**Structural Molecular Biology -- small angle x-ray scattering, X-ray
absorption spectroscopy, and X-ray Footprinting -- at NSLS-II**

Draft by Jen Bohon and other members of the Mark Chance

group at the Case Center for Synchrotron Biosciences

12 June 2007

**Objective:**

Our objective is to establish three premier beamlines for structural
molecular biology (small angle x-ray scattering or SAXS, X-ray
absorption spectroscopy or XAS, and X-ray Footprinting) on the floor of
the NSLS-II in time to begin operations immediately upon ring start up.
This is intended to create a seamless transition for the user population
dependent upon these resources to the new facility with improved
capabilities. Alongside parallel protein crystallography (PX) and
biological imaging programs, these beamlines and their associated
programs are intended to establish NSLS-II as a world leader in the
synchrotron life sciences.

**Rationale:**

***Overview:*** The approved CD0 proposal clearly underscores the
importance of the biological sciences to the mission of NSLS-II, yet no
beamlines dedicated to the life sciences are included in the initial
program. Since the life-science sector represents the largest single
block of users, it is imperative to ensure continuity in beamtime
availability and offer state-of-the-art facilities for this community.
One of the major objectives of structural molecular biology research is
the study of the structure and dynamics of biomolecules in solutions and
membranes, for which the SAXS, XAS, and Footprinting techniques are
recognized to be particularly well suited (CD0, section 2.2). We propose
an initial occupation of three NSLS-II beamlines from two ports: one
undulator in the first port hosting a beamline for SAXS, and one canted
damping wiggler in second port to host separate beamlines for XAS and
Footprinting. A second phase effort would be pursued to develop bending
magnet beamlines using three-pole-wigglers at a later time, coupled to a
transfer of compatible existing components at the NSLS. SAXS would
benefit from the high brightness delivered by an NSLS-II undulator,
while the canted wigglers would provide the high flux density necessary
for time-resolved EXAFS and Footprinting experiments.

**2. *SAXS*:** It is generally recognized that an undulator provides the
best source of radiation for brightness-limited techniques such as SAXS.
This beamline would be designed to have a range of optical
characteristics available, including the smallest possible microfocus,
accompanied by a convergence of \< 1 mrad (5-10µm). It would be set up
with multiple options for micro-focusing and control of beam convergence
(KB mirrors, graded multilayers, refractive lenses, or elliptical
capillaries), and a monochromator that can produce higher harmonics to
narrow the bandwidth.

**3. *Footprinting: ***The upstream device in the canted wiggler pair
would host the X-ray Footprinting beamline. This technique requires
polychromatic light (optimal energies ranging from \<5keV to \>20keV)
and would use either a single focusing mirror or set of Kirkpatrick-Baez
(KB) Optics to focus the beam to the desired size and shape as well as
to tailor the spectrum (remove high photon energies). The high flux of
the damping wiggler would enable this experimental technique to push the
envelope in time-resolved structural biology. The strictly white-beam
set-up in an up-stream position would simplify space concerns that are
expected to accompany the use of the canted devices.

**4. *XAS: ***The downstream device in the canted wiggler pair would
host the biological EXAFS beamline. The damping wiggler source would
provide access to energies from 5-35keV, satisfying the requirements of
80-90% of expected analyses (facilities for low-energy EXAFS are under
discussion and are likely to be handled in phase-II development). Due to
the high power of the damping wiggler source, low-energy absorptive
carbon filters would be used in front of the Beryllium window (or a
diamond window would be used). The beamline would include a
harmonic-rejection/collimating mirror to condition the beam for the
monochromator for elimination of the higher harmonics and reduction of
the vertical diverence, allowing maximum energy resolution from the
double-crystal water- or cryogenically-cooled monochromator.
Macro-focusing optics would be used to collect 2 mrad of radiation,
focused to \~1mm at the sample, resulting in a long beamline to
accommodate geometric considerations due to the proximity of two canted
wiggler beamlines. Focusing would be achieved by sagittal focusing of
the second monochromator crystal. Micro-focusing optics would consist of
advanced KB mirror optics or hard x-ray zone plates. If necessary, the
beamline would include additional optics to allow horizontal beam
steering in order to overcome any difficulties due to space
restrictions. Variable focus (\~10 to \~1μm) would tailor resolution to
accommodate a variety of sample types as well as fluorescence detection.

**Funding strategy:**

The scientific case for x-ray biosciences at NSLS-II has been described
and reviewed in the approved CDR and CD0 proposals. An abbreviated
version of this case, including new developments and programs tailored
to take advantage of the increased capabilities that the NSLS-II would
provide, is presented below. We believe that these technologies are
crucial to the successful establishment of a life-sciences program at
NSLS-II; the user community would approach every possible funding agency
in order to ensure that these facilities become a reality. Pro-active
consultation with the appropriate agencies, such as NIH, NSF, and OBER
would allow successful funding strategies to be developed as the
planning and design process for the ring proceeds.

The Center for Synchrotron Biosciences (CSB) has a proven track record
of attracting funding for these technologies over many years, thus we
intend to provide leadership in approaching relevant funding agencies to
help secure funding for these and future efforts. Synchrotron
biosciences boast the fastest-growing user population in the synchrotron
community. A full spectrum of life science beamlines, including PX,
SAXS, EXAFS, soft x-ray imaging, x-ray footprinting, IR
microspectroscopy, and VUV spectroscopic methods would establish the
NSLS-II as a world leader in synchrotron life sciences, and we would
support a concerted effort within the bioscience community to make
NSLS-II a premier life science facility.

**Construction:**

The design and construction of the beamlines would be carried out by a
team of experienced CSB/NSLS/NSLS-II staff with proven capabilities in
these areas who would work under the guidance of an NSLS-II beamline
advisory team. The CSB has a long and successful record of cooperation
and collaboration with the NSLS in the design and construction of
beamlines.

**Operation:**

The core operations staff would include experienced members of the CSB,
NSLS, NSLS-II and other interested organizations, formed into two
separate teams. Each team would be responsible for one port; one team
for the administration of the SAXS beamline, and one that includes the
EXAFS and Footprinting pair. These staff members would provide
high-quality beamline support for users as well as develop beamline
upgrades as necessary to maintain cutting-edge scientific instruments.

**Relation to existing life sciences/structural molecular biology
programs:**

In order to take full advantage of the capabilities of NSLS-II, most of
the components for the new beamlines would have to be tailored to the
new accelerator and ring systems, and thus would be almost entirely new
equipment. Although this would increase the cost of the beamlines, it
has the advantage of ensuring uninterrupted user access during the
transition. Once the NSLS is shut down, much of the high-quality
equipment at existing life sciences/structural molecular biology
beamlines can be transferred to three-pole-wiggler stations at NSLS-II.
The capabilities of these devices offer a modest improvement over those
of the current NSLS, but are similar enough to allow the employment of
current equipment for many high-throughput albeit standard experiments.

**Access modes:**

Access to the beamlines is expected to be relatively unchanged from the
system currently administered by the NSLS. General users would submit
proposals through the PASS system, with the bulk of the available
beamtime dedicated to general user time and set asides for facility
staff usage. Collaborative access modes are likely to be developed as
NSLS-II evolves.

**Phase-II development:**

Once the initial suite of cutting-edge beamlines is in place, additional
funding sources would be sought for the transfer of current life
sciences/structural molecular biology beamlines to three-pole wigglers.
This would enable the premier beamlines to be dedicated to solving
difficult scientific problems requiring the full capabilities of the
NSLS-II while still offering beam properties surpassing those of the
NSLS for high-throughput standard experiments at these work-horse
beamlines.

**Summary of the scientific case:**

Macromolecular crystallography has been enormously successful in
elucidating the structure of proteins and other biomolecules. An
increasingly complete collection of atomic-resolution protein structures
is becoming available, and a growing catalog of cellular assemblies has
been identified. These data are being combined with emerging genetic and
biochemical information on pathways to suggest temporal, spatial, and
functional relations controlling cellular function. However, the central
question in biophysics still remains: What is the connection between
structure and function of biological macromolecules such as protein,
DNA, RNA, polysaccharides, and their complexes? Answering this question
requires understanding the dynamics of macromolecular structures in
their natural environment. Such studies are poised to provide new
insights into the function, control, and dynamics of individual
macromolecules and large molecular complexes. Techniques such as
solution scattering, spectroscopy, and footprinting provide valuable
information regarding how biomolecules and molecular machines function.
The high brightness and flux density delivered by NSLS-II combined with
the advanced technology of ultra-fast continuous-flow mixers would give
X-ray scattering, spectroscopy and footprinting studies of biomolecules
and membranes unparalleled sensitivity and time resolution, enabling
more precise structure determinations and extending measurements of
dynamics down to the microsecond time range.

***X-ray scattering:*** X-ray scattering can be used to study protein
folding to understand how the protein performs its biological functions.
Time-resolved solution scattering now allows researchers to follow the
structural changes of the protein as folding proceeds. The time scale
that is characteristic of the folding process varies from picoseconds to
nanoseconds, when the initial secondary structure starts to form, to
milliseconds to seconds, when the folding process is completed. The high
brightness of NSLS-II would extend the time resolution of
solution-scattering measurements down to this time scale. Computations
and experiments would then overlap and provide better tests of our
understanding of the physics of the underlying interactions. Combined
with complementary methods, especially NMR, solution scattering and
computer simulation would provide a complete time course for how
proteins fold.

Applications of SAXS have been enormously successful in elucidating the
structure-function relations of skeletal muscle. To understand the
molecular basis of muscle contraction further, however, kinetic/dynamic
behavior of the contractile proteins in living muscle cells at
millisecond or even sub-millisecond time scale must be explored. 
Furthermore, structures of the heart muscle cells are only beginning to
be studied. Because of the branching of the muscle fibers in the
myocardium, to obtain high resolution structures of the cardiac muscle,
diffraction from single cells (myocytes) would have great advantages. 
Because of its small size (10 -- 20μm in width and 100μm in length), a
microfocus and bright beam intensity would be required.  Establishing
SAXS at NSLS-II would greatly facilitate research in this clinically
relevant and yet unexplored field. 

High-resolution methods, both X-ray crystallography and NMR, have
difficulties dealing with macromolecular complexes, where changes in
shape may accompany the biological function that is under study. In
contrast, small angle solution scattering can provide structural
information that reveals global conformation changes in biomolecules and
molecular complexes in solution, and is thus complementary to protein
crystallography. This approach also holds great potential for resolving
the structural kinetics of macromolecular complexes using time resolved
solution scattering to produce low-resolution movies of events such as
the assembly and operation of molecular machines. The high brightness of
NSLS-II would provide high intensity, highly collimated, and small X-ray
beams for SAXS measurements, resulting in high quality data necessary
for accurate shape determinations. Together with the flow-cell mixers
and fast detectors, NSLS-II would also enable time resolved measurements
at microsecond time resolution to become routinely achievable, as well
as the extension of solution scattering to membrane proteins to provide
information about the structural transitions occurred within membrane,
such as the conformation change of receptors upon binding of a ligand.  

***Study of Metalloproteins with X-ray Absorption Spectroscopy:*** X-ray
absorption spectroscopy (XAS) can be used to measure the transition of
core electronic states of a metal atom to excited electronic states or
continuum states. Spectral analysis near the electronic transition,
so-called X-ray absorption near-edge structure, or XANES, provides
information on the metal's charge state and geometry. Spectral analysis
above the absorption edge (from the edge to 10-15% above the edge in
energy), so called Extended X-ray Absorption Fine Structure, or EXAFS,
provides complementary structural information, such as numbers, types,
and distances of ligands or neighboring atoms. Both spectroscopies are
valuable techniques for studying a variety of metal sites in biological
systems. Recent experiments where X-ray crystallography and X-ray
spectroscopy are used in combination have provided information that was
not available from either technique alone.

Traditionally, XAS, like most other spectroscopies, has been used as a
static probe of structure. NSLS-II provides the opportunity to extend
X-ray absorption measurements into the time resolved realm, potentially
revolutionizing the study of biochemical and bioinorganic systems. Since
the biomolecular system being studied evolves continuously during the
reaction, with many different species potentially being present, one
cannot take a literal "snapshot" of intermediates that form. However,
use of sophisticated mathematical approaches such as principal-component
analysis, allows one to come close to this ideal. Combining the
information from tens or hundreds spectra, each measured at a different
time after mixing, makes it possible for one to deconvolve the component
spectra, even for relatively minor components. With the bright X-ray
beams provided by NSLS-II, continuous-flow mixing technology would
enable time resolution close to one microsecond.

***X-ray Footprinting:*** Hydroxyl radicals cleave the phosphodiester
backbone of nucleic acids and covalently modify amino acid side chains
in proteins. In sections where a molecule is folded, however, these
sites are inaccessible to solvent, and therefore protected. X-ray
footprinting employs the very intense and ionizing white (polychromatic)
X-ray beam to generate hydroxyl radicals. For nucleic acids, one then
analyzes the pattern of fragments after X-ray exposure by gel
electrophoresis; the protected sections that are not cleaved yield a
\"footprint\". For proteins, the exposed samples are digested with
proteases and analyzed by LC- and tandem-mass spectrometry to determine
the extent and sites of modification. One can follow the protections as
a function of time, showing tertiary contacts formed during folding
events or enzymatic processing.

Time-resolved footprinting measurements can be achieved with a solution
mixer to induce cleavage after a delay relative to the triggering of a
dynamic event. This is an ideal method for gaining insight into dynamic
processes of large RNA and protein assemblies. It provides detailed
structural information (at the single-nucleotide and single side-chain
level) on biologically relevant timescales. Current time-resolved
footprinting measurements are limited to millisecond time resolution.
The high beam brightness provided by NSLS-II would make the time scale
that is accessible by this technique significantly shorter, certainly by
a factor of ten. This improvement is not only because of the better time
resolution of the smaller-scale solution mixers enabled by NSLS-II, but
also owing to the shorter X-ray exposure time that is necessary to
generate the hydroxyl radicals that cleave the biomolecules.

All of this would benefit from use of microcapillary focusing that could
produce a 10μm spot with high intensity. To achieve an understanding of
conformational dynamics in macromolecules and their complexes during
processes as diverse as ligand binding, folding, catalysis, and
macromolecular assembly is a challenging and important step towards
providing detailed molecular descriptions of biological systems.

**Present and Future Impact:**

The continuously expanding synchrotron life sciences/structural
molecular biology community has a strong record of achievement, both in
scientific publications and in securing funding sources. In response to
the overwhelming demand for bioscience beamtime, the incorporation of
remote users (mail-in systems) and increased collaborations similar to
the template already in place for the PX community, is expected to
result in a significant increase in scientific productivity. In
addition, educational resources, such as the current EXAFS user
workshop, would continue to be provided and expanded further to increase
the size of the proficient user community, and to promote the
development of projects tailored to take advantage of the unique
capabilities offered by NSLS-II.

We believe that the NSLS-II can become a nexus of synchrotron
biosciences in the US. Life science is the fastest-growing area in the
synchrotron community, and a strong concentration of structural biology
centers and resources in the northeast region inherently complements the
establishment of this facility as a world leader in this area.