From: Dean Derbyshire
Date: 17 November 2011 11:09
----------
From: Eleanor Dodson
Yes - I have seen something similar at a lower resolution, but very ugly! I tried to model it as a solvent molecule - possible but not too convincing..
Cl should give an anomalous signal - try a DANO map and see what it shows..
Eleanor
----------
From: Jacob Keller
I also have seen similar. I was thinking it was potentially some kind
of radiation damage? Is there a good paper which examines what
chemistries are seen in rad damage?
Jacob
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
*******************************************
----------
From: Sean Seaver
Hi Jacob,
A couple of years ago, I put together a list of papers that touch on radiation damage:
http://www.p212121.com/2009/11/17/ultimate-list-on-cryocrystallography-radiation-damage/
To answer your question more directly - see pg. 40 of the following paper:
http://www.px.nsls.bnl.gov/courses/papers/Thiel-Garman2006.pdf
I hope it helps!
Take Care,
Sean
P212121
http://store.p212121.com/
----------
From: James Holton
An excellent review of the specific damage reactions observed in cryo-cooled MX is Burmeister (2000) http://dx.doi.org/10.1107/S0907444999016261
There is also a "Beginner's Guide to Radiation Damage" that was published in JSR a few years ago.
Burmeister's Fig 3 shows difference density appearing on an Arg that is involved in a salt bridge with a Glu that is decarboxylating. He interpreted this as a conformational change in response to the damage on the Glu, but whether or not you call such a movement "damage to the Arg" is probably a matter of semantics. It is also entirely possible that Burmeister's "mechanism" is wrong, and there really is some chemistry going on at the Arg, but since Arg by itself (not in a salt bridge) has never been reported as getting "damaged", any plausible chemical mechanism of Arg damage would have to involve the presence of a negative charge (like Glu, or perhaps Cl). In the end, all we can see is the difference density, and there are very few published observations.
The biggest problem with assigning weird density to "radiation damage" is that it is generally not possible to unambiguously distinguish the disorder created by rad dam with the "usual" disorder found all over protein crystals. After all, Arg is a rather flexible side chain. The second weak Cl position is strong evidence that the Arg is in more than one conformer. If you lower the contour of your electron density map you might get a better idea of what is going on.
If you want to blame rad dam as the source of this disorder, then you can cite Burmeister (2000) as a precedence for difference density appearing on Arg associated with a negative charge. However, the hallmark of rad dam is that it changes with time, so if you want to graduate from "suggestive" to "conclusive" then you must demonstrate somehow that the density of you "missing atom" was there before you gave the crystal "significant exposure".
How much exposure is "significant"? Depends on the reaction, and on the beamline! I know that most of us still think about our exposure times in "seconds", and that is all well and good if everyone is doing their experiments with the same machine. Problem is, rad dam is only reproducible when you normalize it to "dose" (MGy), and the beamline-to-beamline variation in dose rate (MGy/s) ranges over a factor of 10,000. I tried to tabulate as many of the world's unattenuated dose rates here:
http://bl831.als.lbl.gov/damage_rates.pdf
However, the short answer is that most beamlines are attenuated so that they deliver about 1 MGy/minute.
As for the reaction type? Current standing "world records" for lowest tolerable dose (in MGy) are:
5 Se-Met Holton (2007)
~3 S-S Murray et al. (2002)
1 Br-RNA Olieric et al. (2007)
0.5 Mn4Ca reduction (photosystem II) Yano et al. (2005)
0.02 Fe reduction (myoglobin) Denisov et al. (2007)
So, at 1 MGy/min, you have 5 min fo SeMet, 1 min for Br popping off nucleic acids and ~1 second before the Fe in myoglobin is reduced. Yes, there are plenty of other damage reactions, but as far as I know these are the only ones that have reported values for how "fast" they can go. If you stay below these exposure times (or attenuate), then you can be reasonably confident that the relevant reaction has not gone beyond 50% completion. By comparison, the "half-life" of the spots you can see on the detector is about 20-45 MGy (Henderson, 1990; Owen et al. 2006). Another good way to estimate your MGy/min is by watching the scaling B-factor, which will change about 1 "B-factor unit" per MGy (Kmetko et al. 2006).
-James Holton
MAD Scientist
Date: 17 November 2011 11:09
Hi all,
Has anyone observed 'odd' arginine residues, missing the NH1 atom; and possibly related.. very close Chlorine-arginine (NH1 again) distances.
I have a 1.3Å structure and am having trouble getting my head around some very odd density features.
I have 2 molecules in the assym. with the equivalent arg residues showing the same odd feature.
Seems to be a chlorine binding site – typical mix of hydrophobic and proton rich side chains – but, the NH1 of the particular arginine residue concerned (as I say in both NCS copies) seems to have only 50% occupancy and the chlorine appears to have a 2nd distinct position closer to the arginine, such that if the NH1 atom were present it would be only 2Å away!
?
Cheers in advance
Dean
----------
From: Eleanor Dodson
Yes - I have seen something similar at a lower resolution, but very ugly! I tried to model it as a solvent molecule - possible but not too convincing..
Cl should give an anomalous signal - try a DANO map and see what it shows..
Eleanor
----------
From: Jacob Keller
I also have seen similar. I was thinking it was potentially some kind
of radiation damage? Is there a good paper which examines what
chemistries are seen in rad damage?
Jacob
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
*******************************************
----------
From: Sean Seaver
Hi Jacob,
A couple of years ago, I put together a list of papers that touch on radiation damage:
http://www.p212121.com/2009/11/17/ultimate-list-on-cryocrystallography-radiation-damage/
To answer your question more directly - see pg. 40 of the following paper:
http://www.px.nsls.bnl.gov/courses/papers/Thiel-Garman2006.pdf
I hope it helps!
Take Care,
Sean
P212121
http://store.p212121.com/
----------
From: James Holton
An excellent review of the specific damage reactions observed in cryo-cooled MX is Burmeister (2000) http://dx.doi.org/10.1107/S0907444999016261
There is also a "Beginner's Guide to Radiation Damage" that was published in JSR a few years ago.
Burmeister's Fig 3 shows difference density appearing on an Arg that is involved in a salt bridge with a Glu that is decarboxylating. He interpreted this as a conformational change in response to the damage on the Glu, but whether or not you call such a movement "damage to the Arg" is probably a matter of semantics. It is also entirely possible that Burmeister's "mechanism" is wrong, and there really is some chemistry going on at the Arg, but since Arg by itself (not in a salt bridge) has never been reported as getting "damaged", any plausible chemical mechanism of Arg damage would have to involve the presence of a negative charge (like Glu, or perhaps Cl). In the end, all we can see is the difference density, and there are very few published observations.
The biggest problem with assigning weird density to "radiation damage" is that it is generally not possible to unambiguously distinguish the disorder created by rad dam with the "usual" disorder found all over protein crystals. After all, Arg is a rather flexible side chain. The second weak Cl position is strong evidence that the Arg is in more than one conformer. If you lower the contour of your electron density map you might get a better idea of what is going on.
If you want to blame rad dam as the source of this disorder, then you can cite Burmeister (2000) as a precedence for difference density appearing on Arg associated with a negative charge. However, the hallmark of rad dam is that it changes with time, so if you want to graduate from "suggestive" to "conclusive" then you must demonstrate somehow that the density of you "missing atom" was there before you gave the crystal "significant exposure".
How much exposure is "significant"? Depends on the reaction, and on the beamline! I know that most of us still think about our exposure times in "seconds", and that is all well and good if everyone is doing their experiments with the same machine. Problem is, rad dam is only reproducible when you normalize it to "dose" (MGy), and the beamline-to-beamline variation in dose rate (MGy/s) ranges over a factor of 10,000. I tried to tabulate as many of the world's unattenuated dose rates here:
http://bl831.als.lbl.gov/damage_rates.pdf
However, the short answer is that most beamlines are attenuated so that they deliver about 1 MGy/minute.
As for the reaction type? Current standing "world records" for lowest tolerable dose (in MGy) are:
5 Se-Met Holton (2007)
~3 S-S Murray et al. (2002)
1 Br-RNA Olieric et al. (2007)
0.5 Mn4Ca reduction (photosystem II) Yano et al. (2005)
0.02 Fe reduction (myoglobin) Denisov et al. (2007)
So, at 1 MGy/min, you have 5 min fo SeMet, 1 min for Br popping off nucleic acids and ~1 second before the Fe in myoglobin is reduced. Yes, there are plenty of other damage reactions, but as far as I know these are the only ones that have reported values for how "fast" they can go. If you stay below these exposure times (or attenuate), then you can be reasonably confident that the relevant reaction has not gone beyond 50% completion. By comparison, the "half-life" of the spots you can see on the detector is about 20-45 MGy (Henderson, 1990; Owen et al. 2006). Another good way to estimate your MGy/min is by watching the scaling B-factor, which will change about 1 "B-factor unit" per MGy (Kmetko et al. 2006).
-James Holton
MAD Scientist
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