in numerous substituted calmodulin-peptide complex using a com- studies of IDPs. SAS was found to be a suit- A large number of naturally occurring proteins able tool to study the effects of crowding on RNA are either disordered or contain long stretches of folding. structural components. of the available donor-acceptor pairs. as the scattering profile different un-labeled proteins as crowding agents represents an ensemble of conformations of the IDP under their “contrast matching” conditions indicated in solution.ORG Biological Applications of Small-Angle Solution Scattering . SAXS-based nation with NMR. which is a tal as a probe was successfully used to measure the clear advantage over crystallography. its function in solution. and are generally classified as crowding promotes compactions in the tertiary and intrinsically disordered proteins (IDP).82.100 On the other hand. which was critical for the correct positioning of tive SAXS and NMR based ensemble analyses.102. a left-skewed pair-distribu.82 An IDP or a flexible protein can be minimal effect of crowding on the protein N.97.75–79 WAXS can sense small ampli.105 Under a sensible approach. However. can be obtained from In addition to the global analysis.80.applications of SAXS are limited to detections of crowding on proteins. SAS provides global information on size and shape ble modeling with SAS data yielded structural but no local structural information on a particular information on the spatial occupancy of glycans in site can be generally obtained. ers that are restricted around the F€orster distance degenerative disorder. SAS analysis of the experimental conditions of low concentrations.84.93 In a recent article.104 In Due to the low information content of SAS and another recent SAXS study.86–89 Ensem.94 SAS. gold cluster-labeled a large number of parameters required for an DNA was used to track conformational changes exhaustive description of an IDP. scattering from the electron-dense gold-component circular dichroism. length of DNA using scattering interference. which is recently reviewed else. remarkable structure. nucleic acids and deuterated proteins. such site-specific details can crystallize.95 WAXS data suggested that large movements. protein parts was contrast matched by sucrose. deuterium-labeled protein N in the presence of two ble for studying these IDPs. SAS in combination with suitable be very instructive. use of labeled samples for SAXS analyses of localized Macromolecular crowding conformational changes.98 SAXS-based studies indicated that unstructured regions. well-defined ity. a much larger range of distances.101 Valu- identified from the global features in the SAS data.103 DNA coupled to gold nanocrys- ble proteins under different conditions. dynamic light scattering and dominated the total scattering.104 270 PROTEINSCIENCE. used SAXS with NMR on SAXS/WAXS and NMR residual dipolar coupling lanthanide-labeled proteins to determine the most measurements. for certain glycosylated proteins that are notoriously difficult to biological applications. able new insights on the effects of crowding on such as the Kratky plot. IDPs is typically complemented with solution NMR. exhaus.86–89 These methods typically involve computational generations of multiple conformers followed by their selections based on their compati. a recent. especially tant but often neglected effect of macromolecular coupled with time-resolved SAXS experiments.70 Unlike the F€orster reso- together with extensive cross-validations. rich elements.92 Bertini et al. were used nance energy transfer or FRET-based molecular rul- to predict conformations of IDPs relevant to neuro. SAXS-based molecular ruler bility with the experimental SAS data. One way to obtain local informa- computational methods can provide potentially unre. will continue to play a pivotal molecular rulers can be potentially used to measure role in providing key structural descriptions of IDPs. bination of sucrose contrast matching. lenge to structural biologists.83 The dis. this ing partners(s) and play crucial role in molecular compaction correlates with higher enzymatic activ- recognition.100 Quite significantly.83 Due to a lack of rigid. molecular recognition proc- SAS provides an elegant way to examine the impor. tion from SAXS is to label the protein with electron- stricted access to the conformational spaces of flexi. characterization of the IDPs pose a chal.99.90 Thus. combining SAS induced by a DNA mismatch repair protein. leading with additional complementary experimental data is to new insights into the repair mechanism.85 and intrinsically disordered. lead- which might be needed for a better explanation of ing to the heavy-atom dominated scattering profile.104 Therefore. conventional where.70 Scattering contribution from the abundant conformers of highly flexible calmodulin.82. SAS is naturally suita.82. unlabeled protein crowding agents that generate an ensemble of protein conformers from realistically mimics the intracellular environment.

111.112 In an ray laser pulses are opening up the first-time possi- extension of this coupled approach. recently published a tallographic results.116 and the protein segment is usable.electron microscopy.12 In recent times. which might one component from the mixture. nated by each of the constituents. such as a “protein-quake. ditional means. while effectively excluding any biological processes.120–124 SANS provides another avenue for time-resolved studies.115 However. models as well as the association constants and ing sites within parvalbumin and the center of mass assembling pathway from the same experi- of the protein. valuable tool for investigating a range of processes For a separable mixture.127 measured using SAXS-SEC in a buffer saturated with Time-resolved SAS is suitable for probing many this peptide ligand. SAXS at a powerful synchrotron source time-scale) as well as fast folding kinetics (second to coupled with in-line size-exclusion column chromatog. 10 to 100 fs X- individual entities while being eluted.104 and contrast matching. may alleviate a biological reaction/process in terms of its constitu- this problem for ASAXS studies. which was consistent with the crys. Computational approaches were developed for which are routinely exploited in multiwavelength extracting individual scattering profiles of the con- anomalous dispersion phasing methods in macromo. cess can be initiated by a number of ways. observe the time progressions of SAXS/WAXS pro- lar recognition processes may become routine. SAXS is the nature and time-scale of the process under one of the very few structural techniques available investigation.114 Sokolova fibrillation pathways that are difficult to study by tra- et al.116–118 Petoukhov et al. 100 ms) and ultra-fast protein movements in the sub- raphy (SAXS-SEC) allows an elegant way to study nanosecond region. powerful syn. which can be judi- Sample heterogeneity pose challenges to routine ciously combined with contrast matching for a two- SAS data analysis that generally assumes stable component system. and WAXS studies can Theoretical calculations predict that the use of metal provide direct mechanistic insights on the course of clusters. However. Above computational approaches involving urements at a tunable-wavelength. and concomitant development of theories.106–109 Strong anomalous solution homo.119 However.”124. Mixed systems albeit at a longer time scale. has been used to a limited set obtained from mixed systems involving weak extent in SAXS. of oligomeric assemblies from condition-dependent tion scattering data at the Fe K edge to determine scattering curves for a variety of user-defined mixed the distance between centers of mass of hemoglobin/ system scenarios. such as ing solution state. powerful synchrotron ces.110 sources equipped with better detectors and better applications of ASAXS-based molecular rulers to methods to trigger an event make it possible to probe intricate conformational changes and molecu. Examples include movements in Chaudhuri PROTEIN SCIENCE VOL 24:267—276 271 . requiring very careful meas. stituents from concentration-dependent SAXS data- lecular crystallography. Data collection capability up to about 100 ps atomic clusters as labels to measure specific distan. such as a monomer with including the slow assembling (second or longer a stable dimer. noninteract. light-triggered proc- low-affinity complex of actin with a peptide ligand was esses.or hetero-oligomers co-existing with mono- X-ray scattering (ASAXS) of terbeium at the LIII mers. scattering from a bility to observe even faster. ment.126 The target biological pro- molecular species of one kind in dilute. such as capsid maturation and scattering from the higher aggregates.125. equilibrium mixtures can be potentially used in con- chrotron X-ray source. modeled intermediate filament assembling path.120–124 Thus. Anomalous scattering properties of metal ions.113.109 Time-resolved SAXS.70 will way from SAXS data by finding out conditions domi- almost certainly become more common in future. files of a variety of evolving systems. such as a gold nanocrystal.107 Makowski et al. time-resolved SAS is a to study these mixed systems. changes upon binding can make simple interpreta- bution due to the metal label to the total scattering tions of SAS data obtained from mixed systems diffi- is typically quite small. Although small anomalous junction with time series of SAXS to provide a rich contribution due to the cross-term between the label variety of information on the mixed system. conformational myoglobin and the metal ion. separation of individual entities in a mixture may not Anomalous solution X-ray scattering be possible or desirable in all cases. recently method for composition analysis and shape modeling used anomalous contribution from iron in the solu. depending upon include weak and transient interactions. scattering contri- bution due to the interference within the anomalous Time-resolved SAS to track biological processes scattering metal component itself is negligible. including the pathway intermedi- explorations of dissimilar anomalous scattering ates. SANS. many interesting biolog.109 Anomalous contri.110 With further ent components. time resolution at the modern. laser light for a light-initiated process (pump-probe) ical phenomena take place in a mixture of or by using a mixing device or by quickly removing interacting particles in solution. cult.116–118 These approaches are original in a sense edge was exploited to measure the mean distance that they allow derivations of low-resolution shape between the terbiums substituting the calcium bind.

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