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Ahh yes, the ol' "beam heating hypothesis". I used to believe in it too...
James Murray just cited studies that cast a great deal of doubt on the
idea that there is any significant heating of protein crystals by x-ray
beams. I don't want to steal all the thunder form the RD4 meeting last
month, but Eddie Snell and Michael Kazmierczak HAVE done heat transfer
calculations and compared their predictions to the results of thermal
imaging experiments.
http://www.spring8.or.jp/en/users/meeting/rd4/rd4 (last two talks)
Very nice work IMHO. And I think they have put the last nail in the
coffin of the "beam heating hypothesis" of radiation damage.
If you think about it, a 30-degree gradient over 100 microns is 3000
degrees/cm. That's like poking a red-hot nail into ice and expecting it
to keep glowing. There are very few substances that have a low enough
thermal conductivity to maintain such a strong thermal gradient with
only a few milliwatts being deposited at the "hot" end. None of the
many phases of water are that good an insulator. Even the most powerful
x-ray beams in the world are only milliwatt-class sources of energy, and
typically, only about 2% of the energy from the beam is deposited in the
sample.
Then again, it is always easier to rationalize things once you know the
right answer...
I don't think the impact of solutes on the phase transition between
glassy water, low-density amorphous water and the ultraviscous deeply
supercooled water phase between 136 and 160K is something that has been
studied all that much. Although there is a very instructive table
summarizing much of the work on amorphous water phases here:
http://www.lsbu.ac.uk/water/amorph.html
One thing I can tell you is that I did an experiment looking at the
diffraction pattern of a MPD-PEG8K-water solution as I slowly ramped up
the cryostream temperature. I saw a fairly clear "elbow" in the graph
of "correlation coefficient to the first image" vs temperature at ~135K,
exactly where the glass transition of water is supposed to be. A
singular result, I admit, but I think it supports the hypothesis that
our cryostream temperature readings are indicative of the sample
temperature.
-James Holton
MAD Scientist
Ian Tickle wrote:
Unless of course the crystal isn't as cold as you think it is!
Essentially all of the energy of absorbed X-ray photons must ultimately
be degraded to thermal energy, and unless this is efficiently conducted
to the surface where it can be removed by the cold stream, it's going to
produce some 'hot spots' which given sufficient X-ray flux may be
sufficient to cause local melting of the water glass. There could well
be a steep temperature gradient, liq N2 temperature at the surface and
much warmer towards the centre. Also if low molecular weight solutes
are present the melting point could be locally depressed way below that
of pure water, so there wouldn't be quite so far to go to cause melting.
Admittedly I haven't looked at any calculations of what order of
magnitude the heating and heat conduction effects might be for a typical
flux and I could be way off beam but it's a least a theoretical
possibility.
-- Ian
-----Original Message-----
From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On
Behalf Of James Holton
Sent: Tuesday, April 25, 2006 3:11 PM
To: Udupi Ramagopal
Cc: Uhnsoo Cho; [EMAIL PROTECTED]
Subject: Re: [ccp4bb]: Radiation damage problem
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It is definitely true that some sensitive residues decay quite a bit
faster than the high-resolution spots. This has been repoted
in several
papers now. What has not been repoted (to my knowledge) is a
convincing
connection between the decay of side chains involved in a crystal
contact and the loss of high-resolution data. In all of the
studies I
have seen that measure the rate of loss of resolution, it is always
tightly related to dose. If you know of a study where it has
been shown
to be related to something else, I would like to hear about it.
I, personally, do not think that there is a connection between
specific damage near crystal contacts and loss of resolution. My
reasoning is that a cryo-cooled crystal is permeated by a matrix of
glassy water. Glassy water is a SOLID. So, once cooled, the crystal
lattice is supported by a lot more than just crystal
contacts. Kind of
like an insect preserved in amber. Breaking bonds near the
contacts are
not going to do much more distortion to the overall lattice
than broken
bonds in the core or even in the solvent. Diffraction is a
process that
occurs over dozens of unit cells. There must be long-range
distortions
to impact it. Site-specific damage, formally, only impacts
the B-factor
of that site.
-James Holton
MAD Scientist
Udupi Ramagopal wrote:
I think some times the sensitivity of residues involved in critical
cystal contacts
can disturb these valuable suggestions and calculations.
Ramu
On Mon, 24 Apr 2006, James Holton wrote:
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Disclaimer:
Radiation damage is a complex phenomenon, and many aspects
of radiation
damage at low temperature are still hard to explain, let
alone predict.
You DEFINITELY need to know things like the beamline's
brightness and
the concentrations of any atoms in your sample heavier
than oxygen to
even have a ghost of a chance of understanding the origin of your
problem.
However...
If you are used to this beamline and you "feel" that these
crystals are
decaying faster than "normal", then the most likely
problem is that you
have a heavy atom in your sample. This will cause your
crystal to absorb
a lot more x-rays than normal (doing more damage).
Anything heavier than
oxygen is a potential problem, and stuff in the solvent counts! The
"rule of thumb" is that the "absorptivity" of an atom is roughly
proportional to the atomic number. So, try doing things
like replacing
potassium with lithium or iodide with fluoride (each a
factor of 6).
Obviously, the more concentrated a heavy atom is, the
bigger the problem
will be.
Since beamline brigthnesses vary by a factor of 30,000
from place to
place, and crystal absorptivity can vary by a factor of 10 or more,
radiation damage questions can be hard to answer without
knowing flux,
beam size and sample composition.
But, if you do know these things...
The decay in protien crystals is generally proportional to
absorbed dose
(Joules/kg or "Gray"). Dose, in turn is proportional to fluence
(photons/mm^2) and the "extinction coefficient" of your
crystal in the
x-ray range. You get fluence (photons/mm^2) from flux
(photons/s) by
dividing by the beam size to get brightness (photons/mm^2/s) and
multiplying by the total exposure time (in seconds). Given
the beamline
flux, wavelength, beam size, and crystal composition the expected
lifetime of your crystal can be computed with a program
called RADDOSE
(Murray et. al. JSR (2005), 12, 268-75). Or you can "google" for
RADDOSE.
For example, you can expect the lifetime of your crystal will be
(roughly) cut in half by the addition of 4M NaCl, 5M
NH4SO4, 1 Se per 30
residues (at 0.97A), or 150 mM CsI.
-James Holton
MAD Scientist
Uhnsoo Cho wrote:
Dear bulletin members,
Sorry for non-CCP4 related questions.
Recently, I've got crystals which have a radiation damage problem.
It also has a weak diffraction because of the big unit
cell dimension
(about 300A in one direction), so I have to overexpose
crystals even
with the synchrotron beam.
The problem is that during the data collection, the
resolution decaded
gradually from 3 to 6A after certain number of frames
(about 30 or 40
frames).
Do you have any experiences or any suggestions that I can
evade this
radiation damage?
Any suggestions will be grateful.
Thank you.
Best regards,
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Uhn-Soo,Cho
Graduate student in Biological Structure Department
University of Washington.
HSB G514
1959 NE Pacific St.
Seattle, WA 98195-7420
Box. 357420
Fax #206-543-1524
Tel # 206-221-2435
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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