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Archives of Biochemistry and Biophysics 602 (2016) 80e94

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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
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 SAD phasing offers some distinct advantages over other phasing
phase of a given reection is lost. This is known as the Phase methods presented above. First the technique does not require
Problem in X-ray crystallography. However, this lost phase infor- multiple data sets as in the case of MIR, SIR and MAD. The SAD data
mation is contained in the ensemble of reections, which makes up set is generally collected from a single crystal containing either an
the data set and if the resolution of the data is high enough the anomalous scatterer such as iron which is naturally present in the
phase information for a given reection can be extracted from the protein, selenomethionine which has been incorporated into the
native data set itself. This process is known as Direct Methods [1,2]. protein during expression [7] or an anomalous scatterer that has
Unfortunately, crystals of most macromolecules do not diffract well been introduced into the crystal via solution diffusion similar to
enough to use Direct Methods to accurately estimate phases. Thus, preparing heavy atom derivative crystals [8]. Since all data is
other approaches such as Multiple Isomorphous Replacement generally collected from a single crystal, crystal nonisomorphism is
(MIR) [3], Single Isomorphous Replacement (SIR) [4], Single- not a problem. Another advantage of SAD phasing is that unlike
wavelength Anomalous Scattering (SAS now called SAD) [4,5] and MAD, SAD does not dependent on the data collection spanning an
Multi-wavelength Anomalous Dispersion (MAD) [6] have been X-ray absorption edge of the anomalous scatterer, which precludes
developed for de novo phase estimation. These approaches are elements such as cadmium, iodine, xenon, cesium and sulfur from
limited by the requirement of the presence of one or more phasing being used as MAD phasing probes. SAD phasing also does not
probes in the protein or crystal lattice; heavy atoms in the case of require the subelectron volt energy resolution of the MAD experi-
MIR or SIR phasing or anomalous scatterers in the case of SAD or ment and the wavelength dependent nature of the anomalous
MAD phasing. This review will focus on the history of SAD phasing, scattering signal means that for most elements an anomalous
its impact on current structural biology and future application that scattering signal sufcient to solve the structure can be obtained
extend beyond structure solution. within the tunable wavelength range (0.70 to 2 ) of most syn-
chrotron beamlines.
This article is part of a Special Issue entitled Protein Crystallography, edited by
Ana Camara-Artigas and Jose Antonio Gavira.
1.2. A brief history on the evolution of SAD phasing
* Corresponding author.
E-mail addresses: (J.P. Rose), (B.-C. Wang). SAD phasing had its beginnings nearly 70 years ago. The
0003-9861/ 2016 Elsevier Inc. All rights reserved.
J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94 81


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 Fig. 1. A Harker construction illustrating the SIR protein phase ambiguity. Here the SIR
SSGCID Seattle Structural Genomics Center for Infectious phase (fSIR) is the average of the true and false phases.
XFEL X-ray Free Electron Laser
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.
anomalous scattering effect which causes the breakdown of Frie- The elimination of the need for preparing an isomorphous heavy
del's law was rst demonstrated when two groups of investigators, atom derivative was an obvious advantage for SAD compared to SIR,
S. Nishikawa and K. Matukawa in 1928 [9] and D. Coster, K.S. Knol but there were other reasons. To appreciate these other advantages,
and J. Prins in 1930 [10] tried to determine which face, the shiny let's compare the information content in SIR and SAD data from our
face or the dull face, corresponded to the Zn-layer in the zincblende current point of view (Fig. 2a and b). Although both SIR and SAD
(sphalerite, ZnS). They carried out diffraction studies on the consist of two sets of measurements (i.e. jFPHj & jFPj for SIR and
opposite [(111) and (-1-1-1)] faces of the zincblende crystal using jF()j & jF()j for SAD), using SIR data alone one may not easily
Au La1 radiation, whose wavelength is slightly shorter than the Zn K recover the anomalous scattering contributions of the heavy
absorption edge. Based on the differences in diffraction intensity atom(s) if these anomalous contributions (differences) were not
observed for the two faces the S-layer, not the Zn-layer, was found experimentally measured. However, for SAD data one can easily
to correspond to the shiny side of zincblende crystals. calculate the diffraction vector (intensity and phases) of the real
Although an anomalous scattering effect was clearly demon- part of the scattering factor of the heavy atom, once the locations of
strated, it was not until some 20 years later that Bijvoet and his the heavy atom sites are determined. As illustrated in Fig. 2b, the
colleagues realized that anomalous scattering techniques might aid calculated diffraction vector FH lies orthogonal to the FH00 vectors
in the phase determination process [11] in addition to its routine obtained from the measured anomalous scattering data (provided
use for determining absolute molecular conguration [12]. The use that heavy atoms are of the same kind). Thus, in principle this
of anomalous scattering for phasing soon attracted considerable orthogonal information which can be extracted from the SAD
interest [13e15]. Various approaches of using anomalous scattering data could provide the third piece of information needed to
were postulated, including the combined use of anomalous scat- partially break (resolve) the phase ambiguity in SAD data. If one
tering with isomorphous replacement for phasing macromolecules applies a similar approach to SIR data this critical third piece of
[16,17]. information which lies orthogonal to the heavy atom vector in the
The idea of the combined use of anomalous scattering and SAD case now lies parallel to it and thus cannot be used to break
isomorphous replacement data for phasing macromolecules the SIR phase ambiguity. This unique characteristic of SAD data
signied the recognition of phase ambiguity problems (Fig. 1) perhaps was the basis behind continued efforts in early SAD
associated with the use of either single wavelength anomalous phasing development by this small group of scientists.
scattering (SAD) data or single isomorphous replacement (SIR) There was also considerable interest in using Direct Methods
data, and that with a proper combination of the SAD and SIR data [19] to address the phase ambiguity problem in SAD or SIR data by
sets, the phase ambiguity problem could be resolved. But, what adding probability constrains based on phase relationships. How-
happens, in the case where we have only SIR or SAD data available, ever, the atomic resolution data needed for successful Direct
how can we overcome the phase ambiguity problem in this case? Methods phasing limited the use of this approach for macromole-
It was soon recognized that if the anomalous scatters were cules, since even today few protein crystals diffract beyond 1
heavy atoms (they were at the time), the phase ambiguity problem resolution. This approach has also been referred to in the literature
in SAD data might be resolvable [14,18] and would be advantageous as the reciprocal space approach. As will be described in a later
over the use of only SIR data. However, at the time it was not easy to section, new developments in the reciprocal space approaches have
measure accurate anomalous scattering data. This begs the ques- now been incorporated in several modern crystallography software
tion as to why there was interest that using SAD data alone might packages to enhance the phasing results [20,21]. The early devel-
be advantageous for phasing compared with using SIR data? 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. Looking back, this heavy atom correlation may be understood
Now back to the historical trail. Concerning the real part of the from the point of view of the Heavy Atom method that was well
heavy atom scattering contribution to SAD data, one knew that it used at the time for small molecule structures such as vitamin B12
could potentially play a role in resolving the SAD phase ambiguity, [27] or organic salts which contained a heavy atom. This was based
but how far could this approach go? Some thought that basically on the idea that the phase of the total structure (fT) will tend to
the phasing power of single wavelength (SAD) data might not be move toward the phase of its imbedded heavy atoms (fH) when the
sufcient for phase resolution and others thought that the phase heavy atom's size (number of electrons) or the mean fractional
ambiguity problem could be resolved if diffraction data were contribution of the heavy atom to the structure increases. Thus,
measured at several frequencies across the absorption edge of an quasianomalous-dispersion synthesis is in principle similar to the
anomalous scatter [23]. A more advanced use of this multi wave- Heavy Atom phasing method where the phases of the heavy
length approach, incorporated in MAD phasing [6], has certainly atom(s) are taken as the initial phases of the overall structure. There
had a signicant impact on protein crystallography for the past two is however an important difference between these two methods in
decades. Here, we are tracing the ideas and efforts of those who that the calculated heavy atom phase is not directly used in the
believed in SAD and how their accumulated improvements to the quasianomalous-dispersion synthesis, but is used instead as a
use of the heavy atom contribution for phase resolution, although reference phase for selecting a probable true phase from the false
not totally successful at the time, stimulated SAD to continue to phase. That is the phase closest to the heavy atom phase is selected
advance to nally become a major method for macromolecular as the probable true phase in the initial map calculation. So, phase
crystallography today. selection in quasianomalous-dispersion synthesis was actually an
Through a series of studies using known and unknown small improvement over the traditional Heavy Atom method.
molecule structures, Ramachandran and colleagues, including The quasianomalous-dispersion synthesis (also referred to as
Raman, Srinivasan and Parthasarathy [18,24,25] tested the role of the Bijvoet-Ramachandran-Raman method [28] has been used to
the heavy atom scattering contribution to phasing success and determine a number of structures, including the crystal structure of
summarized their results in a table showing that the probability of Factor V1a, an aquocyanide B12 variant using the anomalous scat-
resolving the SAD phase ambiguity would increase with the in- tering of cobalt [29]. Notably, the approach was proposed by
crease of s21 (the fractional mean contribution (real part) of the Ramachandran and Parthasarathy in 1965 as a method for protein
heavy contribution to the overall structure) (Table 1). For example, structure determination [26], which is unfortunately seldom cited
with s21 of 0.2 the success rate for identifying the true phases from in the literature. This possibility of protein phasing was further
the false phases is over 70% [26], sufcient for structure determi- discussed by Argos and Mathews in 1973 using the anomalous
nation. They called their approach quasianomalous-dispersion scattering signal of the heme iron in cytochrome b3 (PDB entry
synthesis. 1CYO) [30].
Another theoretical development aimed at handling of noise
in diffraction data was the introduction of a phase probability
Table 1
approach, which used best phases and gure-of-merits for
Percentage of Reections Selected Correctly as a Function of s21. electron density map calculations [16,17,31,32]. Maps that were
calculated using this approach were proven to be superior to ones
s21 1 Atom 2 Atoms Many atoms acentric Many atoms centric
generated by selecting the most probable phases on the basis of
0.2 76.0% 73.0% 72.0% 69.5% which phase lies closer to the heavy atom phase. This approach has
0.4 88.5% 82.3% 81.5% 77.5%
been successfully applied in the calculation of protein phases using
0.6 96.0% 80.0% 88.5% 83. 0%
0.8 99.8% 93.5% 94.5% 89. 0% the multiple isomorphous replacement (MIR) method, where three
or more sets of diffraction data from heavy atom derivatives are
Table adapted from Ramachandran & Parthasarathy, Science 150, 212e214, 1965.
Calculations based on phase differences less than 90 from the Heavy Atom phases. required [28].
s21 is the mean fractional intensity contribution of the anomalous scatters to the In 1980, Hendrickson and Teeter [5] applied phase probability
intensity of the total structure.
J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94 83

methods to SAD phasing by replacing the phase selection process anomalous scattering data and was called Iterative Single Anoma-
used in quasianomalous-dispersion synthesis. They termed this lous Scattering (ISAS) method. A software package (the ISIR/ISAS
approach Resolved-Anomalous Phasing (RAP). Although their program suite) [4] was developed and its Fortran code was
approach was successful in determining the structure of crambin, distributed freely to the crystallographic community upon request.
Protein Data Bank (PDB) entry 1CRN [33] (the rst protein SAD Wang's overall procedure is commonly known as solvent at-
structure), a 46-residue protein containing 6 sulfur atoms, RAP did tening and was the key advance in removing two of the critical
not become a general method for SAD structure determination. The bottlenecks for the phasing of macromolecular structures: 1) the
incorporation of phase probability evaluation over strict phase se- phase ambiguity problem in using SIR or SAD data and 2) the
lection in their approach improved phase accuracy, however the requirement that the protein must have a large heavy atom (or
fundamental phasing concept was similar to that of the sulfur) content as required by the RAP method used to determine
quasianomalous-dispersion synthesis, which required a signicant the S-SAD structure of crambin. The effective removal of these two
heavy atom scattering contribution (sulfur in the case of crambin) bottlenecks was demonstrated by Wang who used his ISIR/ISAS
to the overall structure. Because of this inherent limitation of the program suite to determine the SIR structure of the Bence Jones
approach, RAP was unable to eliminate the need of a large heavy protein Rhe (PDB entry 2RHE) using a data collected from a single
atom scattering contribution (~20%) to overall structure and typical gold derivative, and the S-SAD structure of Rhe (113 residues, 2
proteins have a much smaller sulfur content. sulfur atoms) using error free simulated data [4]. In terms of
In early 1980s, Wang took a different approach by using a rough method development, this approach differs from the earlier
structural model of the total protein structure instead of the partial methods in that it was not developed for the exclusive use of SAD
structure of the anomalous scatterers, to provide the initial infor- data.
mation needed to distinguish the true and false phases [4]. Thus, in The method proved to be generally applicable to all SIR or SAD
principle this approach uses the protein structure itself to provide data, including sulfur-SAD and provided the foundation for routine
nearly 100% of the information that is needed for the phase selec- structure determination of proteins having more typical sulfur
tion process in the quasianomalous-dispersion synthesis. Wang's content. Notable early ISIR structures include the structure of
approach was originally developed for the use of SIR data where, as troponin C (PDB entry 4TNC) [36], the DNA-Eco RI endonuclease
noted above, the calculated heavy atom contribution cannot be recognition complex (PDB entry 1ERI) [37] and the histone octamer
used to resolve the SIR phase ambiguity. Because of this a search for (PDB entry 1HIO) [38,39]. The rst example of using the Wang's
an alternative method for resolving SIR phase ambiguity was process for a de novo structure determination from SAD data did not
needed. The initial rough structural model was obtained by come until 1989 with the determination of the bovine neurophysin
applying an automated protein-solvent boundary determination II dipeptide complex (PDB entry 2BN2) [40,41]. The structure was
algorithm [4] to dene the shape of the molecule, its location and determined by I-SAD using a bound iodinated hormone analogue
orientation (equivalent to a molecular replacement solution [34,35] (p-iodo-Phe-Tyr amide), which mimics hormone binding in the
although no search model was needed). In addition, a repeated active site. To end this summary of early SAD history we present
cycling process between reciprocal space for probability combina- below a brief description of the rst ISAS structure determination of
tion and direct space for noise ltering (solvent attening) was the bovine neurophysin II dipeptide complex (the second SAD
used to gradually make the initial SAD phases converge toward the protein structure determined) to illustrate the practical aspects of
correct protein phases (Fig. 3). The approach was named the Iter- then newly developed SAD phasing method, its capabilities, its
ative Single Isomorphous Replacement (ISIR) method. The ISIR routine determination of the handedness of the anomalous sub-
method was then extended for the use of single wavelength structure and its phase extension capabilities in combination with

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.
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 that the hand of the anomalous substructure is . The
using I-SAD data collected using 5 kW CuKa X-rays (Df00 (I) 6.8 for resulting ISAS phased electron density map was of excellent quality
CuKa X-rays) generated by a rotating anode generator (Rigaku) and and manually traced (388 resides) in 3e4 days.
focused by mirror optics (Charles Supper) with the diffraction It is interesting to note that the 1989 SAD structure determi-
pattern recorded on an X100 imaging proportional counter nation of neurophysin used many of the techniques still in use
(Siemens). Data were collected at 4  C using a crystal today, including labeled amino acids to introduce an anomalous
(0.75  0.35  0.25 mm) mounted in a glass capillary and adjusted scatterer, crystal translation during data collection to mitigate ra-
using the arcs on the goniometer head so that one of the crystal's diation damage, an inverse beam data collection strategy to reduce
symmetry axes (space group P212121) was along the rotation-axis of systematic error, ne slicing and multi dataset averaging to in-
the goniometer. The inverse beam method (20 wedges) using a crease signal to noise and a photon counting detector.
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- 1.3. Current impact
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 Over the past two decades tremendous advances in diffraction
crystal translated after the rst experiment to place a fresh crystal hardware, crystallographic software, data collection methods and
volume in the beam. After data reduction and scaling [42] the nal data collection strategies have been made which has resulted in
data set consisted of 58,983 observations of 12,840 Bijvoet pairs SAD becoming a rst choice phasing method. The introduction of
and gave an Rsym(I) of 0.069. Matthews analysis [43] suggested MAD phasing in the mid 1990's coupled with selenomethionyl
that there were four neurophysin molecules (~40 kDa) per asym- protein labeling and undulator based 3rd generation synchrotron
metric unit and analysis of the Bijvoet difference Patterson maps beamlines revolutionized macromolecular crystallography. Sele-
conrmed this giving the position of the four iodine sites. Using the nomethionine labeling [7] provided both a strong (Df00 (Se) 3.89
2.8 SAD data set, the iodine substructure and a 60% solvent e) anomalous phasing probe (at high occupancy) whose anoma-
content the ISAS phasing process was carried out. The phasing lous scattering electron density could also be used as guide points
process used 20 cycles of iteration and three lters (see Table 2). during the tting process. Because of these advantages, selenomet-
The initial lter and four phasing cycles of iteration were used to MAD quickly became the method of choice for de novo macromo-
resolve the phase ambiguity using 3.5 data. A new lter was then lecular structure determination. However, beginning in 2000 SAD
generated followed again by four phasing cycles and four additional phasing began to gain ground. Today, SAD has become the method
cycles of phase extension to 2.8 resolution. A third lter was then of choice for de novo macromolecular structure determination with
generated followed by a nal round of four phasing cycles and four around 80% of de novo macromolecular structures being deter-
phase extension cycles to give the nal phases. During the phasing mined by the method (see Fig. 4). A survey of Protein Data Bank
process both the and - enantiomers of the heavy entries [33] in the period spanning 1979 to 2015 (Sept 2015)
atom substructure were analyzed to determine the correct hand- showed that 5736 structures were determined using MAD phasing
edness of the sites (Table 2). The nal phase extension cycle had an compared to 7549 determined using SAD with 35 MAD and 221
average gure of merit of 0.67, a map inversion R factor of 0.286 and SAD structures being deposited to date in 2015.
a correlation coefcient of 0.974 for the -handed enan- This article will focus on advances in technology and method-
tiomer and an average gure of merit of 0.64, a map inversion R ology that have caught the community's attention. It will highlight
factor of 0.329 and a correlation coefcient of 0.960 for the 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-
Table 2 fully will be a future follow-up article with additional information
ISAS phasing cycles and corresponding handedness test parameters. contributed from the community.
ISAS () Hand () Hand
1.4. Choice of anomalous scatterer
Filter Cycle m R CC m R 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
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 85

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 collected in the home lab using copper (l 1.5418 ;
the criteria that an element must meet or exceed the limit of Df00 (Se) 1.15) [52] or chromium (l 2.2909 ; Df00 (Se) 2.30) [53]
(Df00 0.70), thus most of the elements listed in Table 3 can (in X-rays.
theory) provide the needed anomalous scattering signal for struc-
ture determination, provided that the data are measured 1.4.2. M-SAD
accurately. M-SAD exploits the anomalous scattering signal of metals (V,
The SAD phasing experiment itself can be grouped into four Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, W) found in metalloproteins, which
general classes depending on how the anomalous scatterer is represent over 30% of the proteins encoded by the human genome.
introduced into the crystal: Se-SAD, M-SAD, X-SAD and Native-SAD. Most of these metals have a convenient absorption edge (Table 3) or
A short description of RIP (Radiation Damage Induced Phasing) will strong (Df00 greater than 3.00) anomalous scattering signal in
also be described below, since it uses only single crystal with data wavelength range from 0.73 to 2.00 (17 keV - 6.2 keV), common
collected at single wavelength. to most macromolecular synchrotron beamlines, which can be
exploited for SAD structure determination. In addition, several
1.4.1. Se-SAD Table 3 elements (V, Mn, Fe, Co, Mo, Cd) produce a signicant
Se-SAD is the most common phasing method for de novo anomalous scattering signal in home source data collected with Cu
structure determination. The Se-SAD experiment uses selenium radiation and several early M-SAD structures such as ferrochelatase
labeled proteins produced by expressing the protein in a methio- solved by Fe-SAD (l 1.5418 ; Df00 (Fe) 3.18; PDB entry 1HRK)
nine auxotrophic E. coli strain and adding L-seleno-methionine to [54] and oxalate decarboxylase solved by Mn-SAD (l 1.5418 ;
the growth media after induction [7]. Similar systems have also Df00 (Mn) 2.79; PDB entry 1UW8) [55] were determined from
been developed for the eukaryotic expression of selenomethionyl home source data.
proteins [49e51] and commercial kits are available from several
vendors. 1.4.3. X-SAD
The Se-SAD experiment is similar to the Se-MAD experiment X-SAD represents the case where the anomalous scattering
except that only one data set is collected at or near (on the higher probes, such as iodine, bromine, or metal ions are introduced into
energy side) the Se absorption maxima (l 0.9795 ) where the Se the crystal by 1) co-crystallization, 2) soaked into the crystal by
anomalous scattering signal is greatest (Df00 3.85). Since the traditional methods [8,56] or 3) introduced into the crystal via high
wavelength of the selenium absorption edge can be inuenced by pressure gas incubation. Bovine neurophysin II, the rst SAD
its environment, a good strategy is to always perform a wavelength structure determined by Wang's ISAS method [4] was determined
dispersive uorescence scan of the crystal to determine the exact by I-SAD using home source data (l 1.5418 ; Df00 (I) 6.8; PDB
wavelength of the Se absorption edge prior to data collection. entry 2BN2) [40,41]. The study used a bound iodinated oxytocin
While synchrotron data collection at the Se absorption edge is the analogue (p-iodo-Phe-Tyr amide) which was co-crystallized with
most common source of data used for Se-SAD phasing, successful the protein. Iodine has proven to be a popular X-SAD phasing probe
Se-SAD structure determinations have been reported for data 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 (
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 Amino-2,4,6-triiodoisophthalic acid) [60] and clusters containing
range from 0.98 (Se absorption edge) to 2 . A method of preparing tantalum (Ta6Br12) [61,62] and tungsten (Na3[PW12O40]$H2O,
iodinated and brominated derivatives by adding the halogenated Na6[H2W12O40]$H2O and [NH4]10[H2W12O42]$H2O) [63] from Jena
salt to the cryoprotectant drop prior to cryoprotection is available Bioscience are commercially available. Both tantalum
[57,58] which simplies the derivation process. Brominated de- (Df00 (Ta) 12.22 at l 1.0247 ) and tungsten (Df00 (W) 12.16 at
rivatives can be exploited for SAD data collection at the Br ab- l 1.0247 ) have X-ray absorption edges close to the selenium
sorption edge (l 0.9202 ; Df00 (Br) 3.83). A method employing absorption edge, which can be exploited for SAD phasing. In
longer iodine/bromine soaks (5 mine2 h) has been adapted to high addition, at low resolution (4 e 6 ) these large tantalum and
through workows by researchers at the Seattle Structural Geno- tungsten clusters produce an anomalous super-atom that can be
mics Center for Infectious Disease (SSGCID) who were successful in readily identied in Bijvoet difference Patterson maps.
determining the structure of 16 of 17 targets during its initial 12 The Nobel gases krypton and xenon introduced into the crystal
month trial [59]. under high pressure can also serve as anomalous scattering probes
Metal ions introduced by soaking can also serve as phasing [64e67]. Krypton produces an anomalous scattering signal at its X-
probes providing that the binding is specic and their occupancy is ray absorption edge (Df00 (Kr) 3.81 at l 1.0247 ) similar to
high. Heavy metal (Ag, Cd, La, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Yb, W, Re, selenium signal used in Se-SAD/MAD experiments and xenon
Os, Ir, Pt, Au, Hg) derivatization kits from Hampton Research and produces a large anomalous scattering signal at longer wavelengths
specialized phasing probes such as the IC3 Magic Triangle (5- (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 87

from xenon pressurized crystals has also been used to trace sub- goniometer [83]. Loop mounting reduces stress on the crystal
strate/solvent channels within the protein structure [68,69]. during the mounting process and can be used to harvest and mount
crystals from microliter to nanoliter crystallization setups. How-
1.4.4. Native-SAD ever, the cryocooling process can introduce noise into the SAD
Native-SAD uses the anomalous scattering signal of sulfur, experiment in the form of higher image backgrounds, reection
phosphorous or other light atoms (Z less than 21) which are present crowding (due to high mosaicity) and the presence of ice rings in
in the crystalline sample (e.g. sulfur or phosphorus present in the the pattern. Two approaches have been developed to limit this
biological sample from protein, DNA or RNA, and other ions inad- phenomenon: the use of cryoprotectants and high-pressure cry-
vertently introduced during protein purication or crystallization ocooling under helium gas.
process) as phasing probes [70]. Native-SAD represents the ulti- The use of small molecule cryoprotectants such as 20e30%v/v
mate goal in SAD phasing since if the anomalous scattering signal of glycerol is the most common means of cryoprotecting the crystal
sulfur can be routinely used for macromolecular structure deter- during the ash cooling process and several excellent reviews have
mination, then virtually all elements listed in Table 3 can be used. been written on this subject [84,85]. High-pressure (200 MPa)
cryocooling has been shown to provide excellent diffraction from
1.4.5. MR-SAD non-cryoprotected crystals [86,87]. This approach is based on
Molecular replacement assisted SAD uses molecular replace- myoglobin cryoprotection studies [88] and the phenomenon that
ment to provide initial phases for determining the anomalous water under high pressure freezes as ice III which contracts upon
substructure and thus bootstrap the phasing process [71,72]. This is freezing compared to ice I which expands. The technique is less
critically important for phasing using weak anomalous scatterers common since it requires specialized apparatus. However, two
such as sulfur and phosphorous. The technique also provides a commercial units are available: the HPC-201 from Advanced Design
simple method of overcoming model bias common to molecular Consulting USA ( and HPM-010 system from
replacement structure determination and in cases where the BAL-TEC AG ( The technique has been recently
experimental phase information is weak. MR-SAD also assists in used in the structure determination of bovine enterovirus 2 from a
model building by providing marker atoms and validate the iden- single high pressure frozen crystal (PDB entry 1BEV) [89].
tity of anomalous scatterers for renement.

1.4.6. Radiation damage induced phasing

Although not strictly an anomalous scattering technique Radi- 2.3. Mounting pins & loops
ation Damage Induced Phasing (RIP) [73] shares many of the ad-
vantages of SAD in that: 1) all necessary data can be collected from Several loop-pin designs are commercially available for har-
one crystal, 2) the technique does not require data collected at an X- vesting and mounting crystals for data collection at cryogenic
ray absorption edge and 3) the data is collected at a single wave- temperatures but care must be taken when choosing a specic pin-
length. However, the technique is more similar to SIR requiring two loop design. The Recent reports have shown that choice of pin-loop
distinct (before and after radiation damage) data sets. It differs design can effect data quality [90,91]. The studies revealed that the
from SIR in that both data sets are collected on the same crystal. The loop stem (the area between the pin head and loop) and diameter
before radiation damage data set (native data) is collected rst. The of the loop are the most important factors when choosing a pin/
crystal is then exposed to high dose X-rays or UV [74,75] to loop design. It is recommended that the stem be as short as possible
essentially denature the substructure in the protein and the after and the loop diameter be chosen to t the size of the crystal (to
radiation damage data set is collected. Today, many common minimize the amount of mother liquor in the loop). For nylon loops
phasing packages (SHARP [76], SHELX C/D/E [77], CCP4 [78] and they recommend reinforcing the loop stem with epoxy or grease to
PHENIX [79]) can be used to generate phases form the RIP data sets. reduce or eliminate vibration of the loop in the cold stream during
data collection. This is especially important when the anomalous
2. Sample preparation signal is low, such as Native-SAD experiments or when data is
collected at high speed (rates > 2 Hz).
The SAD experiment is critically dependent on maximizing the The scatter from the loop and the mother liquor surrounding the
anomalous signal-to-noise ratio in the data. A source of noise that is crystal is another source of noise in the experiment. Several
often overlooked in the experiment is the sample itself: the crystal, methods have been developed to address this source of noise,
its cryoprotection and the loop/pin assembly used to mount it. including the loopless mounting method [92,93], graphene wrap-
ping [94] and the use of gel beads [95]. In loopless mounting the
2.1. Crystals crystal is harvested using the loop and then by using a special pin
the mother liquor is removed by aspiration and the crystal ash
Generally speaking better crystals give better data in terms of cooled in a nitrogen gas cryostream. Once frozen, the loop is
resolution, mosaicity, data accuracy and anomalous signal; all carefully removed using small hook, forceps or laser leaving the
important to successful SAD phasing. Thus, a little time spent crystal mounted directly on the pin ready for data collection. Gra-
producing better crystals (and their cryoprotectant cocktails) can phene wrapping as the name implies wraps a thin multilayer (3e5
often reduce the amount of time and effort needed to solve the layers) of grapheme around the loop mounted crystal trapping a
structure. Protein crystallization and optimization has been the small amount of mother liquor together with the crystal. The
focus of several recent reports by McPherson [80,81] and D'Arcy multilayer layer graphene sandwich is essentially transparent to X-
[82], which can be used for reference. rays and produces signicantly lower scatter compared to loop
mounted crystals. Gel bead mounting uses crystals grown in ioni-
2.2. Cryoprotection cally cross-linked polysaccharide gel beads by the microbatch un-
der oil technique. The approach reduces mechanical damage to
Most SAD data are collected at cryogenic temperatures to reduce crystals during mounting and the porous nature gel bead also al-
radiation damage during data collection using crystals mounted in lows for cryoprotectection, ligand soaking and heavy atom deri-
pin/loops assemblies which are magnetically attached to the 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 or near this wavelength. However, for the other elements in Table 3
whose maximum Df00 value lies outside this wavelength range the
As mentioned above, successful SAD phasing is dependent on choice of wavelength is dependent on several factors including the
maximizing the anomalous signal to noise ratio in the data. effects of X-ray absorption and beam stability at longer wave-
Experimentally, this can be done by either increasing the anoma- lengths. These effects introduce noise into the experiment and can
lous signal (mainly by increase wavelength used) or reducing noise impact the accuracy of the data and the measured anomalous
by various means. Our discussion will start with the use of Cu Ka scattering signal, especially when the anomalous scattering signal
home X-ray sources and then extend to the use of synchrotron X- is weak, as in the case of Native-SAD or when the occupancy of a
rays as outlined below. strong anomalous scatterer is low. A more detailed analysis of an
elements strength of the anomalous scattering (Df00 ) as a function of
3.1. X-ray source wavelength can be found on the University of Washington's
anomalous scattering web site (
It is no surprise that in-house generated Cu Ka radiation, which scatter). Collecting data at longer wavelengths is a general
provides single wavelength X-rays, played a key role in the devel- approach of increasing the anomalous signal for elements in Table 3
opment and early success of SAD structure determination. These that lack an absorption edge since the magnitude of Df00 tends to
early structures include, crambin determined by sulfur-SAD increase with wavelength. X-ray absorption also increases with
(Df00 (S) 0.56 e) data collected using a sealed tube X-ray source wavelength but can be minimized by careful crystal mounting
(Ni ltered) and a Picker FACS-1 4-axis diffractometer [5]; neuro- (described below) and the use of a helium lled beam path be-
physin determined by iodine-SAD (Df00 (I) 6.91 e) data collected tween crystal and detector.
using mirror focused (Supper) 5 kW rotating anode X-rays and a Beam stability is more problematic since it can result from a
Siemens X100A multiwire area detector [41]; and ferrochelatase combination of several factors: 1) positional instability of the syn-
(PDB entry 1HRK) determined by iron-SAD (Df00 (Fe) 3.18 e) data chrotron's electron orbit [100], 2) X-ray beam drift due to thermal
collected from three crystals using focused (Rigaku Osmic copper deformation of optical components as the energy changes and 3)
confocal optics) 6 kW rotating anode X-rays on two different R-Axis mechanical vibrations of components in the optical chain especially
IV image plate area detector systems [54]. in the case of microbeam/microcrystal experiments.
Obelin, the second de novo sulfur-SAD structure (PDB entry Since selenium and approximately 20% of the other elements
1EL4) was one of the rst SAD structures determined using syn- listed in Table 3 have an X-ray absorption edge in the range of
chrotron X-rays. The structure was determined using data collected 0.77e1.3 , beam stability at these wavelengths should not present
on beamline 17ID, Advanced Photon Source with 1.74 X-rays a serious problem. For the other elements that require data
(Df00 (S) 0.70 e) by a MAR Research MX165 CCD detector [96]. collection at longer wavelengths, the ideal solution would be a
The introduction of chromium confocal optics (Rigaku Osmic) in purpose-built, dedicated soft-X-ray beamline. Such facilities have
2002 provided a stable home source soft-X-ray platform for SAD been built at the Photon Factory (BL-A1) in Japan, and at the Dia-
data collection [70,97]. Since then 31 Native-SAD structures have mond Light Source (I23) in the United Kingdom. Lacking a dedi-
been determined using chromium Ka (l 2.29 , Df00 (S) 1.14 e) cated soft X-ray beamline, beam instability due to thermal effects at
X-rays. longer wavelengths can be easily addressed by simply waiting until
Although copper and chromium rotating anodes provide a the thermal drift stabilizes after an energy change. Unfortunately,
highly stable X-ray source, today most SAD structure de- this process could take from several minutes to several hours
terminations are carried out using synchrotron data since this depending on the beamline design. Thus, the time required for
approach offers two distinct advantages over in-house data beamline thermal equilibration after energy changes (at both the
collection: 1) the ability to tune the wavelength to enhance the low and high energy end points) should be considered when
anomalous scattering signal and 2) small high intensity X-ray applying for beam time.
beams that allow efcient data collection on crystals too small for Based on experiments conducted at wavelengths ranging from
the home source. However, one must remember that most syn- 0.80 to 2.65 , Mueller-Dieckmann et al. have proposed that the
chrotron beamlines were designed and optimized to support Se- optimal wavelength for sulfur-SAD data collection is 2.1 [101].
MAD data collection at the selenium absorption edge However, few structures have been reported using synchrotron
(l 0.9795 ) and for the collection of high-resolution data. If data data collected at wavelengths above 2 . Instead, the majority of
collection at longer wavelengths is required, then beam stability Native-SAD structures determined with synchrotron X-rays used
and X-ray absorption can become problematic, especially in the data collected at wavelengths in the range of 1.7e1.8 [70]. James
case where the anticipated anomalous signal is weak. Holton has also proposed collecting SAD data using 1.714 X-rays
Finally two recent papers using serial femtosecond crystallog- (for Table 3 elements that lack an X-ray absorption edge) to reduce
raphy (SFX) to determine the Gd-SAD phased [98] (PDB entry systematic errors related to detector pixel nonuniformity issues
4N5R; LCLS; l 1.46 ; 60,000 images) and Native-SAD phased (termed Spatial Heterogeneity in Sharp Spot Sensitivity) that are
[99] (PDB entry 4YOP; SACLA; l 1.74 ; 150,000 images) struc- not addressed by current calibration procedures [102]. However, 31
tures of lysozyme have been reported which illustrate the feasi- Native-SAD structures have been determined using home source
bility of determining de novo protein structures using SAD and 4th chromium X-rays (l 2.29 ) and an image plate detector tted
generation X-ray sources. Importantly the SFX experiment pro- with a helium beam path.
duces data that are free of radiation damage - another source of
error in the SAD experiment. 3.3. Goniometer

3.2. Wavelength Collecting accurate data requires keeping the crystal centered in
the X-ray beam during data collection. This task becomes more
For those elements in Table 3 that have an X-ray absorption edge challenging as crystal size and/or beam size become smaller as
close (0.3 ) to the selenium edge at 0.9795 the choice of careful crystal centering is essential to keeping the crystal in the
optimal wavelength is straightforward, since most synchrotron beam during data collection. Modern goniometers such as the
beamlines have been designed and optimized for data collection at MD2/MD3 microdiffractometers developed at the ESRF provide on-
J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94 89

axis crystal viewing, which makes crystal centering much easier millisecond or better readout time for these detectors enables
[103]. These systems are found at many beamlines around the shutterless data collection, reducing noise associated with shutter
world and are available from Bruker/ARIMAX (http://www.bruker- jitter and synchronization error, and also allows for efcient ne- In addition to on-axis cameras, these systems also provide slice data collection reducing background fog on the image,
a set of user selectable pinholes to dene beam size and other tools which increases the anomalous signal-noise in the data.
to help the user verify both beam focus and that the crystal is
properly centered in the beam. 3.5. Data collection strategies for increasing signal to noise
Automated diffraction-based centering (raster centering) is also
available at many beamlines. During the rastering process the Once source and hardware considerations have been taken into
crystal is translated in an x, y, z grid (step size dependent on beam account the most general approach of increasing the anomalous
size) and a diffraction image is recorded at each position using a signal-to-noise of the data is to collect data sets with high multi-
highly attenuated X-ray beam [104]. High speed detectors make plicity (redundancy) from one or more crystals. This is because the
this a quick and efcient process. The images are then used to error associated with a measurement decreases as the square of the
determine the point (or points) of optimal diffraction, which can number of observations. The technique is routinely used in the
then be used to dene the crystal's center or hot spots for data Native-SAD experiment where the anomalous scattering signal is
collection. Modern goniometers coupled with small beams and weak, to increase the anomalous signal-to-noise in the data
virtual motors offer the ability to translate the crystal during data [111,112]. Increased redundancy also improves the accuracy of the
collection with the aim of continually introducing a fresh portion of measurements, which in turn produces more accurate anomalous
the crystal into the beam to minimize radiation damage [105,106]. differences, better phases and electron density maps showing
Generally, two translational data collection modes are provided, greater detail. Together this increases the success rate of SAD
translational (or segmented mode) and helical mode. In segmented phasing, aids in model building and produces structures of higher
mode, the crystal is divided into domains (dependent on crystal quality [113]. However, adding images to increase multiplicity also
dimensions and beam size) with the length and direction of increases the likelihood of radiation damage even at cryogenic
translation determined by centering the crystal at its two ends (or temperatures [84,114].
the ends of the desired translation vector). Data is then collected In the diffraction experiment, the loss of diffraction intensity
sequentially from domain 1 to domain N with the number of im- during the course of the data collection is an indication of radiation
ages determined based on both the total number of images desired damage. Structurally speaking radiation damage is manifested by
and the number of domains available. A similar approach can be the breaking of disulde bonds, loss of CO2 from aspartic and glu-
used to center multiple crystals mounted on a grid or in small wells tamic acid side chains, loss of the tyrosine hydroxyl group and the
on a micro crystallization plate such as the X-Chip [107]. In helical formation of free radicals. Thus, crystal lifetime must be taken into
scan mode, the length and direction of the translation vector is consideration when designing a data collection strategy. Based on
again dened by centering the crystal at the two end points. The the theoretical X-ray dose of 2.2  107 Gy (J/kg) [115] needed to
crystal is then slowly translated along this vector during data reduce diffraction power of a protein by 50%, a crystal lifetime of
collection. These data collection methods are very attractive since ~17 s is obtained for a typical insertion device beamline at the
SAD phasing generally requires data sets with high reection Advanced Photon Source using 12 keV X-rays (data taken from
multiplicity and methods to minimize radiation damage are In addition,
benecial. an estimate of the crystal's theoretical half-life can be calculated
Finally, some facilities offer multi-axis goniometers, which can using RADDOSE-3D [114] based on beam parameters (size, shape,
take advantage of crystal symmetry and/or habit to optimize the ux and energy) and the crystal's composition, which will allow
anomalous signal-to-noise ratio during data collection [45,108]. experiments to be designed to limit the effect of radiation damage.
These systems include the Bruker/ARIMAX MD2/MD3 equipped Finally, some success has been reported for the use of radial scav-
with a MK3 mini-Kappa goniometer and the PRIGo multi-axis engers such as sodium ascorbate (a hydroxyl radical OH scavenger)
goniometer recently developed at the Swiss Light Source [109]. and sodium nitrate (an electron scavenger) in signicantly pro-
With respect to SAD data collection, multi-axis goniometers allow longing crystal lifetime [116,117].
the crystal to be positioned such that Bijvoet mates can be recorded How the data is collected can also affect SAD phasing since
at the same time from the same image, critical to reducing errors in missing data will impact data scaling, map connectivity and auto
the SAD experiment. Multi-axis goniometers can also be used to tracing results. One generally should strive to collect the unique
reduce the number of images need for a complete data set by data rst and then add more data to increase multiplicity. This
rotating the crystal about one of its crystal rotation axes, reducing approach will allow the data set to be properly scaled and the effect
X-ray exposure and limiting radiation damage. In addition, they can on data quality of adding more data can be analyzed. Over the past
be used to reduce reection crowding in cases where one-unit cell 30 years SAD data collection strategies have evolved from carefully
axis is signicantly longer than the other two by orientating this designed protocols requiring data collection from aligned crystals
axis parallel to the spindle during data collection. using multi-axis goniometers to much simpler experiments carried
out on randomly orientated crystals with data collected around a
3.4. Detector xed rotation axis. This trend was due in part to advances in protein
production (recombinant proteins & selenomethionine labeling),
Successful SAD structure determination has been carried out crystal mounting (loops), cryogenic data collection, diffraction
using data recorded with scintillation counters, multiwire propor- hardware (synchrotron sources, X-ray optics, goniometry, detectors
tional counters, imaging plates and CCD cameras of various con- and computers) and better software for data reduction (Fourier
gurations (single chip or multichip arrays). The recent indexing) and structure determination that made routine Se-SAD
introduction of detectors with 10 Hz plus readout speeds such as phasing a reality.
the hybrid photon counting detectors produced by Dectris [110] Early experiments generally employed the inverse beam (or
( and the high speed CCD based detectors pro- Friedel ip) data collection strategy [40,118]. Here, the crystal is
duced by Rayonix ( have signicantly impacted mounted along a symmetry axis and data is collected in small
the ease with which SAD data collection can be carried out. The 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 orientations further reducing systematic error. Data were pro-
the technique used (with an aligned crystal) for the I-SAD structure cessed with XDS [122]. The anomalous substructure was deter-
determination of the neurophysin p-iodo-Tyr-Phe amide complex mined using SHELXD [123] and expanded using PHASER [124]. The
(the rst ISAS phased structure) in the late 1980's [41]. However, structures were autobuilt using either BUCCANEER [125] or PHENIX
with the advent of Fourier indexing in the Denzo/Scalepack pack- [79]. The structures reported included membrane proteins, DNA-
age [119] crystal alignment using a multi-axis goniometer was no protein complexes and the 266 kDa T2R-TTL multiprotein com-
longer needed since the crystal could be indexed from a single plex (a complex between ab-tubulin, stathmin-4 and tubulin-
image. tyrosine ligase), PDB entry 4WBN, phased from 118 S, 13 P, 3 Ca2
However, as targets become more challenging (e.g. membrane and 2 Cl ions that represents the largest Native-SAD structure
proteins and large multi protein complexes) and protein expression determined to date. The recent advances in fast detectors and
utilizing insect and mammalian cell lines makes selenomethionine shutterless data collection methods have made the MDS method
labeling problematic, there is a growing interest in Native-SAD practical for Native-SAD in reducing both data collection time and
phasing as described above [70]. Native-SAD represents the ulti- experimental effort.
mate SAD experiment requiring only crystals of the native protein, a
stable tunable X-ray source, precision goniometry and a large high 3.7. The multi-crystal approach
speed detector. Since the sulfur anomalous scattering signal Df00
ranges from 0.24 to 1.14 in the wavelength range spanning 1.0 to MCA as the name implies is based on averaging data sets
2.3 , all sources of noise in the system must be identied and collected from a number of different crystals in order to limit ra-
mitigated. Furthermore, to increase the anomalous signal-to-noise diation damage and increase the redundancy of the nal averaged
ratio, data sets of high multiplicity are required. The trick is to in- data set. The strategy requires collecting complete data sets having
crease redundancy without incurring signicant radiation damage low multiplicity on a number of different crystals to minimize ra-
(a current bottleneck of the approach). diation damage. Cluster analysis of the individual processed data-
Recently two data collection strategies have been reported that sets in terms of unit cell deviation, diffraction dissimilarity
addresses this bottleneck, single-crystal low-dose multi-data set (resolution and mosaicity) and the calculated relative anomalous
summation (MDS) [44,120] and multi-crystal averaging (MCA) [46]. correlation coefcient (RACC) is then used to identify outlier
Although these approaches were developed for the collection of datasets and exclude from the analysis [47]. MCA has been incor-
Native-SAD data they are equally applicable to any SAD analysis porated into both CCP4 (Blend) [78] and PHENIX (
where crystal quality or radiation damage is a problem. 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
3.6. The single-crystal approach
I. loihiensis (data from 6 crystals; PDB entry 3TX3), the 132 kDa
TorT/TorSS complex from V. parahaemolyticus (data collected from
The MDS approach uses dose-slicing to conserve the life-time
13 crystals; PDB entry 3O1I) and the 134 kDa chaperone protein
of the crystal. In this approach multiple data sets are collected using
DnaK from E. coli (data collected from 5 crystals; PDB entry 4JN4)
one crystal (or one location on the same crystal) with either an
[126]. The approach has been recently used to determine the
attenuated X-ray beam or reduced exposure time. The degree of
Native-SAD structure of the Hepatitis C virus envelope protein E1
attenuation or exposure time reduction is inversely proportional to
N-terminal ectodomain domain (data collected from 32 crystals;
the number of data sets to be collected (generally 3 or 4). The
PDB entry 4UOI) [127] using 4.2 data. Using the MCA data set
following example illustrates this concept. Consider a crystal hav-
(~31,646 Bijvoet pairs) phenix.autosol employing 6-fold non-
ing an optimal exposure time of 3 s and one wished to collect a
crystallographic symmetry was used to determine the structure,
three data set ensemble. The exposure time used for each image
which was then rened against a native data set to 3.5 . This case
would be 1 s. The total X-ray dose accumulated for the three (1 s
is signicant since it represents the lowest resolution Native-SAD
exposure) data sets is the same as the dose received for the data set
structure determined to date.
collected using the optimal 3 s exposure time. The three datasets
are then merged together to yield a nal dataset that will have an
3.8. Data reduction
improved signal-to-noise ratio than the data set collected using the
optimal exposure time. This is because MDS data set will give a
The current generation of fast detectors can collect data at an
smaller s(I) value as indicated by the equation below.
astonishing rate producing 10 to 100 images per second allowing
h m i m K=A2 I 2 for efcient ultra-ne data slicing (e.g. 0.05 per image). These data
s2total G Is Ibg Ibg rates can make data storage, data transfer and data reduction
n N difcult in the home lab. A typical SAD data set (180 of data)
Note that the second term of the conventional sigma equation collected using these fast detectors and ultra ne slicing could
(above) is divided by the number of data sets N collected [44]. This contain thousands of images depending on the rotation slice used.
improvement in I/s will further improve the D(I)/s values of the These large data sets can represent hundreds of Gbytes of data that
strong reections, which are essential for Native-SAD phasing. This places demands on both disk space and processor speed. In addi-
approach can easily be combined with ne-slicing and helical (or tion, transferring this large volume of data over the internet is also
segmented) data collection to further improve I/s and mitigate challenging. Thus, many facilities hosting these fast detectors are
radiation damage, respectively. Since the data have been collected providing computational resources for data reduction (either while
from the same crystal the datasets should be isomorphous, if on-site or remotely) to eliminate the need for extensive computing
exposure is kept below the Garman limit [121]. This is another and data storage resources at the user's home site. In addition,
advantage of the MDS. Using the MDS approach and the PRIGo these beamlines, in many cases auto-process the data while it is
multi-axis crystal positioner, a group from the Swiss Light Source being collected [128]. However, it is important to remember that
has recently reported 11 Native-SAD structures determined on although auto-processing is fast and efcient, care must be used in
beamline X06DA using 2.066 X-rays [45]. The PRIGo crystal the data reduction process to ensure that the various processing
positioner was used to collect data at 3e5 different crystal 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/ of the anomalous substructure. One way to do this is to calculate
HKL3000 [119], MOSFLM [129]and XDS [122] can handle the ultra protein phases based on the anomalous substructure ( ) and its
ne slice data sets produced by these new fast detectors. XDS offers enantiomorph (- - -) and compare the resulting map inversion
the additional advantage of parallel data processing on systems Rfactor and the map correlation coefcient [4].
having multiprocessors speeding up the data integration. 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)
3.9. Phasing & structure solution
while the incorrect hand will produce a map that is non-
interpretable. If both maps can not be interpreted, then the
The SAD phasing process can be broken down into three steps.
anomalous substructure is not correct. Several software packages
The rst step is determining the anomalous substructure (the po-
such as CCP4, the SHELX-CDE suite [123], Auto-Rickshaw [130],
sition of each anomalous scatterer producing a signal). It requires
IPCAS (Iterative Protein Crystal structure Automatic Solution) [21]
only the scaled structure factors containing the Bijvoet pairs and
and PHENIX can be used to carry out the SAD phasing process.
some idea of the composition of the anomalous contributors. For
The bottleneck of the phasing process is the identication of the
Se-SAD, this would be the expected number of selenomethione
correct anomalous substructure and cases where tens of thousands
residues based on the contents of the crystal's asymmetric unit.
of SHELX-D trials have been need to nally achieve the correct
Once the anomalous substructure has been determined the next
solution [45]. To address this, both SHELXD and phenix.HYSS can
step is to generate protein phases and determine the correct hand
92 J.P. Rose, B.-C. Wang / Archives of Biochemistry and Biophysics 602 (2016) 80e94

take advantage of multiprocessor or cluster-based systems to probes and 2) the reduction of error needed to make Native-SAD
signicantly speed up the search for anomalous scatterers. In routine will increase the accuracy of all data.
addition, recent improvements to solution ranking and phase
generation have been reported. These include the inclusion of a Acknowledgements
SAD likelihood function to rank possible solutions in phenix.HYSS
[131] and the introduction of a direct phase-selection step prior to The authors would like to thank Professor Gary Newton
density modication (using RESOLVE or DM) [20]. Both approaches (deceased February 22, 2013) for his foresight in reproducing
were shown to signicantly improve the nal experimental phases Wang's original gures from his 1985 article, some of which are
and the quality of the resulting electron density maps. included in this review. We also acknowledge the University of
Finally, SAD phasing is critically dependent on having the cor- Georgia and the Ramsey-Georgia Research Alliance endowment
rect anomalous substructure. The Native-SAD structure of the West funds (to BCW) for providing partial support for the preparation of
Nile Virus NS1 protein (PDB entry 4O6C) [132] provides a good the manuscript.
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