CHESS X-ray Runs:
2017: Jan 25 - Mar 7
2017: Mar 15 - Apr 24
2017: May 17 - Jun 29
2017: Oct 11 - Dec 21
How big is your molecule? This is an important
question in BioSAXS. The maximum diameter of your molecule will
determine how long the sample-to-detector distance needs to be. This
can be a limiting factor.
The Shannon Sampling Theorem suggests that objects having a diameter
should be measured down to at least q = π/Dmax,
preferably lower. When computing Rg
directly from a Guinier plot it is customary to use data with
q<1.3/Rg. Guinier and Fournet, in their 1955 book "Small-Angle
Scattering of X-rays", recommend that qmin<1.3/(2Rg). For some shapes, such as
rods, it may be necessary to collect data to even lower q values to
get an accurate Rg, but since the shape is
not known a priori, the 1.3 convention is widely accepted.
The current beamline configuration at G1 (as of 3/2014) allows
for the determination of objects having Rg < 100 Å
and Dmax <450 Å (qmin > 0.007 Å).
Users with larger
systems and 400 Å should consult MacCHESS staff for details.
MacCHESS staff can teach you how
to create scattering profiles, evaluate data quality, and compute
Rg on site.
But, just like protein crystallography, there is a lot to learn …
than you can absorb in one sitting. We strongly recommend that new users
take a training course, if possible. Such 1-2 day courses are offered by a
number of synchrotron sources. MacCHESS periodically offers an introductory
BioSAXS Essentials. To receive notification of the next
scheduled course offering, please visit this link.
MacCHESS provides several protein standards for use in
your SAXS experiments. It is important to run at least one
standard so that you have a way to estimate molecular mass,
but also a way of being confident that the beamline is
running properly. Standards also serve as a reminder of
what good monodisperse sample *should* look like.
40 mM NaOAc pH 4.0
50 mM NaCl
1% glycerol v/v
Glucose Isomerase Buffer:
10 mM HEPES pH 7.0
1 mM MgCl2
Protein concentrations vary from run to run, but will be approximately
4 mg/ml for lysozyme and 0.4 mg/ml for glucose isomerase. A more
complete list of standards can be found in:
Mylonas, E., and D. I. Svergun. 2007. Accuracy of molecular mass
determination of proteins in solution by small-angle X-ray scattering.
J. Appl. Cryst. 40:S245-S249.
Kozak, M. 2005. Glucose isomerase from Streptomyces rubiginosus -
potential molecular weight standard for small-angle X-ray scattering.
J. Appl. Cryst. 38:555-558.
Users must provide a matched buffer solution for
This should be the exact same buffer used in the original
sample preparation if possible. Change buffer (by centrifugal
concentrator, dialysis, or SEC) if you don't know the original
composition exactly. Prepare plenty of extra buffer for sample
dilutions and for rinsing sample cells (bring at least 10 ml
if you can).
Protein solutions should be monodisperse and concentrated to
at least 5-10 mg/ml if possible without aggregation. It is a good
idea to bring at least some unconcentrated sample to test for
concentration-induced aggregation. You should check monodipsersity
using dynamic light scattering (DLS), if available. Purifying your
sample on a size-exclusion column is highly desirable. Even with
pure proteins it may be necessary to experiment to find the best
conditions under which the protein is monodisperse and well folded.
Sometimes proteins will aggregate rapidly after purification and
must be purified on site. Protein samples have been frozen
successfully for shipment, but users should not assume their
proteins will necessarily freeze well.
A small amount of glycerol (1-5 %) is advisable for protection
from radiation damage, but more than 15% will degrade the strength
of your scattering signal and should be avoided. In general, high
salt (> 1M) should be avoided for the same reason. Preservatives
like DTT and TCEP are known to reduce radiation damage in small
amounts and are therefore recommended.
We strongly suggest that you measure your concentration as
accurately as possible! Molecular weight estimates based on
known concentration are valuable indicators of sample monodipsersity,
oligomeric state, and can help diagnose problems with mixtures. A280
UV sample measurements are possible at the beamline, if needed.
Individual exposures can be done with sample volumes as small
as 20 microliters, if loaded by hand. But for easy and reliable
sample loading, we recommend using the sample loading robot with
at least 30 microliters if possible. Larger sample volumes ensure
easy and rapid positioning of sample in the beam and a generous
oscillation size which will eliminate potential radiation damage.
Since BioSAXS requires multiple dilutions (at least on some
representative samples), it is advisable to bring more than
the minimum volume. A sample size of 50 microliters, for example,
would allow just enough to prepare a minimum series of 3 dilutions
of 30 microliters each: full strength (30 ul sample), 66% (20 ul
sample + 10 ul buffer), and 33% (10 ul sample + 20 ul buffer).
More dilutions are desirable, if possible. Many users prefer to
prepare dilutions by halving: 1.0, 0.5, 0.25 0.125 etc.
With a flow cell, the more protein you bring, the better the
signal will be. For lysozyme-sized proteins (14 kDa), don't expect
to get much usable signal below 1 mg/ml. For larger proteins,
the low concentration limit will improve. Glucose isomerase,
for example, (MWt = 173 kDa) gives good data at 0.3 mg/ml and is
essentially at the infinite dilution limit.
Solutions that are too concentrated exhibit concentration-related
distortions of the small-angle part of the scattering curve. You
can see this in lysozyme stronger than 10 mg/ml and Glucose Isomerase
stronger than 0.5 mg/ml.
It is advisable to collect data at several (at least 3) different
concentrations and extrapolate to infinite dilution if necessary.
Alternatively, you can combine a dilute curve (small-angle part)
with a concentrated curve (wide-angle part). Concentration does
not effect the wide angle part of the scattering curve.
The concentration of protein necessary to get a
good signal in BioSAXS depends inversely on molecular weight. For small proteins like lysozyme
collected on F2 station using typical exposure times, 4 mg/ml will usually produce a profile that
is dilute enough to avoid interparticle interactions, but strong enough to give a good low-noise
Guinier plot with accurate radius of gyration. Larger proteins like glucose isomerase (173 kDa)
need only reach 0.3 mg/ml to give the same strength of signal in the low-angle region.
Actual time for data collection is hard to estimate
because there are so many variables. With our new capillary-flow cell, we can
now take much longer exposures than before without radiation damage. This can
dramatically improve data quality, but it also will reduce the number of samples
that can be examined in the time available. Currently we are recommending at
least ten 2 second exposures per sample, so the total exposure would be 20 seconds.
Each protein you examine will include a buffer exposure of equal length and
at least 3 concentrations. You may use the same buffer profile for multiple
proteins, if you like. But, it is wise to re-take the buffer periodically
after each re-filling of the synchrotron storage ring. You may wish to expose
dilute solutions for longer using larger sample volumes. You should also factor
in sample cell rinse and dry time between different proteins, which will add 2 min. Spinning samples prior to data collection
requires 10 min at 14,000 rpm, but sample spinning and dilution can be done
simultaneously with data collection.
We strongly recommend reading these recently published guidelines:
"Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution." David A. Jacques, J. Mitchell Guss, Dmitri I. Svergun and Jill
Trewhellaa, Acta Crystallographica Section D, Biological Crystallography, Vol 68, Part 6, pp. 620-626, 2012.
E = 9.968 keV (1.257 A) (as of 10/18/2013)
Beam diameter 250 um x 250 um
Flux = 1.6 x 10**11 photons/sec (as of 10/18/2013)
Minimum sample volumes:
using the robot 25 ul
manual loading 20 ul
recommended working volume 30 ul
Detector: dual Pilatus 100K-S SAXS/WAXS
qmin = 0.006 A-1
qmax = 0.8 A-1
Recommended sample exposure: 10 frames @ 2 sec each (20 sec total).
E = 9.881 keV (decommissioned 6/13/2013)
Beam diameter: 250 μm × 250 μm
Flux = 2×1010 photons/sec
Minimum sample volumes:
using the robot: 15 μL
manual loading: 5 μL (practical limit)
recommended working volume: 30 μL
Detector: dual Pilatus 100K-S SAXS/WAXS
qmin = 0.007 Å<sup>-1</sup>
qmax = 0.7 Å-1
Total exposure times: 300-600 sec
80 sample plugs per 24 hours
(about 20 protein samples including buffer, dilutions, and cleaning).
Direct questions, suggestions or problems to