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So, you are a macromolecular
crystallographer and have crystals of an interesting protein. You know you
can get a set of native diffraction data, but phasing is a problem. You
can't do molecular replacement because this is a brand new protein, and you
haven't been able to find any usable isomorphous heavy atom derivatives.
However, you can make a non-isomorphous derivative, or you know you can get
cloned protein incorporating selenomethionine - looks like a case for MAD
(multiple wavelength anomalous diffraction). |
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In order for MAD phasing to be successful,
the signal from the anomalous scatterers must be sufficiently large relative
to the total scattering from anomalous plus non-anomalous scatterers. The
anomalous signal from each heavy atom depends on what that atom is, and you
must also consider the available x-ray flux at the wavelength of the
appropriate absorption edge. A
database of absorption edges is available,
courtesy of Ethan Merritt.
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At the CHESS F2 station, with the standard Si (111)
monochromator crystals, the range of possible
energies is 7.9 - 14 keV (1.57 - 0.89 A). This allows use of the K edges of
elements 28 - 36 (including
Cu, Se, Br, etc.) or the L-III edges of elements 67 - 84 (including Hg, Pt,
Au, some lanthanides, etc.).
The total signal depends on the number of (well-ordered) anomalous
scatterers as well as on their
nature. As an example, consider a 100 residue protein containing 2 Se atoms.
The total number of
electrons is approximately 5200. Of this, the two selenium atoms contribute
68. The single-wavelength
anomalous (Bijvoet) signal depends on the imaginary component of the
scattering factor, f'', while the
multiple-wavelength (dispersive) signal depends on the difference in
scattering factors at the two
wavelengths, f'(1)-f'(2). These factors vary with the environment of the Se
atoms, but typical values are
at most 3.7 and 6.6 electrons, respectively [1], assuming suitable selection
of wavelengths. The
resulting ratio of anomalous signal to total structure factor may then be
calculated according to the method of Hendrickson [2], and is at most 4.0% for the Bijvoet and 3.5% for
the dispersive case.
Table 1 gives the maximum number of residues per heavy atom for a reasonable
signal (i.e. 3%
Bijvoet differences and 2% dispersive differences) from some representative
anomalous scatterers,
using f' and f'' values determined in protein crystals. Your crystals may
have a larger f'', and you may
be able to get useful information from a smaller signal, if you are very
careful in collecting data, but
this table is a good place to start in deciding whether a MAD experiment is
likely to succeed.
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Table 1 |
Maximum Amino Acid Residues per
Heavy Atom for Good MAD Signal |
| Heavy atom |
Absorption edge |
Maximum f" |
Maximum delta (f') |
Max. no residues
per heavy atom |
| Cu |
1.381 ? (K) |
4.2 |
5.0 |
90 |
| Se |
0.980 ? (K) |
3.7 |
6.6 |
90 |
| Br |
0.920 ? (K) |
3.4 |
5.5 |
75 |
| Hg |
1.009 ? (L-III) |
10.2 |
13.3 |
640 |
| Ho |
1.536 ? (L-III) |
25.7 |
19.9 |
1430 |
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The last column of this table was calculated
using an inversion of the equation given by Hendrickson in Reference 2 for
calculating anomalous signal ratios. The f" and delta (f') values are taken
from References 3 (Cu), 1 (Se), 2 (Br), 4 (Hg), and 5 (Ho). |
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Because the MAD signal is so
small, it is especially important to minimize errors in intensity
measurements. This means: use your best crystals, freeze them, and be
careful in selecting data collection parameters. It is worth spending
considerable time optimizing crystallization and freezing conditions (before
coming to CHESS, of course). During data collection, select each crystal
carefully and optimize exposure time, oscillation range, etc. for it - see
information on
"Efficient collection of oscillation data"
for hints.
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Having decided to attempt a MAD experiment at
CHESS, follow the
usual procedures to request
beam time. As with any experiment involving heavy atoms, you must consider
the total amount and
form of heavy atom compounds to be brought to CHESS. If heavy atoms are only
present in
prederivatized crystals, the experiment is non-hazardous - just check the
appropriate place on the
proposal form. If you are bringing unfrozen crystals in a soak solution
containing heavy atoms, you
will need to include a "Heavy Atom Compounds Declaration" describing the
type and amount of
heavy atom compounds that you will be bringing. The small amount of heavy
atoms present in the
crystals themselves plus a few milliliters of millimolar soak solutions
poses no hazard and no
administrative complications beyond supplying the Declaration. If you expect
to bring concentrated
stock solutions of toxic compounds (not recommended but occasionally
necessary), you will also
need to complete a "Hazardous Materials Declaration". See the
heavy atom guidelines for more
detail.
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You will need a sample containing your heavy
atom for taking reference energy scans (see below).
CHESS has reference samples for a number of commonly used heavy atoms on
hand; check with
the staff to see if they have (or can make) one for your experiment. If
necessary, make up a standard
and bring it along. A simple and satisfactory method is to sandwich a pinch
of powdered material
(e.g. mercuric acetate for a mercury sample) between two pieces of x-ray
transparent sticky tape
(Kapton tape is good), and mount the sandwich in a slide mount. The area
covered by the powder
should be at least a centimeter or so square, so that it is easy to be sure
the x-ray beam is passing
through it. If you are bringing a reference sample containing toxic
material, such as a mercury
compound, be sure to include it on the Heavy Atom Compounds and Hazardous
Materials
Declarations. If you think you might need to make up another sample while at
CHESS and are
bringing a bottle of material to do so, include that also. When you are at
CHESS, handle any
reference samples you bring with care - label them, try not to spill them,
know how to clean them up
safely if you do spill them, and don't bring any more than necessary in the
first place.
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Energy scans are essential to select the
energies (wavelengths) at which to collect data and to check
for drifting of the monochromator during the experiment. These are taken by
scanning the
monochromator through the appropriate energy range and monitoring the x-ray
fluorescence from the
sample. CHESS staff will set the station monochromator at the absorption
edge of the element you
are using; scans may then be easily performed in the vicinity of this
energy. Reference samples
containing high concentrations of commonly used elements are provided (see
above for how to
make your own if you are using something unusual). The reference sample is
mounted towards the
front of the hutch; the "refscan" command moves it into the beam, scans the
energy over a range of
about 40 eV (specified using the "madsetup" command), lists and plots the
resulting fluorescence
spectrum, moves the sample out of the beam, and resets the energy to its
initial value. Reference
scans are run periodically during the experiment to check, and if necessary
recalibrate, the
monochromator. In most cases, you will also want to take an energy scan on
your crystal; this can be
a little more tricky.
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Ideally, you take an energy scan on the
crystal you will be using for data collection. However, if this
crystal is too small to give a usable fluorescence signal, another (bigger)
crystal, or multiple crystals,
may be used. In order to get the best signal-to-noise ratio, the
fluorescence detector should be
placed at 90 degrees to the beam and in the plane of the ring, i.e. pointing
along the spindle axis. As
normally configured, the cold stream nozzle comes in at an angle and does
not interfere with the
fluorescence detector, so you may do a crystal scan on a frozen crystal. If
you prefer to have the cold
stream coming in along the spindle axis, you will have to temporarily remove
the cooling nozzle and
use unfrozen crystal(s) for the crystal scan. Unfrozen crystals are
perfectly satisfactory for this
purpose, as the environments of the anomalously scattering atoms change very
little on freezing.
Here is a good place to use those junky crystals that are not suitable for
diffraction - pack a bunch of
them into a capillary and do a crystal scan with them. If possible, tilt the
capillary relative to the spindle
axis, so that the fluorescent x-rays don't have to go through too much
glass. Once you have a sample
in the beam, the "xtal_scan" command runs the scan and outputs the results.
Panels a - c of the figure
below show scans across the Se edge. In panel a is a reference scan taken on
a foil. The scan in
panel b was taken using a medium-sized crystal with the detector poorly
positioned; the panel c scan
used a large crystal sample with the detector correctly placed. Panel d of
the figure shows a
reference scan for Hg. It is typical of this element that the L-III edge is
rather broad, with no "white line".
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Experimenters have also found empirically
that there is very little variation in the edge from one
sample to another, so this is a case where you may get away without a
crystal scan at all, if you are
sure there is mercury in the crystal and are pressed for time.
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Select your desired
wavelengths from the crystal scan. For a 4-energy MAD experiment, use (1) a
point slightly below the edge, (2) the inflection point of the edge, (3) the
top of the "white line" peak,
and (4) a point about 200 eV above the edge. For a typical 3-energy
experiment, omit energy (1). For
some elements, such as Hg, it may be better to omit energy (3) instead. In
case of severe time
limitations, useful data may be obtained using just two energies, e.g. (2)
and (3) or (1) and (4). An
alternative to a full MAD experiment is the single wavelength anomalous
diffraction (SAD) technique;
this method uses only the Bijvoet differences collected at a single
wavelength. For SAD, the preferred
energy is (3), at which f" is largest. Look at the f' and f'' curves for
your element (see Ref. 4 for how to
calculate these curves from an x-ray absorption spectrum) to decide what
selection of energies will
give you the best signal in case you can't collect all the data you would
like to. It is generally
preferable to have complete data at fewer wavelengths rather than partial
data at more wavelengths.
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Note that a
reasonable anomalous signal can be obtained over a range of energies above
the
absorption edge, and this range can be quite large for some elements. Hence
SAD data may be
collected at A1 for Se-containing samples (station tuned 40 eV above the Se
edge) and at F1 for
Br-containing samples (station tuned 40 eV above the Br edge). At F2, a SAD
experiment may be
possible even if the station energy cannot be tuned as low as the desired
absorption edge. Consult
CHESS staff about this possibility if you are working with an element such
as I or Xe.
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Geometry |
Once you have selected the
wavelengths for data collection, get a good crystal
mounted and determine the appropriate oscillation range and exposure time.
Typical values are 1 degree and 1 minute, but these depend heavily on your
particular crystal. Decide whether you will be using "mirror, "inverse
beam", or no
special geometry. In "mirror" geometry, anomalous pairs appear on each
image,
and in the "inverse beam" case anomalous mates appear on pairs of images
collected with spindle angles differing by 180 degrees. Mirror geometry
requires
the crystal to be oriented with a mirror plane perpendicular to the spindle
axis. At
CHESS, the only means for getting this orientation is the use of the arcs on
the
goniometer head, so that it may not be possible to get a mirror where you
would
like it. There can also be a problem in scaling images from a precisely
oriented
crystal together, particularly in the case of a monoclinic crystal of low
mosaicity.
Nonetheless, mirror geometry is clearly preferable in terms of reducing the
number
of exposures and insuring good scaling between anomalous pairs, and should
generally be used when possible, especially when another data set is
available for
scaling purposes. For inverse beam collection, software settings allow
collection of
wedges of data separated by 180 degrees. It is also possible to collect data
in a
single sweep, from a randomly oriented crystal, as for a monochromatic
experiment; this can be perfectly satisfactory for a robust crystal of
relatively high
symmetry.
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CCD detector |
MAD users at CHESS
usually use a CCD detector. Considerations in your planning due to the
detector include: |
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You will need to take a
background image every time you change the exposure time. This doesn't take
long, but is important. |
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The readout time, including overhead, is
about 3 seconds for the ADSC Q-210
detector (at F2 and A1) and about 11 seconds for the ADSC Q-4 detector (at
F1); you must include this time when calculating how many exposures you can
get per hour. |
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The detector area is limited. To get high
resolution data, it may be necessary
to offset the detector, so that the direct beam position is no longer in the
center
of the detector. For mirror geometry, a vertical offset, i.e. perpendicular
to the
spindle, will still allow recording of anomalous pairs on each image; a
horizontal offset will eliminate some pairs. In the inverse beam case,
corresponding reflections appear in opposite quadrants on the paired images,
and any offset will eliminate some of them.
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Timing |
Data collection
proper consists of a series of energy changes, oscillation exposures, and
crystal rotations. A sequence for a 3-wavelength experiment using inverse
beam geometry, for example, might be: |
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go to wavelength 1, take 5
degrees of data, rotate crystal -5 degrees, |
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go to wavelength 2, take 5
degrees of data, rotate crystal -5 degrees, |
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go to wavelength 3, take 5
degrees of data, rotate crystal (180 - 5 = 175) degrees, |
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go to wavelength 1, take 5
degrees of data, rotate crystal -5 degrees, |
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go to wavelength 2, take 5
degrees of data, rotate crystal -5 degrees, |
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go to wavelength 3, take 5
degrees of data, rotate crystal -180 degrees.
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Repeat until data
set is complete, crystal dies, or fill ends.
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The major contributor to data
collection time is the actual exposure time (at least at
F2), which can vary from a few seconds to a few minutes, depending on the
crystal
quality. Use a series of snapshots to determine the optimal exposure time.
Knowing
the detector readout time, the time to change wavelengths (about 3 seconds),
and
the time to rotate the spindle 180 degrees (about 2 seconds) , it is then
straightforward to calculate how much data you can collect per hour. Adding
in the
time to refill CESR (about 10 minutes every 2 - 4 hours), will you have time
to get
your data? If not, consider reducing the number of wavelengths or the
resolution, or
plan to come back another time.
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You need to monitor the data collection -
sorry, it's not safe to set up an 8-hour data collection and
take the day off. It is particularly important to keep an eye on the images
being produced to be sure
that the crystal is still diffracting. It is also highly desirable to pause
data collection whenever the ring
is being refilled. The data collection software will halt collection if
collimator counts fall too low, but
even if this does not happen the beam motion that occurs during filling
makes images collected at
that time of dubious quality. If collection is halted automatically, you
will need to restart it once beam
returns. Reference scans should be taken at least once per fill, to check
for any monochromator
drifting. Once some data have been collected, start processing them to be
sure that the quality is
sufficient to be worth continuing with the current crystal. Processing as
you go will also let you know
when enough data have been collected, and may even let you solve a structure
at the beamline.
Don't forget to keep up with data backup, so as not to be left with several
hours worth of file transfer to
do at the end of your run.
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A suitable molecular system with
enough expected signal for phasing. |
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Good freezable
crystals, and (preferably) a suitable sample for a crystal scan. |
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Beam time at CHESS. |
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Well-thought out strategy for
data collection, with contingency plans. |
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Careful experimental technique,
with due consideration for safety. |
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Good bookkeeping skills. |
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A sense of humor to get you
through the inevitable equipment failures. |
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1 |
Yang, W., Hendrickson, W. A.,
Crouch, R. J., and Satow, Y. "Structure of RNase H Phased at 2A Resolution
by MAD Analysis of the Selenomethionyl Protein", Science, 249,
1398-1405 (1990)
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2 |
Hendrickson, W. A.
"Determination of Macromolecular Structures from Anomalous Diffraction of
Synchrotron Radiation", Science 254, 51-58 (1991)
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3 |
Walter, R. L., Ealick, S. E., Friedman, A.
M., Blake, R.C., II, Proctor, P., and Shoham, M. "Multiple Wavelength
Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly
Oxidizing Cupredoxin with Extreme Acid Stability", J. Mol. Biol. 263,
730-51 (1996).
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4 |
Tesmer, J. J. G., Stemmler, T.
L., Penner-Hahn, J. E., Davisson, V. J., and Smith, J. L. "Preliminary X-ray
Analysis of Escherichia coli GMP Synthetase: Determination of
Anomalous Scattering Factors for a Cysteinyl Mercury Derivative",
Proteins: Structure, Function, Genetics 18, 394-403 (1994)
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5 |
Weis, W. I., Kahn, R., Drickamer, K., and
Hendrickson, W. A. "Structure of the Calcium-Dependent Lectin Domain from a
Rat Mannose-Binding Protein Determined by MAD Phasing", Science 254,
1608-15 (1991).
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