Archives of Biochemistry and Biophysics 602 (2016) 80e94

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Archives of Biochemistry and Biophysics

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SAD phasing: History, current impact and future opportunities*

John P. Rose, Bi-Cheng Wang*
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA

a r t i c l e i n f o a b s t r a c t

Article history: Single wavelength anomalous diffraction (SAD) can trace its beginnings to the early 1950s. Researchers at
Received 23 December 2015 the time recognized that SAD offers some unique features that might be advantageous for crystallo-
Received in revised form graphic phasing, despite the fact that at that time recording accurate SAD data was problematic. In this
17 March 2016
review we will follow the trail from those early days, highlighting key advances in the eld and inter-
Accepted 19 March 2016
preting them in terms on how they stimulated continued phasing development that produced the
Available online 30 March 2016
theoretical foundation for the routine macromolecular structure determination by SAD today. The
technological advances over the past three decades in both hardware and software, which played a
Keywords:
Single wavelength anomalous diffraction
signicant role in making SAD phasing a rst choice method, will also be described.
Phasing 2016 Elsevier Inc. All rights reserved.
Early work
Current methods
Hardware and software
Native SAD

1. Introduction 1.1. Advantages of SAD phasing

In the X-ray diffraction experiment, information related to the
phase of a given reection is lost. This is known as the Phase
Problem in X-ray crystallography. However, this lost phase infor-
mation is contained in the ensemble of reections, which makes up
the data set and if the resolution of the data is high enough the
phase information for a given reection can be extracted from the
native data set itself. This process is known as Direct Methods [1,2].
Unfortunately, crystals of most macromolecules do not diffract well
enough to use Direct Methods to accurately estimate phases. Thus,
other approaches such as Multiple Isomorphous Replacement
(MIR) [3], Single Isomorphous Replacement (SIR) [4], Single-
wavelength Anomalous Scattering (SAS now called SAD) [4,5] and
Multi-wavelength Anomalous Dispersion (MAD) [6] have been
developed for de novo phase estimation. These approaches are
limited by the requirement of the presence of one or more phasing
probes in the protein or crystal lattice; heavy atoms in the case of
MIR or SIR phasing or anomalous scatterers in the case of SAD or
MAD phasing. This review will focus on the history of SAD phasing,
its impact on current structural biology and future application that
extend beyond structure solution.
*
This article is part of a Special Issue entitled Protein Crystallography, edited by
Ana Camara-Artigas and Jose Antonio Gavira.
* Corresponding author.
E-mail addresses: jprose@uga.edu (J.P. Rose), bcwang@uga.edu (B.-C. Wang).
SAD phasing offers some distinct advantages over other phasing
methods presented above. First the technique does not require
multiple data sets as in the case of MIR, SIR and MAD. The SAD data
set is generally collected from a single crystal containing either an
anomalous scatterer such as iron which is naturally present in the
protein, selenomethionine which has been incorporated into the
protein during expression [7] or an anomalous scatterer that has
been introduced into the crystal via solution diffusion similar to
preparing heavy atom derivative crystals [8]. Since all data is
generally collected from a single crystal, crystal nonisomorphism is
not a problem. Another advantage of SAD phasing is that unlike
MAD, SAD does not dependent on the data collection spanning an
X-ray absorption edge of the anomalous scatterer, which precludes
elements such as cadmium, iodine, xenon, cesium and sulfur from
being used as MAD phasing probes. SAD phasing also does not
require the subelectron volt energy resolution of the MAD experi-
ment and the wavelength dependent nature of the anomalous
scattering signal means that for most elements an anomalous
scattering signal sufcient to solve the structure can be obtained
within the tunable wavelength range (0.70 to 2 ) of most syn-
chrotron beamlines.
1.2. A brief history on the evolution of SAD phasing
SAD phasing had its beginnings nearly 70 years ago. The

http://dx.doi.org/10.1016/j.abb.2016.03.018
0003-9861/ 2016 Elsevier Inc. All rights reserved.
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
Abbreviations
s21 The fractional mean contribution (real part) of the
heavy contribution to the overall structure
ESRF European Synchrotron Research Facility
Gy Gray, a measure of absorbed X-ray dose,
ISAS Iterative Single Anomalous Scattering
ISIR Iterative Single Isomorphous Replacement
LCLS Linac Coherent Light Source
MCA Multi crystal data averaging
MDS low-dose multi-data set summation
MIR Multiple Isomorphous Replacement
RACC relative anomalous correlation coefcient (RACC)
RAP Resolved-Anomalous Phasing
SACLA SPring-8 Angstrom Compact Free Electron Laser
SAD Single Wavelength Anomalous Diffraction
SAS Single Wavelength Anomalous Scattering
SECSG Southeast Collaboratory for Structural Genomics
SFX Serial femtosecond crystallography
SIR Single Isomorphous Replacement
SSGCID Seattle Structural Genomics Center for Infectious
Disease
XFEL X-ray Free Electron Laser
anomalous scattering effect which causes the breakdown of Frie-
del's law was rst demonstrated when two groups of investigators,
S. Nishikawa and K. Matukawa in 1928 [9] and D. Coster, K.S. Knol
and J. Prins in 1930 [10] tried to determine which face, the shiny
face or the dull face, corresponded to the Zn-layer in the zincblende
(sphalerite, ZnS). They carried out diffraction studies on the
opposite [(111) and (-1-1-1)] faces of the zincblende crystal using
Au La1 radiation, whose wavelength is slightly shorter than the Zn K
absorption edge. Based on the differences in diffraction intensity
observed for the two faces the S-layer, not the Zn-layer, was found
to correspond to the shiny side of zincblende crystals.
Although an anomalous scattering effect was clearly demon-
strated, it was not until some 20 years later that Bijvoet and his
colleagues realized that anomalous scattering techniques might aid
in the phase determination process [11] in addition to its routine
use for determining absolute molecular conguration [12]. The use
of anomalous scattering for phasing soon attracted considerable
interest [13e15]. Various approaches of using anomalous scattering
were postulated, including the combined use of anomalous scat-
tering with isomorphous replacement for phasing macromolecules
[16,17].
The idea of the combined use of anomalous scattering and
isomorphous replacement data for phasing macromolecules
signied the recognition of phase ambiguity problems (Fig. 1)
associated with the use of either single wavelength anomalous
scattering (SAD) data or single isomorphous replacement (SIR)
data, and that with a proper combination of the SAD and SIR data
sets, the phase ambiguity problem could be resolved. But, what
happens, in the case where we have only SIR or SAD data available,
how can we overcome the phase ambiguity problem in this case?
It was soon recognized that if the anomalous scatters were
heavy atoms (they were at the time), the phase ambiguity problem
in SAD data might be resolvable [14,18] and would be advantageous
over the use of only SIR data. However, at the time it was not easy to
measure accurate anomalous scattering data. This begs the ques-
tion as to why there was interest that using SAD data alone might
be advantageous for phasing compared with using SIR data?
81
Fig. 1. A Harker construction illustrating the SIR protein phase ambiguity. Here the SIR
phase (fSIR) is the average of the true and false phases.
Since this review is focused on SAD, we shall follow the evolu-
tion of the ideas behind this interest and how they have led to the
methods used today for SAD phasing.
The elimination of the need for preparing an isomorphous heavy
atom derivative was an obvious advantage for SAD compared to SIR,
but there were other reasons. To appreciate these other advantages,
let's compare the information content in SIR and SAD data from our
current point of view (Fig. 2a and b). Although both SIR and SAD
consist of two sets of measurements (i.e. jFPHj & jFPj for SIR and
jF()j & jF()j for SAD), using SIR data alone one may not easily
recover the anomalous scattering contributions of the heavy
atom(s) if these anomalous contributions (differences) were not
experimentally measured. However, for SAD data one can easily
calculate the diffraction vector (intensity and phases) of the real
part of the scattering factor of the heavy atom, once the locations of
the heavy atom sites are determined. As illustrated in Fig. 2b, the
calculated diffraction vector FH lies orthogonal to the FH00 vectors
obtained from the measured anomalous scattering data (provided
that heavy atoms are of the same kind). Thus, in principle this
orthogonal information which can be extracted from the SAD
data could provide the third piece of information needed to
partially break (resolve) the phase ambiguity in SAD data. If one
applies a similar approach to SIR data this critical third piece of
information which lies orthogonal to the heavy atom vector in the
SAD case now lies parallel to it and thus cannot be used to break
the SIR phase ambiguity. This unique characteristic of SAD data
perhaps was the basis behind continued efforts in early SAD
phasing development by this small group of scientists.
There was also considerable interest in using Direct Methods
[19] to address the phase ambiguity problem in SAD or SIR data by
adding probability constrains based on phase relationships. How-
ever, the atomic resolution data needed for successful Direct
Methods phasing limited the use of this approach for macromole-
cules, since even today few protein crystals diffract beyond 1
resolution. This approach has also been referred to in the literature
as the reciprocal space approach. As will be described in a later
section, new developments in the reciprocal space approaches have
now been incorporated in several modern crystallography software
packages to enhance the phasing results [20,21]. The early devel-
opment of this approach has been well documented by Woolfson
82 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
Fig. 2. a. The Harker construction for SIR phase calculation (adapted from Wang, 1985). Note that the FH vector lies parallel to the fSIR vector. In this case the heavy atom
contribution can not be used to resolve the SIR phase ambiguity. Also, the SIR map can be considered as a superposition of two Fourier maps, one map being correctly phased and
the other map being noise. b. The Harker construction for SAD phase calculation (adapted from Wang, 1985). Note that the FH vector now lies orthogonal to the fSAS vector. In this
case the heavy atom contribution can be used to resolve the SAD phase ambiguity. Similar to the SIR case the SAD map can be considered as a superposition of two maps, one
generated from the true phases and the other being noise.
and Fan [22] and will not be repeated here.
Now back to the historical trail. Concerning the real part of the
heavy atom scattering contribution to SAD data, one knew that it
could potentially play a role in resolving the SAD phase ambiguity,
but how far could this approach go? Some thought that basically
the phasing power of single wavelength (SAD) data might not be
sufcient for phase resolution and others thought that the phase
ambiguity problem could be resolved if diffraction data were
measured at several frequencies across the absorption edge of an
anomalous scatter [23]. A more advanced use of this multi wave-
length approach, incorporated in MAD phasing [6], has certainly
had a signicant impact on protein crystallography for the past two
decades. Here, we are tracing the ideas and efforts of those who
believed in SAD and how their accumulated improvements to the
use of the heavy atom contribution for phase resolution, although
not totally successful at the time, stimulated SAD to continue to
advance to nally become a major method for macromolecular
crystallography today.
Through a series of studies using known and unknown small
molecule structures, Ramachandran and colleagues, including
Raman, Srinivasan and Parthasarathy [18,24,25] tested the role of
the heavy atom scattering contribution to phasing success and
summarized their results in a table showing that the probability of
resolving the SAD phase ambiguity would increase with the in-
crease of s21 (the fractional mean contribution (real part) of the
heavy contribution to the overall structure) (Table 1). For example,
with s21 of 0.2 the success rate for identifying the true phases from
the false phases is over 70% [26], sufcient for structure determi-
nation. They called their approach quasianomalous-dispersion
synthesis.
Table 1
Percentage of Reections Selected Correctly as a Function of s21.
s21 1 Atom 2 Atoms Many atoms acentric Many atoms centric
0.2 76.0% 73.0% 72.0% 69.5%
0.4 88.5% 82.3% 81.5% 77.5%
0.6 96.0% 80.0% 88.5% 83. 0%
0.8 99.8% 93.5% 94.5% 89. 0%
Table adapted from Ramachandran & Parthasarathy, Science 150, 212e214, 1965.
Calculations based on phase differences less than 90 from the Heavy Atom phases.
s21 is the mean fractional intensity contribution of the anomalous scatters to the
intensity of the total structure.
Looking back, this heavy atom correlation may be understood
from the point of view of the Heavy Atom method that was well
used at the time for small molecule structures such as vitamin B12
[27] or organic salts which contained a heavy atom. This was based
on the idea that the phase of the total structure (fT) will tend to
move toward the phase of its imbedded heavy atoms (fH) when the
heavy atom's size (number of electrons) or the mean fractional
contribution of the heavy atom to the structure increases. Thus,
quasianomalous-dispersion synthesis is in principle similar to the
Heavy Atom phasing method where the phases of the heavy
atom(s) are taken as the initial phases of the overall structure. There
is however an important difference between these two methods in
that the calculated heavy atom phase is not directly used in the
quasianomalous-dispersion synthesis, but is used instead as a
reference phase for selecting a probable true phase from the false
phase. That is the phase closest to the heavy atom phase is selected
as the probable true phase in the initial map calculation. So, phase
selection in quasianomalous-dispersion synthesis was actually an
improvement over the traditional Heavy Atom method.
The quasianomalous-dispersion synthesis (also referred to as
the Bijvoet-Ramachandran-Raman method [28] has been used to
determine a number of structures, including the crystal structure of
Factor V1a, an aquocyanide B12 variant using the anomalous scat-
tering of cobalt [29]. Notably, the approach was proposed by
Ramachandran and Parthasarathy in 1965 as a method for protein
structure determination [26], which is unfortunately seldom cited
in the literature. This possibility of protein phasing was further
discussed by Argos and Mathews in 1973 using the anomalous
scattering signal of the heme iron in cytochrome b3 (PDB entry
1CYO) [30].
Another theoretical development aimed at handling of noise
in diffraction data was the introduction of a phase probability
approach, which used best phases and gure-of-merits for
electron density map calculations [16,17,31,32]. Maps that were
calculated using this approach were proven to be superior to ones
generated by selecting the most probable phases on the basis of
which phase lies closer to the heavy atom phase. This approach has
been successfully applied in the calculation of protein phases using
the multiple isomorphous replacement (MIR) method, where three
or more sets of diffraction data from heavy atom derivatives are
required [28].
In 1980, Hendrickson and Teeter [5] applied phase probability
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
methods to SAD phasing by replacing the phase selection process
used in quasianomalous-dispersion synthesis. They termed this
approach Resolved-Anomalous Phasing (RAP). Although their
approach was successful in determining the structure of crambin,
Protein Data Bank (PDB) entry 1CRN [33] (the rst protein SAD
structure), a 46-residue protein containing 6 sulfur atoms, RAP did
not become a general method for SAD structure determination. The
incorporation of phase probability evaluation over strict phase se-
lection in their approach improved phase accuracy, however the
fundamental phasing concept was similar to that of the
quasianomalous-dispersion synthesis, which required a signicant
heavy atom scattering contribution (sulfur in the case of crambin)
to the overall structure. Because of this inherent limitation of the
approach, RAP was unable to eliminate the need of a large heavy
atom scattering contribution (~20%) to overall structure and typical
proteins have a much smaller sulfur content.
In early 1980s, Wang took a different approach by using a rough
structural model of the total protein structure instead of the partial
structure of the anomalous scatterers, to provide the initial infor-
mation needed to distinguish the true and false phases [4]. Thus, in
principle this approach uses the protein structure itself to provide
nearly 100% of the information that is needed for the phase selec-
tion process in the quasianomalous-dispersion synthesis. Wang's
approach was originally developed for the use of SIR data where, as
noted above, the calculated heavy atom contribution cannot be
used to resolve the SIR phase ambiguity. Because of this a search for
an alternative method for resolving SIR phase ambiguity was
needed. The initial rough structural model was obtained by
applying an automated protein-solvent boundary determination
algorithm [4] to dene the shape of the molecule, its location and
orientation (equivalent to a molecular replacement solution [34,35]
although no search model was needed). In addition, a repeated
cycling process between reciprocal space for probability combina-
tion and direct space for noise ltering (solvent attening) was
used to gradually make the initial SAD phases converge toward the
correct protein phases (Fig. 3). The approach was named the Iter-
ative Single Isomorphous Replacement (ISIR) method. The ISIR
method was then extended for the use of single wavelength
Fig. 3. A schematic diagram of the dual space ISIR/ISAS phasing system developed by Wang for resolving the phase ambiguity in SIR/SAD data.
83
anomalous scattering data and was called Iterative Single Anoma-
lous Scattering (ISAS) method. A software package (the ISIR/ISAS
program suite) [4] was developed and its Fortran code was
distributed freely to the crystallographic community upon request.
Wang's overall procedure is commonly known as solvent at-
tening and was the key advance in removing two of the critical
bottlenecks for the phasing of macromolecular structures: 1) the
phase ambiguity problem in using SIR or SAD data and 2) the
requirement that the protein must have a large heavy atom (or
sulfur) content as required by the RAP method used to determine
the S-SAD structure of crambin. The effective removal of these two
bottlenecks was demonstrated by Wang who used his ISIR/ISAS
program suite to determine the SIR structure of the Bence Jones
protein Rhe (PDB entry 2RHE) using a data collected from a single
gold derivative, and the S-SAD structure of Rhe (113 residues, 2
sulfur atoms) using error free simulated data [4]. In terms of
method development, this approach differs from the earlier
methods in that it was not developed for the exclusive use of SAD
data.
The method proved to be generally applicable to all SIR or SAD
data, including sulfur-SAD and provided the foundation for routine
structure determination of proteins having more typical sulfur
content. Notable early ISIR structures include the structure of
troponin C (PDB entry 4TNC) [36], the DNA-Eco RI endonuclease
recognition complex (PDB entry 1ERI) [37] and the histone octamer
(PDB entry 1HIO) [38,39]. The rst example of using the Wang's
process for a de novo structure determination from SAD data did not
come until 1989 with the determination of the bovine neurophysin
II dipeptide complex (PDB entry 2BN2) [40,41]. The structure was
determined by I-SAD using a bound iodinated hormone analogue
(p-iodo-Phe-Tyr amide), which mimics hormone binding in the
active site. To end this summary of early SAD history we present
below a brief description of the rst ISAS structure determination of
the bovine neurophysin II dipeptide complex (the second SAD
protein structure determined) to illustrate the practical aspects of
then newly developed SAD phasing method, its capabilities, its
routine determination of the handedness of the anomalous sub-
structure and its phase extension capabilities in combination with
84 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94

solvent attening. - -handed enantiomer. These statistics gave a clear indication

The structure of neurophysin II dipeptide complex was solved
using I-SAD data collected using 5 kW CuKa X-rays (Df00 (I) 6.8 for
CuKa X-rays) generated by a rotating anode generator (Rigaku) and
focused by mirror optics (Charles Supper) with the diffraction
pattern recorded on an X100 imaging proportional counter
(Siemens). Data were collected at 4  C using a crystal
(0.75  0.35  0.25 mm) mounted in a glass capillary and adjusted
using the arcs on the goniometer head so that one of the crystal's
symmetry axes (space group P212121) was along the rotation-axis of
the goniometer. The inverse beam method (20 wedges) using a
step size of 0.25 and an exposure time of 180 s was employed to
improve the anomalous signal-to-noise in the data, with the de-
tector placed at a 2q of 15 to enable data to 2.8 resolution to be
recorded. Two inverse beam experiments were carried out with the
crystal translated after the rst experiment to place a fresh crystal
volume in the beam. After data reduction and scaling [42] the nal
data set consisted of 58,983 observations of 12,840 Bijvoet pairs
and gave an Rsym(I) of 0.069. Matthews analysis [43] suggested
that there were four neurophysin molecules (~40 kDa) per asym-
metric unit and analysis of the Bijvoet difference Patterson maps
conrmed this giving the position of the four iodine sites. Using the
2.8 SAD data set, the iodine substructure and a 60% solvent
content the ISAS phasing process was carried out. The phasing
process used 20 cycles of iteration and three lters (see Table 2).
The initial lter and four phasing cycles of iteration were used to
resolve the phase ambiguity using 3.5 data. A new lter was then
generated followed again by four phasing cycles and four additional
cycles of phase extension to 2.8 resolution. A third lter was then
generated followed by a nal round of four phasing cycles and four
phase extension cycles to give the nal phases. During the phasing
process both the and - enantiomers of the heavy
atom substructure were analyzed to determine the correct hand-
edness of the sites (Table 2). The nal phase extension cycle had an
average gure of merit of 0.67, a map inversion R factor of 0.286 and
a correlation coefcient of 0.974 for the -handed enan-
tiomer and an average gure of merit of 0.64, a map inversion R
factor of 0.329 and a correlation coefcient of 0.960 for the
Table 2
ISAS phasing cycles and corresponding handedness test parameters.
ISAS () Hand () Hand
Filter Cycle m R CC m R
that the hand of the anomalous substructure is . The
resulting ISAS phased electron density map was of excellent quality
and manually traced (388 resides) in 3e4 days.
It is interesting to note that the 1989 SAD structure determi-
nation of neurophysin used many of the techniques still in use
today, including labeled amino acids to introduce an anomalous
scatterer, crystal translation during data collection to mitigate ra-
diation damage, an inverse beam data collection strategy to reduce
systematic error, ne slicing and multi dataset averaging to in-
crease signal to noise and a photon counting detector.
1.3. Current impact
Over the past two decades tremendous advances in diffraction
hardware, crystallographic software, data collection methods and
data collection strategies have been made which has resulted in
SAD becoming a rst choice phasing method. The introduction of
MAD phasing in the mid 1990's coupled with selenomethionyl
protein labeling and undulator based 3rd generation synchrotron
beamlines revolutionized macromolecular crystallography. Sele-
nomethionine labeling [7] provided both a strong (Df00 (Se) 3.89
e) anomalous phasing probe (at high occupancy) whose anoma-
lous scattering electron density could also be used as guide points
during the tting process. Because of these advantages, selenomet-
MAD quickly became the method of choice for de novo macromo-
lecular structure determination. However, beginning in 2000 SAD
phasing began to gain ground. Today, SAD has become the method
of choice for de novo macromolecular structure determination with
around 80% of de novo macromolecular structures being deter-
mined by the method (see Fig. 4). A survey of Protein Data Bank
entries [33] in the period spanning 1979 to 2015 (Sept 2015)
showed that 5736 structures were determined using MAD phasing
compared to 7549 determined using SAD with 35 MAD and 221
SAD structures being deposited to date in 2015.
This article will focus on advances in technology and method-
ology that have caught the community's attention. It will highlight
both de novo SAD structures, as well as recent structures that were
key to methods development. Page limits prevents a fully
comprehensive review, which is in the planning stage and hope-
fully will be a future follow-up article with additional information
contributed from the community.
1.4. Choice of anomalous scatterer
CC

1 1 Phase 0.58 0.507 0.814 0.57 0.522 0.803 One advantage of SAD phasing is that it is not dependent on a
1 2 Phase 0.67 0.373 0.900 0.66 0.392 0.889
1 3 Phase 0.72 0.317 0.929 0.71 0.337 0.921
tunable X-ray source or access to an X-ray absorption edge, thus
1 4 Phase 0.74 0.286 0.944 0.73 0.308 0.935 almost every element with a Z greater than 14 can currently serve
2 1 Phase 0.59 0.496 0.824 0.57 0.517 0.808 as an anomalous phasing probe, the technique becomes even more
2 2 Phase 0.68 0.358 0.909 0.66 0.389 0.892 powerful when X-ray sources with longer wavelengths (for
2 3 Phase 0.73 0.303 0.936 0.71 0.337 0.922
example ~ 3.1) are used (see Table 3). The recent advances in data
2 4 Phase 0.75 0.276 0.948 0.73 0.310 0.935
2 1 Extend 0.47 0.500 0.954 0.49 0.510 0.942 collection strategies (discussed in detail below) such as single
2 2 Extend 0.57 0.410 0.964 0.59 0.418 0.952 crystal low-dose multi-dataset summation (MDS) data collection
2 3 Extend 0.63 0.335 0.969 0.63 0.359 0.956 [44,45] and multi crystal data averaging (MCA) [46,47] have shown
2 4 Extend 0.66 0.395 0.972 0.65 0.328 0.958 that large structures such as the 266 kDa T2R-TTL multiprotein (ab-
3 1 Phase 0.59 0.493 0.824 0.57 0.514 0.809
3 2 Phase 0.68 0.356 0.909 0.66 0.388 0.893
tubulin, stathmin-4 and tubulin-tyrosine ligase) complex [45] and
3 3 Phase 0.73 0.310 0.936 0.71 0.336 0.922 the 132 kD histidine kinase TorT/TorSS complex [48] can be solved
3 4 Phase 0.75 0.271 0.949 0.73 0.309 0.937 using the weak anomalous scattering signal from sulfur and other
3 1 Extend 0.47 0.498 0.956 0.48 0.511 0.944 light atoms found in the crystal. The T2R-TTL complex (PDB entry
3 2 Extend 0.57 0.408 0.966 0.57 0.421 0.954
4WBN) was phased from 118 S, 13 P, 3 Ca2 and 2 Cl ions using SAD
3 3 Extend 0.64 0.330 0.973 0.62 0.360 0.958
3 4 Extend 0.67 0.287 0.974 0.64 0.329 0.960 data collected from a single crystal by MDS approach while the
TorT/TorSS complex (PDB entry 3O1I) was phased from 28S and
Key:
m e gure of merit.
3SO 4 ions using SAD data collected on 13 crystals by the MCA
R e map inversion R factor. approach. These experiments were carried out using 2.06
CC e map correlation coefcient. (Df00 (S) 0.90) and 1.74 (Df00 (S) 0.70) X-rays, respectively to
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
Fig. 4. A plot of the ratio of PDB entries determined by MAD phasing compared to those determined by SAD phasing for the period spanning 1995 to 2015 (as stated in the PDB
REMARK 200 records). The sharp rise in SAD structures beginning in 2000 can be attributed in part by the focus by the Southeast Collaboratory for Structural Genomics [135] on
making SAD phasing the rst choice method for macromolecular structure determination.
enhance the anomalous scattering signal of sulfur. Thus, based on
the criteria that an element must meet or exceed the limit of
(Df00 0.70), thus most of the elements listed in Table 3 can (in
theory) provide the needed anomalous scattering signal for struc-
ture determination, provided that the data are measured
accurately.
The SAD phasing experiment itself can be grouped into four
general classes depending on how the anomalous scatterer is
introduced into the crystal: Se-SAD, M-SAD, X-SAD and Native-SAD.
A short description of RIP (Radiation Damage Induced Phasing) will
also be described below, since it uses only single crystal with data
collected at single wavelength.
1.4.1. Se-SAD
Se-SAD is the most common phasing method for de novo
structure determination. The Se-SAD experiment uses selenium
labeled proteins produced by expressing the protein in a methio-
nine auxotrophic E. coli strain and adding L-seleno-methionine to
the growth media after induction [7]. Similar systems have also
been developed for the eukaryotic expression of selenomethionyl
proteins [49e51] and commercial kits are available from several
vendors.
The Se-SAD experiment is similar to the Se-MAD experiment
except that only one data set is collected at or near (on the higher
energy side) the Se absorption maxima (l 0.9795 ) where the Se
anomalous scattering signal is greatest (Df00 3.85). Since the
wavelength of the selenium absorption edge can be inuenced by
its environment, a good strategy is to always perform a wavelength
dispersive uorescence scan of the crystal to determine the exact
wavelength of the Se absorption edge prior to data collection.
While synchrotron data collection at the Se absorption edge is the
most common source of data used for Se-SAD phasing, successful
Se-SAD structure determinations have been reported for data
85
collected in the home lab using copper (l 1.5418 ;
Df00 (Se) 1.15) [52] or chromium (l 2.2909 ; Df00 (Se) 2.30) [53]
X-rays.
1.4.2. M-SAD
M-SAD exploits the anomalous scattering signal of metals (V,
Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, W) found in metalloproteins, which
represent over 30% of the proteins encoded by the human genome.
Most of these metals have a convenient absorption edge (Table 3) or
strong (Df00 greater than 3.00) anomalous scattering signal in
wavelength range from 0.73 to 2.00 (17 keV - 6.2 keV), common
to most macromolecular synchrotron beamlines, which can be
exploited for SAD structure determination. In addition, several
Table 3 elements (V, Mn, Fe, Co, Mo, Cd) produce a signicant
anomalous scattering signal in home source data collected with Cu
radiation and several early M-SAD structures such as ferrochelatase
solved by Fe-SAD (l 1.5418 ; Df00 (Fe) 3.18; PDB entry 1HRK)
[54] and oxalate decarboxylase solved by Mn-SAD (l 1.5418 ;
Df00 (Mn) 2.79; PDB entry 1UW8) [55] were determined from
home source data.
1.4.3. X-SAD
X-SAD represents the case where the anomalous scattering
probes, such as iodine, bromine, or metal ions are introduced into
the crystal by 1) co-crystallization, 2) soaked into the crystal by
traditional methods [8,56] or 3) introduced into the crystal via high
pressure gas incubation. Bovine neurophysin II, the rst SAD
structure determined by Wang's ISAS method [4] was determined
by I-SAD using home source data (l 1.5418 ; Df00 (I) 6.8; PDB
entry 2BN2) [40,41]. The study used a bound iodinated oxytocin
analogue (p-iodo-Phe-Tyr amide) which was co-crystallized with
the protein. Iodine has proven to be a popular X-SAD phasing probe
due to its ease of incorporation and large anomalous scattering
86 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94

Table 3
Minimum and maximum Df00 values for elements #12 to 92 in the wavelength range from 0.70 to 3.10.

Element Z Df00 Min Df00 Max lmax () #edges Element Z Df00 Min Df00 Max lmax () #edges

Mg 12 0.04 0.67 3.10 0 I 53 1.77 13.45 (L-I) 2.3898 3

Al 13 0.05 0.91 3.10 0 Xe 54 1.91 13.37 (L-I) 2.2738 3
Si 14 0.07 1.19 3.10 0 Cs 55 2.08 13.34 (L-I) 2.1697 3
P 15 0.09 1.53 3.10 0 Ba 56 2.23 13.25 (L-I) 2.0703 3
S 16 0.12 1.92 3.10 0 La 57 2.40 13.31 (L-I) 1.9786 3
Cl 17 0.16 2.36 3.10 0 Ce 58 2.58 13.15 (L-I) 1.8932 3
Ar 18 0.20 2.88 3.10 0 Pr 59 2.77 13.08 (L-I) 1.8140 3
K 19 0.24 3.43 3.10 0 Nd 60 2.96 13.03 (L-I) 1.7399 3
Ca 20 0.30 4.05 (K) 3.0704 1 Pm 61 3.16 12.98 (L-I) 1.6692 3
Sc 21 0.36 4.03 (K) 2.7596 1 Sm 62 3.37 12.88 (L-I) 1.6022 3
Ti 22 0.44 4.01 (K) 2.4965 1 Eu 63 3.60 12.83 (L-I) 1.5398 3
V 23 0.45 3.99 (K) 2.2689 1 Gd 64 3.64 12.82 (L-I) 1.4803 3
Cr 24 0.46 3.97 (K) 2.0701 1 Tb 65 3.66 12.84 (L-I) 1.4238 3
Mn 25 0.46 3.96 (K) 1.8961 1 Dy 66 3.68 12.84 (L-I) 1.3706 3
Fe 26 0.47 3.95 (K) 1.7433 1 Ho 67 3.69 12.77 (L-I) 1.3198 3
Co 27 0.47 3.94 (K) 1.6083 1 Er 68 3.71 12.77 (L-I) 1.2715 3
Ni 28 0.48 3.92 (K) 1.4879 1 Tm 69 3.73 12.78 (L-I) 1.2257 3
Cu 29 0.48 3.90 (K) 1.3808 1 Yb 70 3.74 12.43 (L-I) 1.1823* 3
Zn 30 0.49 3.90 (K) 1.2837 1 Lu 71 3.76 12.34 (L-I) 1.1406* 3
Ga 31 0.49 3.90 (K) 1.1959 1 Hf 72 3.77 12.29 (L-I) 1.1001* 3
Ge 32 0.50 3.88 (K) 1.1167 1 Ta 73 3.79 12.22 (L-I) 1.0614* 3
As 33 0.50 3.87 (K) 1.0448 1 W 74 3.81 12.16 (L-I) 1.0247* 3
Se 34 0.50 3.85 (K) 0.9795 1 Re 75 3.83 12.25 (L-I) 0.9898* 3
Br 35 0.51 3.83 (K) 0.9202* 1 Os 76 3.84 12.18 (L-I) 0.9561* 3
Kr 36 0.51 3.81 (K) 0.8655* 1 Ir 77 3.86 12.12 (L-I) 0.9240* 3
Rb 37 0.51 3.79 (K) 0.8157* 1 Pt 78 3.88 12.05 (L-I) 0.8933* 3
Sr 38 0.53 3.77 (K) 0.7899* 1 Au 79 3.90 11.97 (L-I) 0.8638* 3
Y 39 0.53 3.75 (K) 0.7277* 1 Hg 80 3.92 11.90 (L-I) 0.8355* 3
Zr 40 0.55 7.26 3.10 0 Tl 81 3.93 11.83 (L-I) 0.8079* 3
Nb 41 0.61 7.98 3.10 0 Pb 82 3.95 11.76 (L-I) 0.7817* 3
Mo 42 0.67 8.73 3.10 0 Bi 83 3.96 11.67 (L-I) 0.7566* 3
Tc 43 0.75 9.52 3.10 0 Po 84 3.98 24.91 (M2) 2.9990* 4
Ru 44 0.82 10.38 3.10 0 At 85 4.00 26.45 (M2) 3.0934 5
Rh 45 0.90 11.28 3.10 0 Rn 86 4.01 26.24 (M2) 2.9811 4
Pd 46 0.99 12.68 3.10 0 Fr 87 4.03 25.70 (M2) 2.8654 3
Ag 47 1.08 13.64 3.10 0 Ra 88 4.04 25.71 (M2) 2.7616* 3
Cd 48 1.17 14.24 (L-I) 3.0857 1 Ac 89 4.05 25.46 (M2) 2.6629* 3
In 49 1.28 13.87 (L-I) 2.9259 1 Th 90 4.07 31.61 (M3) 3.0643 4
Sn 50 1.39 13.70 (L-I) 2.7770 2 Pa 91 4.08 33.10 (M3) 2.9705 4
Sb 51 1.51 13.60 (L-I) 2.6389 3 U 92 4.10 35.01 (M3) 2.8811 4
Te 52 1.64 13.52 (L-I) 2.5102 3

Key: Data from the University of Washington Anomalous Scattering Site (skuld.bmsc.washington.edu/scatter).
Element e Elements name.
Z e Elements Atomic Number.
Df00 Min e Minimum anomalous signal (Df00 ) in the wavelength range from 0.70 to 3.10 (4000 e 17714 eV).
Df00 Max e Maximum anomalous signal (Df00 ) at the peak of the largest absorption edge (if more than 1) or at 3.1 when there is no absorption edge in the 0.7 to 3.1 wavelength
range. The edge of the Df00 Max is listed in parentheses. Note: For example, element #53 (iodine) has three L absorption edges (L-I, L-II and L-III) in the wavelength range from 0.7
to 3.1 .L-I has the largest Df00 and is listed.
lmax () e Wavelength where Df00 is largest in the wavelength range from 0.7 to 3.1 (values less than 3.1 denote an absorption edge in the wavelength range). Star *
indicates that the Df00 value (unspecied) at 3.1 is actually greater than the Df00 Max value of the absorption edge(s) of this element in the 0.7 to 3.1 wavelength range.
#edges e Number of absorption edges the element has in the wavelength range from 0.7 to 3.1.

signal with Df00 (I) ranging from 3.14 to 10.38 in the wavelength
range from 0.98 (Se absorption edge) to 2 . A method of preparing
iodinated and brominated derivatives by adding the halogenated
salt to the cryoprotectant drop prior to cryoprotection is available
[57,58] which simplies the derivation process. Brominated de-
rivatives can be exploited for SAD data collection at the Br ab-
sorption edge (l 0.9202 ; Df00 (Br) 3.83). A method employing
longer iodine/bromine soaks (5 mine2 h) has been adapted to high
through workows by researchers at the Seattle Structural Geno-
mics Center for Infectious Disease (SSGCID) who were successful in
determining the structure of 16 of 17 targets during its initial 12
month trial [59].
Metal ions introduced by soaking can also serve as phasing
probes providing that the binding is specic and their occupancy is
high. Heavy metal (Ag, Cd, La, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Yb, W, Re,
Os, Ir, Pt, Au, Hg) derivatization kits from Hampton Research and
specialized phasing probes such as the IC3 Magic Triangle (5-
Amino-2,4,6-triiodoisophthalic acid) [60] and clusters containing
tantalum (Ta6Br12) [61,62] and tungsten (Na3[PW12O40]$H2O,
Na6[H2W12O40]$H2O and [NH4]10[H2W12O42]$H2O) [63] from Jena
Bioscience are commercially available. Both tantalum
(Df00 (Ta) 12.22 at l 1.0247 ) and tungsten (Df00 (W) 12.16 at
l 1.0247 ) have X-ray absorption edges close to the selenium
absorption edge, which can be exploited for SAD phasing. In
addition, at low resolution (4 e 6 ) these large tantalum and
tungsten clusters produce an anomalous super-atom that can be
readily identied in Bijvoet difference Patterson maps.
The Nobel gases krypton and xenon introduced into the crystal
under high pressure can also serve as anomalous scattering probes
[64e67]. Krypton produces an anomalous scattering signal at its X-
ray absorption edge (Df00 (Kr) 3.81 at l 1.0247 ) similar to
selenium signal used in Se-SAD/MAD experiments and xenon
produces a large anomalous scattering signal at longer wavelengths
(e.g. Df00 (Xe) 11.08 at l 2.00 ). The anomalous scattering signal
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
from xenon pressurized crystals has also been used to trace sub-
strate/solvent channels within the protein structure [68,69].
1.4.4. Native-SAD
Native-SAD uses the anomalous scattering signal of sulfur,
phosphorous or other light atoms (Z less than 21) which are present
in the crystalline sample (e.g. sulfur or phosphorus present in the
biological sample from protein, DNA or RNA, and other ions inad-
vertently introduced during protein purication or crystallization
process) as phasing probes [70]. Native-SAD represents the ulti-
mate goal in SAD phasing since if the anomalous scattering signal of
sulfur can be routinely used for macromolecular structure deter-
mination, then virtually all elements listed in Table 3 can be used.
1.4.5. MR-SAD
Molecular replacement assisted SAD uses molecular replace-
ment to provide initial phases for determining the anomalous
substructure and thus bootstrap the phasing process [71,72]. This is
critically important for phasing using weak anomalous scatterers
such as sulfur and phosphorous. The technique also provides a
simple method of overcoming model bias common to molecular
replacement structure determination and in cases where the
experimental phase information is weak. MR-SAD also assists in
model building by providing marker atoms and validate the iden-
tity of anomalous scatterers for renement.
1.4.6. Radiation damage induced phasing
Although not strictly an anomalous scattering technique Radi-
ation Damage Induced Phasing (RIP) [73] shares many of the ad-
vantages of SAD in that: 1) all necessary data can be collected from
one crystal, 2) the technique does not require data collected at an X-
ray absorption edge and 3) the data is collected at a single wave-
length. However, the technique is more similar to SIR requiring two
distinct (before and after radiation damage) data sets. It differs
from SIR in that both data sets are collected on the same crystal. The
before radiation damage data set (native data) is collected rst. The
crystal is then exposed to high dose X-rays or UV [74,75] to
essentially denature the substructure in the protein and the after
radiation damage data set is collected. Today, many common
phasing packages (SHARP [76], SHELX C/D/E [77], CCP4 [78] and
PHENIX [79]) can be used to generate phases form the RIP data sets.
2. Sample preparation
The SAD experiment is critically dependent on maximizing the
anomalous signal-to-noise ratio in the data. A source of noise that is
often overlooked in the experiment is the sample itself: the crystal,
its cryoprotection and the loop/pin assembly used to mount it.
2.1. Crystals
Generally speaking better crystals give better data in terms of
resolution, mosaicity, data accuracy and anomalous signal; all
important to successful SAD phasing. Thus, a little time spent
producing better crystals (and their cryoprotectant cocktails) can
often reduce the amount of time and effort needed to solve the
structure. Protein crystallization and optimization has been the
focus of several recent reports by McPherson [80,81] and D'Arcy
[82], which can be used for reference.
2.2. Cryoprotection
Most SAD data are collected at cryogenic temperatures to reduce
radiation damage during data collection using crystals mounted in
pin/loops assemblies which are magnetically attached to the
87
goniometer [83]. Loop mounting reduces stress on the crystal
during the mounting process and can be used to harvest and mount
crystals from microliter to nanoliter crystallization setups. How-
ever, the cryocooling process can introduce noise into the SAD
experiment in the form of higher image backgrounds, reection
crowding (due to high mosaicity) and the presence of ice rings in
the pattern. Two approaches have been developed to limit this
phenomenon: the use of cryoprotectants and high-pressure cry-
ocooling under helium gas.
The use of small molecule cryoprotectants such as 20e30%v/v
glycerol is the most common means of cryoprotecting the crystal
during the ash cooling process and several excellent reviews have
been written on this subject [84,85]. High-pressure (200 MPa)
cryocooling has been shown to provide excellent diffraction from
non-cryoprotected crystals [86,87]. This approach is based on
myoglobin cryoprotection studies [88] and the phenomenon that
water under high pressure freezes as ice III which contracts upon
freezing compared to ice I which expands. The technique is less
common since it requires specialized apparatus. However, two
commercial units are available: the HPC-201 from Advanced Design
Consulting USA (www.adc9001.com) and HPM-010 system from
BAL-TEC AG (www.bal-tec.com). The technique has been recently
used in the structure determination of bovine enterovirus 2 from a
single high pressure frozen crystal (PDB entry 1BEV) [89].
2.3. Mounting pins & loops
Several loop-pin designs are commercially available for har-
vesting and mounting crystals for data collection at cryogenic
temperatures but care must be taken when choosing a specic pin-
loop design. The Recent reports have shown that choice of pin-loop
design can effect data quality [90,91]. The studies revealed that the
loop stem (the area between the pin head and loop) and diameter
of the loop are the most important factors when choosing a pin/
loop design. It is recommended that the stem be as short as possible
and the loop diameter be chosen to t the size of the crystal (to
minimize the amount of mother liquor in the loop). For nylon loops
they recommend reinforcing the loop stem with epoxy or grease to
reduce or eliminate vibration of the loop in the cold stream during
data collection. This is especially important when the anomalous
signal is low, such as Native-SAD experiments or when data is
collected at high speed (rates > 2 Hz).
The scatter from the loop and the mother liquor surrounding the
crystal is another source of noise in the experiment. Several
methods have been developed to address this source of noise,
including the loopless mounting method [92,93], graphene wrap-
ping [94] and the use of gel beads [95]. In loopless mounting the
crystal is harvested using the loop and then by using a special pin
the mother liquor is removed by aspiration and the crystal ash
cooled in a nitrogen gas cryostream. Once frozen, the loop is
carefully removed using small hook, forceps or laser leaving the
crystal mounted directly on the pin ready for data collection. Gra-
phene wrapping as the name implies wraps a thin multilayer (3e5
layers) of grapheme around the loop mounted crystal trapping a
small amount of mother liquor together with the crystal. The
multilayer layer graphene sandwich is essentially transparent to X-
rays and produces signicantly lower scatter compared to loop
mounted crystals. Gel bead mounting uses crystals grown in ioni-
cally cross-linked polysaccharide gel beads by the microbatch un-
der oil technique. The approach reduces mechanical damage to
crystals during mounting and the porous nature gel bead also al-
lows for cryoprotectection, ligand soaking and heavy atom deri-
vation of the crystal as required.
88 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
3. The SAD experiment
As mentioned above, successful SAD phasing is dependent on
maximizing the anomalous signal to noise ratio in the data.
Experimentally, this can be done by either increasing the anoma-
lous signal (mainly by increase wavelength used) or reducing noise
by various means. Our discussion will start with the use of Cu Ka
home X-ray sources and then extend to the use of synchrotron X-
rays as outlined below.
3.1. X-ray source
It is no surprise that in-house generated Cu Ka radiation, which
provides single wavelength X-rays, played a key role in the devel-
opment and early success of SAD structure determination. These
early structures include, crambin determined by sulfur-SAD
(Df00 (S) 0.56 e) data collected using a sealed tube X-ray source
(Ni ltered) and a Picker FACS-1 4-axis diffractometer [5]; neuro-
physin determined by iodine-SAD (Df00 (I) 6.91 e) data collected
using mirror focused (Supper) 5 kW rotating anode X-rays and a
Siemens X100A multiwire area detector [41]; and ferrochelatase
(PDB entry 1HRK) determined by iron-SAD (Df00 (Fe) 3.18 e) data
collected from three crystals using focused (Rigaku Osmic copper
confocal optics) 6 kW rotating anode X-rays on two different R-Axis
IV image plate area detector systems [54].
Obelin, the second de novo sulfur-SAD structure (PDB entry
1EL4) was one of the rst SAD structures determined using syn-
chrotron X-rays. The structure was determined using data collected
on beamline 17ID, Advanced Photon Source with 1.74 X-rays
(Df00 (S) 0.70 e) by a MAR Research MX165 CCD detector [96].
The introduction of chromium confocal optics (Rigaku Osmic) in
2002 provided a stable home source soft-X-ray platform for SAD
data collection [70,97]. Since then 31 Native-SAD structures have
been determined using chromium Ka (l 2.29 , Df00 (S) 1.14 e)
X-rays.
Although copper and chromium rotating anodes provide a
highly stable X-ray source, today most SAD structure de-
terminations are carried out using synchrotron data since this
approach offers two distinct advantages over in-house data
collection: 1) the ability to tune the wavelength to enhance the
anomalous scattering signal and 2) small high intensity X-ray
beams that allow efcient data collection on crystals too small for
the home source. However, one must remember that most syn-
chrotron beamlines were designed and optimized to support Se-
MAD data collection at the selenium absorption edge
(l 0.9795 ) and for the collection of high-resolution data. If data
collection at longer wavelengths is required, then beam stability
and X-ray absorption can become problematic, especially in the
case where the anticipated anomalous signal is weak.
Finally two recent papers using serial femtosecond crystallog-
raphy (SFX) to determine the Gd-SAD phased [98] (PDB entry
4N5R; LCLS; l 1.46 ; 60,000 images) and Native-SAD phased
[99] (PDB entry 4YOP; SACLA; l 1.74 ; 150,000 images) struc-
tures of lysozyme have been reported which illustrate the feasi-
bility of determining de novo protein structures using SAD and 4th
generation X-ray sources. Importantly the SFX experiment pro-
duces data that are free of radiation damage - another source of
error in the SAD experiment.
3.2. Wavelength
For those elements in Table 3 that have an X-ray absorption edge
close (0.3 ) to the selenium edge at 0.9795 the choice of
optimal wavelength is straightforward, since most synchrotron
beamlines have been designed and optimized for data collection at
or near this wavelength. However, for the other elements in Table 3
whose maximum Df00 value lies outside this wavelength range the
choice of wavelength is dependent on several factors including the
effects of X-ray absorption and beam stability at longer wave-
lengths. These effects introduce noise into the experiment and can
impact the accuracy of the data and the measured anomalous
scattering signal, especially when the anomalous scattering signal
is weak, as in the case of Native-SAD or when the occupancy of a
strong anomalous scatterer is low. A more detailed analysis of an
elements strength of the anomalous scattering (Df00 ) as a function of
wavelength can be found on the University of Washington's
anomalous scattering web site (www.bmsc.washington.edu/
scatter). Collecting data at longer wavelengths is a general
approach of increasing the anomalous signal for elements in Table 3
that lack an absorption edge since the magnitude of Df00 tends to
increase with wavelength. X-ray absorption also increases with
wavelength but can be minimized by careful crystal mounting
(described below) and the use of a helium lled beam path be-
tween crystal and detector.
Beam stability is more problematic since it can result from a
combination of several factors: 1) positional instability of the syn-
chrotron's electron orbit [100], 2) X-ray beam drift due to thermal
deformation of optical components as the energy changes and 3)
mechanical vibrations of components in the optical chain especially
in the case of microbeam/microcrystal experiments.
Since selenium and approximately 20% of the other elements
listed in Table 3 have an X-ray absorption edge in the range of
0.77e1.3 , beam stability at these wavelengths should not present
a serious problem. For the other elements that require data
collection at longer wavelengths, the ideal solution would be a
purpose-built, dedicated soft-X-ray beamline. Such facilities have
been built at the Photon Factory (BL-A1) in Japan, and at the Dia-
mond Light Source (I23) in the United Kingdom. Lacking a dedi-
cated soft X-ray beamline, beam instability due to thermal effects at
longer wavelengths can be easily addressed by simply waiting until
the thermal drift stabilizes after an energy change. Unfortunately,
this process could take from several minutes to several hours
depending on the beamline design. Thus, the time required for
beamline thermal equilibration after energy changes (at both the
low and high energy end points) should be considered when
applying for beam time.
Based on experiments conducted at wavelengths ranging from
0.80 to 2.65 , Mueller-Dieckmann et al. have proposed that the
optimal wavelength for sulfur-SAD data collection is 2.1 [101].
However, few structures have been reported using synchrotron
data collected at wavelengths above 2 . Instead, the majority of
Native-SAD structures determined with synchrotron X-rays used
data collected at wavelengths in the range of 1.7e1.8 [70]. James
Holton has also proposed collecting SAD data using 1.714 X-rays
(for Table 3 elements that lack an X-ray absorption edge) to reduce
systematic errors related to detector pixel nonuniformity issues
(termed Spatial Heterogeneity in Sharp Spot Sensitivity) that are
not addressed by current calibration procedures [102]. However, 31
Native-SAD structures have been determined using home source
chromium X-rays (l 2.29 ) and an image plate detector tted
with a helium beam path.
3.3. Goniometer
Collecting accurate data requires keeping the crystal centered in
the X-ray beam during data collection. This task becomes more
challenging as crystal size and/or beam size become smaller as
careful crystal centering is essential to keeping the crystal in the
beam during data collection. Modern goniometers such as the
MD2/MD3 microdiffractometers developed at the ESRF provide on-
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
axis crystal viewing, which makes crystal centering much easier
[103]. These systems are found at many beamlines around the
world and are available from Bruker/ARIMAX (http://www.bruker-
est.com). In addition to on-axis cameras, these systems also provide
a set of user selectable pinholes to dene beam size and other tools
to help the user verify both beam focus and that the crystal is
properly centered in the beam.
Automated diffraction-based centering (raster centering) is also
available at many beamlines. During the rastering process the
crystal is translated in an x, y, z grid (step size dependent on beam
size) and a diffraction image is recorded at each position using a
highly attenuated X-ray beam [104]. High speed detectors make
this a quick and efcient process. The images are then used to
determine the point (or points) of optimal diffraction, which can
then be used to dene the crystal's center or hot spots for data
collection. Modern goniometers coupled with small beams and
virtual motors offer the ability to translate the crystal during data
collection with the aim of continually introducing a fresh portion of
the crystal into the beam to minimize radiation damage [105,106].
Generally, two translational data collection modes are provided,
translational (or segmented mode) and helical mode. In segmented
mode, the crystal is divided into domains (dependent on crystal
dimensions and beam size) with the length and direction of
translation determined by centering the crystal at its two ends (or
the ends of the desired translation vector). Data is then collected
sequentially from domain 1 to domain N with the number of im-
ages determined based on both the total number of images desired
and the number of domains available. A similar approach can be
used to center multiple crystals mounted on a grid or in small wells
on a micro crystallization plate such as the X-Chip [107]. In helical
scan mode, the length and direction of the translation vector is
again dened by centering the crystal at the two end points. The
crystal is then slowly translated along this vector during data
collection. These data collection methods are very attractive since
SAD phasing generally requires data sets with high reection
multiplicity and methods to minimize radiation damage are
benecial.
Finally, some facilities offer multi-axis goniometers, which can
take advantage of crystal symmetry and/or habit to optimize the
anomalous signal-to-noise ratio during data collection [45,108].
These systems include the Bruker/ARIMAX MD2/MD3 equipped
with a MK3 mini-Kappa goniometer and the PRIGo multi-axis
goniometer recently developed at the Swiss Light Source [109].
With respect to SAD data collection, multi-axis goniometers allow
the crystal to be positioned such that Bijvoet mates can be recorded
at the same time from the same image, critical to reducing errors in
the SAD experiment. Multi-axis goniometers can also be used to
reduce the number of images need for a complete data set by
rotating the crystal about one of its crystal rotation axes, reducing
X-ray exposure and limiting radiation damage. In addition, they can
be used to reduce reection crowding in cases where one-unit cell
axis is signicantly longer than the other two by orientating this
axis parallel to the spindle during data collection.
3.4. Detector
Successful SAD structure determination has been carried out
using data recorded with scintillation counters, multiwire propor-
tional counters, imaging plates and CCD cameras of various con-
gurations (single chip or multichip arrays). The recent
introduction of detectors with 10 Hz plus readout speeds such as
the hybrid photon counting detectors produced by Dectris [110]
(www.dectris.com) and the high speed CCD based detectors pro-
duced by Rayonix (www.rayonix.com) have signicantly impacted
the ease with which SAD data collection can be carried out. The
89
millisecond or better readout time for these detectors enables
shutterless data collection, reducing noise associated with shutter
jitter and synchronization error, and also allows for efcient ne-
slice data collection reducing background fog on the image,
which increases the anomalous signal-noise in the data.
3.5. Data collection strategies for increasing signal to noise
Once source and hardware considerations have been taken into
account the most general approach of increasing the anomalous
signal-to-noise of the data is to collect data sets with high multi-
plicity (redundancy) from one or more crystals. This is because the
error associated with a measurement decreases as the square of the
number of observations. The technique is routinely used in the
Native-SAD experiment where the anomalous scattering signal is
weak, to increase the anomalous signal-to-noise in the data
[111,112]. Increased redundancy also improves the accuracy of the
measurements, which in turn produces more accurate anomalous
differences, better phases and electron density maps showing
greater detail. Together this increases the success rate of SAD
phasing, aids in model building and produces structures of higher
quality [113]. However, adding images to increase multiplicity also
increases the likelihood of radiation damage even at cryogenic
temperatures [84,114].
In the diffraction experiment, the loss of diffraction intensity
during the course of the data collection is an indication of radiation
damage. Structurally speaking radiation damage is manifested by
the breaking of disulde bonds, loss of CO2 from aspartic and glu-
tamic acid side chains, loss of the tyrosine hydroxyl group and the
formation of free radicals. Thus, crystal lifetime must be taken into
consideration when designing a data collection strategy. Based on
the theoretical X-ray dose of 2.2  107 Gy (J/kg) [115] needed to
reduce diffraction power of a protein by 50%, a crystal lifetime of
~17 s is obtained for a typical insertion device beamline at the
Advanced Photon Source using 12 keV X-rays (data taken from
bl831.als.lbl.gov/~jamesh/ACA2007/damage_rates.pdf). In addition,
an estimate of the crystal's theoretical half-life can be calculated
using RADDOSE-3D [114] based on beam parameters (size, shape,
ux and energy) and the crystal's composition, which will allow
experiments to be designed to limit the effect of radiation damage.
Finally, some success has been reported for the use of radial scav-
engers such as sodium ascorbate (a hydroxyl radical OH scavenger)
and sodium nitrate (an electron scavenger) in signicantly pro-
longing crystal lifetime [116,117].
How the data is collected can also affect SAD phasing since
missing data will impact data scaling, map connectivity and auto
tracing results. One generally should strive to collect the unique
data rst and then add more data to increase multiplicity. This
approach will allow the data set to be properly scaled and the effect
on data quality of adding more data can be analyzed. Over the past
30 years SAD data collection strategies have evolved from carefully
designed protocols requiring data collection from aligned crystals
using multi-axis goniometers to much simpler experiments carried
out on randomly orientated crystals with data collected around a
xed rotation axis. This trend was due in part to advances in protein
production (recombinant proteins & selenomethionine labeling),
crystal mounting (loops), cryogenic data collection, diffraction
hardware (synchrotron sources, X-ray optics, goniometry, detectors
and computers) and better software for data reduction (Fourier
indexing) and structure determination that made routine Se-SAD
phasing a reality.
Early experiments generally employed the inverse beam (or
Friedel ip) data collection strategy [40,118]. Here, the crystal is
mounted along a symmetry axis and data is collected in small
alternating wedges (f and f p) such that Bijvoet mates are
90 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
collected on the same image and close together in time. This was
the technique used (with an aligned crystal) for the I-SAD structure
determination of the neurophysin p-iodo-Tyr-Phe amide complex
(the rst ISAS phased structure) in the late 1980's [41]. However,
with the advent of Fourier indexing in the Denzo/Scalepack pack-
age [119] crystal alignment using a multi-axis goniometer was no
longer needed since the crystal could be indexed from a single
image.
However, as targets become more challenging (e.g. membrane
proteins and large multi protein complexes) and protein expression
utilizing insect and mammalian cell lines makes selenomethionine
labeling problematic, there is a growing interest in Native-SAD
phasing as described above [70]. Native-SAD represents the ulti-
mate SAD experiment requiring only crystals of the native protein, a
stable tunable X-ray source, precision goniometry and a large high
speed detector. Since the sulfur anomalous scattering signal Df00
ranges from 0.24 to 1.14 in the wavelength range spanning 1.0 to
2.3 , all sources of noise in the system must be identied and
mitigated. Furthermore, to increase the anomalous signal-to-noise
ratio, data sets of high multiplicity are required. The trick is to in-
crease redundancy without incurring signicant radiation damage
(a current bottleneck of the approach).
Recently two data collection strategies have been reported that
addresses this bottleneck, single-crystal low-dose multi-data set
summation (MDS) [44,120] and multi-crystal averaging (MCA) [46].
Although these approaches were developed for the collection of
Native-SAD data they are equally applicable to any SAD analysis
where crystal quality or radiation damage is a problem.
3.6. The single-crystal approach
The MDS approach uses dose-slicing to conserve the life-time
of the crystal. In this approach multiple data sets are collected using
one crystal (or one location on the same crystal) with either an
attenuated X-ray beam or reduced exposure time. The degree of
attenuation or exposure time reduction is inversely proportional to
the number of data sets to be collected (generally 3 or 4). The
following example illustrates this concept. Consider a crystal hav-
ing an optimal exposure time of 3 s and one wished to collect a
three data set ensemble. The exposure time used for each image
would be 1 s. The total X-ray dose accumulated for the three (1 s
exposure) data sets is the same as the dose received for the data set
collected using the optimal 3 s exposure time. The three datasets
are then merged together to yield a nal dataset that will have an
improved signal-to-noise ratio than the data set collected using the
optimal exposure time. This is because MDS data set will give a
smaller s(I) value as indicated by the equation below.
h
m i m K=A2 I 2
s
s2total G Is Ibg Ibg
n N
Note that the second term of the conventional sigma equation
(above) is divided by the number of data sets N collected [44]. This
improvement in I/s will further improve the D(I)/s values of the
strong reections, which are essential for Native-SAD phasing. This
approach can easily be combined with ne-slicing and helical (or
segmented) data collection to further improve I/s and mitigate
radiation damage, respectively. Since the data have been collected
from the same crystal the datasets should be isomorphous, if
exposure is kept below the Garman limit [121]. This is another
advantage of the MDS. Using the MDS approach and the PRIGo
multi-axis crystal positioner, a group from the Swiss Light Source
has recently reported 11 Native-SAD structures determined on
beamline X06DA using 2.066 X-rays [45]. The PRIGo crystal
positioner was used to collect data at 3e5 different crystal
orientations further reducing systematic error. Data were pro-
cessed with XDS [122]. The anomalous substructure was deter-
mined using SHELXD [123] and expanded using PHASER [124]. The
structures were autobuilt using either BUCCANEER [125] or PHENIX
[79]. The structures reported included membrane proteins, DNA-
protein complexes and the 266 kDa T2R-TTL multiprotein com-
plex (a complex between ab-tubulin, stathmin-4 and tubulin-
tyrosine ligase), PDB entry 4WBN, phased from 118 S, 13 P, 3 Ca2
and 2 Cl ions that represents the largest Native-SAD structure
determined to date. The recent advances in fast detectors and
shutterless data collection methods have made the MDS method
practical for Native-SAD in reducing both data collection time and
experimental effort.
3.7. The multi-crystal approach
MCA as the name implies is based on averaging data sets
collected from a number of different crystals in order to limit ra-
diation damage and increase the redundancy of the nal averaged
data set. The strategy requires collecting complete data sets having
low multiplicity on a number of different crystals to minimize ra-
diation damage. Cluster analysis of the individual processed data-
sets in terms of unit cell deviation, diffraction dissimilarity
(resolution and mosaicity) and the calculated relative anomalous
correlation coefcient (RACC) is then used to identify outlier
datasets and exclude from the analysis [47]. MCA has been incor-
porated into both CCP4 (Blend) [78] and PHENIX (phenix.sca-
le_and_merge) [79]. The approach has been applied to a wide range
of proteins of varying sizes and complexity solved by Native-SAD
including the 62 kDa integral membrane protein CysZ from
I. loihiensis (data from 6 crystals; PDB entry 3TX3), the 132 kDa
TorT/TorSS complex from V. parahaemolyticus (data collected from
13 crystals; PDB entry 3O1I) and the 134 kDa chaperone protein
DnaK from E. coli (data collected from 5 crystals; PDB entry 4JN4)
[126]. The approach has been recently used to determine the
Native-SAD structure of the Hepatitis C virus envelope protein E1
N-terminal ectodomain domain (data collected from 32 crystals;
PDB entry 4UOI) [127] using 4.2 data. Using the MCA data set
(~31,646 Bijvoet pairs) phenix.autosol employing 6-fold non-
crystallographic symmetry was used to determine the structure,
which was then rened against a native data set to 3.5 . This case
is signicant since it represents the lowest resolution Native-SAD
structure determined to date.
3.8. Data reduction
The current generation of fast detectors can collect data at an
astonishing rate producing 10 to 100 images per second allowing
for efcient ultra-ne data slicing (e.g. 0.05 per image). These data
rates can make data storage, data transfer and data reduction
difcult in the home lab. A typical SAD data set (180 of data)
collected using these fast detectors and ultra ne slicing could
contain thousands of images depending on the rotation slice used.
These large data sets can represent hundreds of Gbytes of data that
places demands on both disk space and processor speed. In addi-
tion, transferring this large volume of data over the internet is also
challenging. Thus, many facilities hosting these fast detectors are
providing computational resources for data reduction (either while
on-site or remotely) to eliminate the need for extensive computing
and data storage resources at the user's home site. In addition,
these beamlines, in many cases auto-process the data while it is
being collected [128]. However, it is important to remember that
although auto-processing is fast and efcient, care must be used in
the data reduction process to ensure that the various processing
parameters are optimally set.
 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94 91

Fig. 5. Representative structures that resulted from the theoretical and technological advances of SAD phasing from crambin by S-SAD in 1981 to the recent structure of the
266 kDa T2R-TTL multiprotein complex (a complex between ab-tubulin, stathmin-4 and tubulin-tyrosine ligase) determined by Native-SAD using MDS in 2014. Also included are
the structure of neurophysin, the rst ISAS structure determined by I-SAD in 1989, obelin the second S-SAD structure determined in 2000 using 1.74 X-rays, ferrochelatase
determined by Fe-SAD in 2000 using CuKa X-rays and the 132 kDa TorT/TorSS complex determined Native-SAD using MCA. Images generated using CHIMERA [136].

Most current data reduction programs such as HKL2000/
HKL3000 [119], MOSFLM [129]and XDS [122] can handle the ultra
ne slice data sets produced by these new fast detectors. XDS offers
the additional advantage of parallel data processing on systems
having multiprocessors speeding up the data integration.
3.9. Phasing & structure solution
The SAD phasing process can be broken down into three steps.
The rst step is determining the anomalous substructure (the po-
sition of each anomalous scatterer producing a signal). It requires
only the scaled structure factors containing the Bijvoet pairs and
some idea of the composition of the anomalous contributors. For
Se-SAD, this would be the expected number of selenomethione
residues based on the contents of the crystal's asymmetric unit.
Once the anomalous substructure has been determined the next
step is to generate protein phases and determine the correct hand
of the anomalous substructure. One way to do this is to calculate
protein phases based on the anomalous substructure ( ) and its
enantiomorph (- - -) and compare the resulting map inversion
Rfactor and the map correlation coefcient [4].
A nal check is to look at the electron density maps themselves.
The correct hand (if anomalous signal is present) should produce an
interpretable map (the presence of helices, strands and side chains)
while the incorrect hand will produce a map that is non-
interpretable. If both maps can not be interpreted, then the
anomalous substructure is not correct. Several software packages
such as CCP4, the SHELX-CDE suite [123], Auto-Rickshaw [130],
IPCAS (Iterative Protein Crystal structure Automatic Solution) [21]
and PHENIX can be used to carry out the SAD phasing process.
The bottleneck of the phasing process is the identication of the
correct anomalous substructure and cases where tens of thousands
of SHELX-D trials have been need to nally achieve the correct
solution [45]. To address this, both SHELXD and phenix.HYSS can
92 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94
take advantage of multiprocessor or cluster-based systems to
signicantly speed up the search for anomalous scatterers. In
addition, recent improvements to solution ranking and phase
generation have been reported. These include the inclusion of a
SAD likelihood function to rank possible solutions in phenix.HYSS
[131] and the introduction of a direct phase-selection step prior to
density modication (using RESOLVE or DM) [20]. Both approaches
were shown to signicantly improve the nal experimental phases
and the quality of the resulting electron density maps.
Finally, SAD phasing is critically dependent on having the cor-
rect anomalous substructure. The Native-SAD structure of the West
Nile Virus NS1 protein (PDB entry 4O6C) [132] provides a good
example. The 2.9 structure was solved using MCA and data
collected from 18 crystals, which yielded a data set containing
65,510 Bijvoet pairs (100-fold multiplicity). Analysis of the data
showed that while 100-fold Bijvoet multiplicity was absolutely
needed to obtain the correct anomalous substructure, only data
sets with 30e40 fold Bijvoet multiplicity were required for phasing.
4. Future opportunities
The growth of SAD over the past 30 plus years is illustrated in
Fig. 5. Beginning with the sulfur-SAD structure of crambin the
complexity and size of SAD structures are seen to increase as new
technology and methods become available. This is exemplied by
the recent Native-SAD structures of the 266 kDa T2R-TTL multi-
protein complex and the 132 kDa TorT/TorSS membrane associated
receptor complex.
Se-SAD is dependent on expressing seleno-methionine labeled
protein in E. coli or S. cerevsiae based expression systems. Unfor-
tunately, many of the most interesting systems such as many
eukaryotic proteins, protein complexes and integral membrane
proteins require expression in cell lines that are not amenable to
the production of selenomethionine labeled proteins in the quan-
tities needed for crystallization. This explains the growing interest
in Native-SAD and facilities worldwide are exploring means of
optimizing their facilities to support this approach. Dedicated long
wavelength beamlines have been built in Europe and Japan and the
use of XFEL enabled serial crystallography (using 1.74 X-rays) for
the Native-SAD phasing has been demonstrated [99]. New de-
tectors with millisecond readout times allow shutterless data
collection and provides a means of efcient ne slice data collection
which improves data accuracy and increases anomalous signal to
noise by removing shutter error and background fog, respectively.
The use of radical scavengers, software simulation (RADOSE-3D)
and improved data collection strategies (MDS, MCA and serial
crystallography) mitigates radiation damage another signicant
source of error in the SAD experiment. Improved software for data
reduction (HKL, MOSFLM and XDS) and phasing (CCP4, IPCAS, and
PHENIX) developed over the last three decades has increased the
success rate of SAD phasing such that SAD has eclipsed MAD as the
rst-choice method for de novo structure determination.
These advances have provided a solid foundation for Native-SAD
as reected by the recent reports on applying this method to large
integral membrane proteins and multiprotein complexes [45,133].
However, for Native-SAD to become a rst choice phasing method
(for those systems which are not amenable to selenomethionine
incorporation), it must meet the following criteria: 1) It must be no
more difcult than Se-SAD, 2) It must not require complicated
specialized hardware setups and 3) A majority of the electron
density map should be auto traced by programs such as CCP4,
PHENIX, SHELX-E or ARP/wARP [134]. In other words, Native-SAD
must be easy to do. The success of Native-SAD will also benet
the community since 1) if Native-SAD becomes routine then
virtually all atoms in Table 3 become viable anomalous phasing
probes and 2) the reduction of error needed to make Native-SAD
routine will increase the accuracy of all data.
Acknowledgements
The authors would like to thank Professor Gary Newton
(deceased February 22, 2013) for his foresight in reproducing
Wang's original gures from his 1985 article, some of which are
included in this review. We also acknowledge the University of
Georgia and the Ramsey-Georgia Research Alliance endowment
funds (to BCW) for providing partial support for the preparation of
the manuscript.
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