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Biodesign
MINI REVIEW P 18-24
Structure determination of biological
macromolecular complexes by small-angle
X-ray scattering (SAXS) combined with
validating tools
Donghyuk Shin, Seungsu Han and Sangho Lee*
Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea. *Correspondence: [email protected]
The molecular world in a cell is operated by the action of biological macromolecular complexes in solution. The past couple
of decades have witnessed an incredible advance in our understanding of atomic details of biological macromolecular
complexes, mainly driven by X-ray crystallography. Despite the wealth of structural information, crystallographic structure
determination of the biological complexes still remains challenging mostly because such complexes often resist to be
crystallized. Small-angle X-ray scattering (SAXS) is emerging as an alternative to provide structural information for the
biological macromolecular complexes at low resolution in solution. SAXS is advantageous in that it does not require
crystalline state of the complexes and that multiple conformational states and/or conformational changes can be observed
in solution. Combined with pre-existing high-resolution structures of components of the macromolecular complexes, SAXS
can be used to build a testable molecular model for the complex. The structural model driven by the SAXS data can then
be validated by biophysical, biochemical and cellular techniques. Here we attempt to review recent advances in application
of SAXS to uncover the molecular basis of macromolecular complexes. Such a hybrid approach equipped with SAXS and
complementary validation tools may be proven useful to obtain structural insights into the function of biological complexes
when no high-resolution structure determination techniques such as crystallography, NMR and electron microscopy are
applicable.
INTRODUCTION
Getting to know structure of a biological molecule can
open a new way to understand and modulate the function
of the molecule. Structural biology, mainly driven by X-ray
crystallography for the past decades, has made tremendous
contributions to modern molecular biology (Shi, 2014).
Elucidation of individual macromolecules spanning from the
historic DNA structure to numerous protein structures shed
enormous light on the molecular mechanisms of such biological
molecules (Watson and Crick, 1953). Although many biological
molecules such as proteins and nucleic acids can work on
their own, most biological processes are mediated by the
action of macromolecular complexes, some notable examples
being ribosomes and complexes involved in transcription and
intracellular signaling (Fields et al., 2015). Revealing molecular
structures of such macromolecular complexes rely on highresolution techniques such as X-ray crystallography and cryoelectron microscopy (cryoEM). However, both crystallography
and cryoEM have some limitations: crystallography requires the
formation of diffraction-quality crystals, which often becomes
the major bottleneck. By contrast, cryoEM does not require any
crystals, but current technology makes it challenging to study
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complexes under 300 kDa in size and demands large-scale
computation to determine the molecular structure. In addition,
a sample must be frozen to be investigated. Small-angle X-ray
scattering (SAXS) provides a complementary tool for investigating
the structures of macromolecular complexes without altering the
sample state.
In a SAXS experiment, structural information can be derived
from a sample in solution state without size restriction. Since
SAXS is a solution-based technique, it can be utilized to
probe structural dynamics. SAXS for studying biological
macromolecules, often called bioSAXS, has been recently
employed in structural biology (Putnam et al., 2007).
Representative examples for SAXS-based structural studies
include inter-domain movement of a multi-domain protein
(Huang et al., 2013) and conformational change of a nucleic
acid (Hura et al., 2013). BioSAXS, however, imposes its own
technical limitation: it basically provides low-resolution structural
information. Primary structural information obtained by bioSAXS
is a molecular envelope analogous to a low-resolution envelope
by cryoEM. Computational modeling is an essential step to fit a
molecular-level structure into the envelope derived by bioSAXS.
Here we attempt to review recent developments to uncover
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Donghyuk Shin, Seungsu Han and Sangho Lee
biological macromolecular complex structures by combining
bioSAXS with supporting experimental techniques.
BIOSAXS FOR BIOLOGICAL
MACROMOLECULAR COMPLEXES
SAXS measures radial scattering of biological macromolecules
in solution near the beam center, thereby named “small-angle”.
Isotropic, radially symmetric profiles of scattering are recorded
as a function of the scattering vector
where θ is the half of the scattering angle and λ the wavelength
of the incident X-ray beam. Basic parameters one can obtain
at the conclusion of data acquisition include radius of gyration,
Rg, and pair distribution function P(r) from which the maximum
distance of a sample, Dmax, can be derived. Molecular mass of
the sample can be also determined by extrapolating scattering
intensity at the zero scattering angle I(0), which can in turn
be utilized to determine oligomeric state of a macromolecule
or stoichiometry of a macromolecular complex. Once basic
parameters are determined, molecular envelope of the sample
can be modelled using a variety of computational algorithms
implemented in SAXS software packages, the most popular
one being ATSAS (Petoukhov et al., 2012). Readers are referred
to excellent reviews on principles of SAXS and computational
modelling methods (Mertens and Svergun, 2010; Putnam et al.,
2007).
Although theories of SAXS have been developed for a
few decades, practical application of SAXS to biological
macromolecules became routine in the advancement of
synchrotron facilities with bright X-ray beams and instrumentation
to deal with automation and sample handling at beamlines.
Synchrotron X-rays much brighter than conventional in-house
X-rays enable data acquisition of biological samples done in
less a second, extended to millisecond scale. One of problems
in measuring SAXS for biological macromolecules is that the
biological macromolecules often exhibit polydispersity and are
only transiently stable in solution. Introduction of a “in-line” or “in
situ” chromatographic system gives researchers opportunities
to separate biological samples into distinct, monodisperse
state in solution so that a sample at a specific physicochemical
state in solution can be investigated using SAXS. For instance,
a protein or protein complex sample is fractionated on a size
exclusion chromatography column that is connected to an
automatic sample loading device at a SAXS beamline. In this
way, the macromolecular sample is subject to SAXS experiments
FIGURE 1 I Overview of the structure determination of macromolecular complexes by bioSAXS. Three main stages in the bioSAXS structure
determination are described. For details, refer to the text.
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Structure determination of biological macromolecular complexes by small-angle X-ray scattering (SAXS) combined with validating tools
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FIGURE 2 I Schematic diagram of bioSAXS molecular envelope derivation and model fitting. The general procedure of molecular envelope derivation
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from bioSAXS data and model fitting is shown for the dimeric form of an intramolecular tandem coiled coil as an example. Part of the figure is reprinted
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as it is being separated in situ. By establishing automatic
sample loading, data acquisition and analysis system, more
conditions and sample types are investigated in a very efficient
way. More bioSAXS synchrotron beamlines are now streamlined
with automatic sample changers and in-line chromatographic
systems, some of them even having stopped-flow system for
kinetic studies. Advancement of X-ray free electron laser (XFEL)
facilities with even brighter X-ray beams than current synchrotron
facilities opens up new opportunities for bioSAXS because the
very bright and coherent properties of XFEL are ideal for probing
dynamic conformational changes of macromolecular samples at
single-molecule level (Li et al., 2015).
Determination of biological macromolecular complexes using
bioSAXS consists of three stages (Figure 1). The first step is
to obtain high-quality samples. Conventional recombinant
protein expression systems such as bacterial, insect and
mammalian cell culture systems are used to express individual
component proteins or a whole protein complex. For the
study of protein:nucleic acid complexes, nucleic acids can be
chemically synthesized. Modern chromatographic platforms
can be employed to purify the components and/or the whole
complexes to the highest homogeneity. Except successful
expression of a whole complex by co-expresssion or a polycistronic system, complex formation using individual component
molecules and subsequent purification and characterization of
the formed complex should be performed. Upon the conclusion
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of the purification of the macromolecular complex of interest,
characterization of the complex in solution should be carried
out to ensure the complex formed in vitro exhibits all the desired
characteristics such as correct stoichiometry, functionality and
structural homogeneity. In the second stage, bioSAXS data
collection and processing will produce key parameters such as
Rg, Dmax and P(r) (“Data collection and processing” and “Guinier
plot” in Figure 2). Molecular envelope for the macromolecular
complex can be derived by inverse Fourier transform. Multiple
ab initio envelope models of a target macromolecular complex
can be generated by either DAMMIN or DAMMIF from the
ATSAS package (Franke and Svergun, 2009; Svergun, 1999)
(“Molecular envelope” in Figure 2). Typically 10-20 envelope
models are generated. The best ab initio envelope model is
selected based on the similarity among the envelope models
generated by comparing normalized-spatial-discrepancy (NSD)
values calculated by DAMAVER (Volkov and Svergun, 2003):
the envelope model with the lowest NSD is chosen as the
final envelope model for the macromolecular complex under
investigation. One should note that output files from DAMAVER
such as damfilt.pdb and damaver.pdb should not be used as the
final envelope model. These files represent filtered and averaged
models, respectively, mainly for providing insights on the overall
feature of the shape of the complex. However, neither of them
is meant to be consistent with the experimental scattering data.
Rigid body fitting of structural models of the components in the
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Donghyuk Shin, Seungsu Han and Sangho Lee
TABLE 1 I Examples of macromolecular complex structures determined by bioSAXS
Macromolecules
Complex
Protein:protein
Protein:nucleic
acid
Validation of SAXS model
References
Approach
Techniques used
Biophysical and
biochemical assay
Pull-down assay, SEC
To validate whether proteins interact
(Ahn et al., 2015;
Kuwabara et al., 2015)
ITC, SPR, BLI
To measure the strength of their interactions
and identify key residues in binding
(Kuwabara et al., 2015)
AUC
To measure molecular weight of
macromolecule complex
(Kosek et al., 2014;
Pietras et al., 2013)
Functional assay
Enzymatic assay
To confirm key residues in the integrity of
the protein complex functionally
(Pietras et al., 2013)
Biophysical and
biochemical assay
Pull-down assay, SEC
To confirm whether the complex is form
in solution
(Chaix et al., 2010;
Hammel et al., 2010;
Mallam et al., 2011;
Meier et al., 2013)
ITC, SPR, BLI
To quantitate the contribution of key
residues in binding
(Kim et al., 2011;
Kulczyk et al., 2012;
Patel et al., 2012)
EMSA
To confirm the interaction of protein with
nucleic acid
(Cordeiro et al., 2011;
Hammel et al., 2010;
Patel et al., 2012)
NMR
To obtain structural information of the
complex at atomic level
(Cordeiro et al., 2011;
Meier et al., 2013;
Ozawa et al., 2013)
Enzymatic assay
To confirm key residues in nucleic acid
recognition
(Mason et al., 2014;
Matot et al., 2012;
Nowak et al., 2013)
Functional assay
Purpose
Abbreviations used: SEC, size exclusion chromatography; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; BLI, bio-layer interferometry; AUC, analytical ultracentrifugation; EMSA, electrophoretic mobility shift assay; and NMR, nuclear magnetic resonance.
macromolecular complex to the final envelope model follows
to yield a testable structural model for the complex (“Model
fitting” in Figure 2). Structural information of the component
molecules can come from available high-resolution structures
from databases such as protein data bank and various modelling
algorithms for molecules with unknown experimental structures.
Once a structural model for the complex is established, validation
of the model at multiple levels should be done. Firstly, quality of
the structural model for the complex should be checked against
SAXS raw data. Algorithms such as CRYSOL (Svergun et al.,
1995) from the ATSAS package and FoxS server (SchneidmanDuhovny et al., 2013) can be employed for this purpose (“Model
fitting” in Figure 2). Technically validated structural models are
then subject to functional and biological validation steps. A
typical approach is to generate a series of mutants identified
from the structural model that seem to be critical in the structural
integrity and/or functionality of the complex. Those mutants are
subsequently investigated for their effects using biochemical,
biophysical and molecular cell biological assays. One should
refine the initial structural model for the complex based on the
validation results. We will cover some examples of biological
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macromolecular complex structure determination by bioSAXS in
the following sections (Table 1).
SAXS AS A MODEL BUILDING TOOL FOR
BIOLOGICAL MACROMOLECULE COMPLEX
Proteins are key players for almost every aspect of cellular
activities. Complexes consisting of proteins often mediate
very complicated biological processes with astonishingly
high efficiency and accuracy. Here are some good examples
that SAXS revealed the structure of protein:protein complex
in solution. CTNNBL1 and CDC5L are the key components
of non-snRNP spliceosome complex. SAXS studies on these
molecules revealed that CTNNBL1 exists as dimer in solution at
physiological NaCl concentration and that CTNNBL1 and CDC5L
interact with each other to form a hetero-tetramer in solution
(Ahn et al., 2015). Apoptosis signal-regulating kinase 1 (ASK1),
regulated by thioredoxin (TRX1), plays an important role in the
pathogenic diseases. Solution structure of the complex of TRX
binding domain in ASK1 (ASK1-TBD) with TRX1 was resolved by
SAXS. This structural model suggests that ASK1-TBD binds to
TRX1 via its N-terminal domain (Kosek et al., 2014). Regulator
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Structure determination of biological macromolecular complexes by small-angle X-ray scattering (SAXS) combined with validating tools
of ribonuclease activity A (RraA) is known to regulate DEAD box
RNA helicase (RhlB) in Escherichia coli. SAXS was used to solve
the solution structures of RraA and two different DEAD box RNA
helicases: RraA:RhlB and RraA:SrmB complexes (Pietras et al.,
2013). Comparison of the two complex structures reveals that
RraA interacts with both helicases via the same binding domain
(Pietras et al., 2013), which implicates two different mechanisms
of RraA. BAG6, a co-chaperone interacting with aggregationprone polypeptides, plays an important role in tail-anchored
(TA) transmembrane protein biogenesis. Crystallographic and
biochemical analyses established that the C-terminal BAG6,
termed BAGS domain, and the C-terminal TUGS domain of
Ubl4a, an ubiquitin-like protein, participate in the heterodimer
(Kuwabara et al., 2015). SAXS solution structure revealed that the
BAGS:full-length Ubl4a features more extended structure than
that of BAGS:TUGS, filling the gap between crystallographic and
solution structural states.
SAXS can be also applied to study protein:nucleic acid
interactions. Proteins with nucleic acid binding domains are
associated with DNA replication (Kulczyk et al., 2012; Mason
et al., 2014; Matot et al., 2012; Ozawa et al., 2013; Tang et al.,
2008), gene regulation (Chaix et al., 2010; Cordeiro et al., 2011;
Kim et al., 2011; Meier et al., 2013; Pendini et al., 2013), DNA
repair/modification (Hammel et al., 2010; Lang et al., 2011; Majka
et al., 2012) and RNA metabolism (Mallam et al., 2011; Nowak et
al., 2013; Patel et al., 2012). In these cases, it is of great interest
to structural biologists to recognize which region of the protein
component is involved in specific interactions with DNA or
RNA. Protein:nucleic acid complexes are usually stable enough
to be investigated by SAXS in vitro. During DNA replication,
DNA structure undergoes significant remodeling in unwinding
double-stranded DNA, reading the sequence information from
template DNA and synthesizing a new complement DNA strand.
The protein:DNA complexes involved in the DNA replication
protein structures are inherently dynamic in their structures.
Highly dynamic property of the protein:DNA complexes is
a big barrier to obtain high-quality crystals leading to highresolution structures. SAXS can be a good alternative to uncover
structural dynamics including multiple conformations in solution.
SMARCAL1 is a DNA modeling protein associated to DNA
replication forks in higher eukaryotes such as mouse and human.
Its deficit induces cell arrest in S-phage during mitosis and
chromosomal DNA instability. M. musculus SMARCAL1 catalytic
domain (SMARCAL1CD) is composed of HARP domain followed
by ATPase domain. The complex of SMARCAL1 CD with DNA
assumes different conformations depending on bound cofactors
in DNA replication steps (Mason et al., 2014). The protein
envelope of SMARCAL1CD with AMP-PNP, which mimics ATPbound form, showed just HARP2 domain bound to one end of
ssDNA-dsDNA junction. However, with ADP BeFx, which mimics
a potential transition state between ATP and ADP, the protein
envelope showed ATPase domain of SMARCALCD also engaged
the middle region of ssDNA-dsDNA substrate. These SAXS
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results supported the DNA-binding function of HARP domain and
how conformational change of SMARCALCD affects the function
as DNA remodeler in presence of ATP and its catalyzed form.
SAXS is an effective tool for probing dynamic conformational
changes of protein:nucleic acid complexes in solution without
the need for crystallization and freezing. DEAD-box proteins play
critical roles in RNA metabolism as general RNA chaperone.
Mss116p of Saccharomyces cerevisiae is a model system of
DEAD-box proteins for studies of its mechanism and highresolution structures of Mss116p are available (Mallam et al.,
2012). However, these structures lack C-terminal tail that its
truncation of Mss116p strongly inhibits its activity as RNAdependent ATPase (Mohr et al., 2008). Although C-tail of
Mss116p should be essential on its activity, the flexibility of the
C-terminal tail of Mss116p might have prevented the formation of
crystals for X-ray crystallography. SAXS experiments using fulllength Mss116p revealed that its conformational change occurs
in presence of RNA and adenosine nucleotides (Mallam et al.,
2011). The flexible C-terminal tail of Mss116p protrudes from
its core domain, rendering it suitable for binding nucleic acid.
Combined with high-resolution crystal structural information
and biochemical studies, SAXS results strongly support that the
C-terminal tail of Mss116p is likely to be involved to bind RNA
and tether the core domain to large RNA substrates.
VALIDATION OF SAXS BASED MODEL
Since bioSAXS provides low-resolution structural information of
the macromolecular complexes in solution, such SAXS-based
complex structural models should be validated biophysically,
biochemically and functionally. Site-directed mutagenesis is
routinely used to validate structural analysis. In the case of a
molecular model fitted to an envelope derived by bioSAXS,
mutagenesis is more than essential for validating the structural
model of the macromolecular complexes. Introducing a single
or multiple mutations into the expected interaction surface
followed by tracking the differences in binding affinities will prove
whether the SAXS structural model is correct. Biophysical and
quantitative techniques such as bio-layer interferometry (BLI),
surface plasmon resonance, and isothermal titration calorimetry
can be used to determine binding affinities among components
of macromolecular complexes. Biochemical and qualitative
pull-down assay provides a quick and convenient measure to
validate the SAXS structural model for the complex. Mutations
predicted by SAXS structural model to be critical in the integrity
of the complex are tested. Size exclusion chromatography (SEC)
provides an estimate for the molecular mass of the complex from
which stoichiometry of the complex can be determined. However,
if the complex subject to investigation assumes non-spherical
shape, SEC results should be interpreted with caution: it may
be necessary to employ a secondary measure such as multiangle static light scattering and analytical ultracentrifugation to
unambiguously determine the molecular size of the complex.
Yeast Doa1 is an adaptor protein for Cdc48 involved in endo-
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Donghyuk Shin, Seungsu Han and Sangho Lee
plasmic reticulum-associated protein degradation pathway and
endosomal sorting. In the endosomal sorting in yeast, Hse1, a
protein of an endosomal sorting complex required for transport
system, recruits Doa1. The protein:protein interactions of these
two proteins were characterized by GST pull-down assay and
BLI experiments (Han et al., 2014). Additionally, some residues
might be important for their interactions and investigated by sitedirected mutagenesis and BLI experiment. The residues, N438 of
Doa1/PFU domain and W254 of Hse1/SH3 domain, were found
to be critical to this interaction. According to these studies, it
was successful to build a solution structure complex model by
SAXS and computational modelling approaches. A low-resolution
structural model for the Rad18:linear di-ubiquitin complex
associated with DNA double-strand damage repair pathway was
derived by SAXS experiments (Thach et al., 2015). The SAXS
model suggested that residues E227 and E228, unrecognized
previously, are crucial in the contact between Rad18 and linear
di-ubiquitin. When the residue E227 and E228 were mutated to
alanine, the binding affinity was significantly reduced, strongly
supporting the conclusion drawn from the SAXS structural
model. In the CTNNBL:CDC5L complex, the complex structural
model also validated by mutagenesis assay. They mutated the
key residues such as L87, I101, G148, Q192, Q199, Q238, E278,
and E282 on the dimer interface and check the ability of dimer
formation (Ahn et al., 2015).
Any structural model for a macromolecule or complex should
be eventually validated from functional aspects. Mutagenesis
is the basis for most molecular and cellular assays to test
the validity of the macromolecular complex structural model
derived by bioSAXS. For Rad18:linear Ub2 case, changes in the
localization of Rad18 at DNA damage sites were monitored using
critical mutants. Single mutants E227A and E228A decreased
the ability of Rad18 to localize at DNA damage sites (Thach et
al., 2015). CTNNBL:CDC5L study demonstrated the complete
reduction of dimerization from octuple mutant while double and
triple mutants still possessed the dimerization state. (double
mutant: L87R/ I101R; triple mutants: G148R/Q192R/Q199R, and
Q238A/E278A/E282A; and octuple mutant: L87R/I101R/G148R/
Q192R/Q199R/Q238A/E278A/E282A) (Ahn et al., 2015).
CONCLUDING REMARKS
Biological macromolecules such as protein, DNA, and RNA often
function as macromolecular complexes. It is extremely important
to know how such complexes are organized and changing their
conformations. BioSAXS has been used as a supplementary
tool for structure determination, mainly combined with
crystallographic studies: SAXS is a low-resolution technique,
but can provide valuable structural information when any highresolution technique such as crystallography cannot cover the
whole region of a macromolecular complex and conformational
changes are believed to be critical in the function of the complex
in solution. Due to the expansion of the number of highresolution structures for components of many macromolecular
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complexes and the advancement of molecular modelling, SAXS
can be a primary structural tool to investigate the structure of
macromolecular complexes. Still the structural model derived
by SAXS should be validated by biophysical, biochemical and
functional assays, which becomes routine for not only SAXSderived structural models, but also many high-resolution
structural models. With emerging technical development in
bioSAXS synchrotron beamlines, SAXS can be deployed for
investigating challenging structural works including obtaining
structural information from non-crystallizable or troublesome
samples upon freezing and probing conformational changes
upon biological processes that macromolecular complexes are
involved.
ACKNOWLEDGEMENT
This work was supported by the Basic Science Research Program through
the National Research Foundation of Korea (NRF) grants funded by the
Ministry of Education (NRF-2013R1A1A2059981) and by the Ministry
of Science, ICT and Future Planning (NRF-2015R1A2A1A15055951),
the Pioneer Research Center Program (2012-0009597) through NRF
grant funded by the Korea government (MSIP), and the Woo Jang Chun
Program (PJ009106) through the Rural Development Agency.
AUTHOR INFORMATION The Authors declare no potential conflicts of interest.
Original Submission: Feb 22, 2016
Revised Version Received: Mar 11, 2016
Accepted: Mar 14, 2016
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