Indeed there are radicals, but they are not free.

Perhaps a better terminology for them would be "fixed radicals".  
Species such as the disulphide radical have been well established by 
spectrophotometric and other evidence (M. Weik et. al. and others).  It 
is quite remarkable actually how long-lived different radical species 
can be a ~100K.  The most extensive literature on radical states at low 
temperature is the ESR field.  Much of it is quite old, but still 
accessible in textbooks, such as Box (1977).

Still, I think my best answer to your first question (Bart) is that 
cryo-cooled samples are solids, and solid-state chemistry is different 
from liquid-state chemistry.  This fact is further supported by 
observations of the effects of so-called "radical scavengers" under 
cryo.  Some of these still work in the cold, others do not, and recently 
it has been reported (In a talk at RD5) that some scavengers can be 
effective against damage to one part of the protein, but not others, 
even for chemically identical sites.  Indeed, chemically identical sites 
in the same protein are often seen to decay at wildly different rates 
with or without scavengers.  Since two disulphide bonds have exactly the 
same likelihood of interacting directly with an x-ray photon (called the 
x-ray absorption cross section), these different decay rates cannot be 
explained this way.  It can also not be explained by "solvent 
accessibility" as first pointed out in Burmeister (2000). 

So, basically, the model that liquid-phase chemistry is still going on 
under cryo is inconsistent with almost every observation made in 
cryo-cooled samples.  The kinetics in particular make no sense at all 
(first pointed out by R. Thorne, and also detailed in Holton, JSR 
2007).  The only thing solid-state and liquid-state radiation damage 
have in common is that some of the chemical consequences (such as 
disulphide breakage) are similar.

The "jumping radicals" you describe actually fall very nicely into the 
quasiparticle category I was talking about.  That is: migration of a 
chemical state with no mass transport.  Believe it or not, the problem 
right now is that there are so many quasiparticle mechanisms that can do 
this we still aren't sure which one is involved.  The only thing that is 
fairly certain is that molecular diffusion is not. 

Most of what we know about solid-state chemistry comes from the 
semiconductor field, where the reactions are VERY well understood.  
Unfortunately, that literature is rather opaque to biologists who aren't 
working at a synchrotron. ;)  The most well-studied quasiparticles in 
silicon are the so-called "electrons" and "holes".  The "electrons" 
aren't really electrons, but rather a charged excited state of a silicon 
atom in  the lattice.  I will admit that the term "diffusion" is used in 
this field.  This is largely because these "jumping" quasiparticle 
entities moving about a silicon crystal lattice actually obey the ideal 
gas law!  However, since these are quasiparticles and not real 
particles, using the term "diffusion" gives biochemists the wrong 
impression of the mechanism.

I know the "primary and secondary" damage formalism, but I think the 
problem these days is that I, for one, am fairly convinced that noone is 
ever going to be able to observe primary damage.  The effects we 
classify as "secondary damage" outnumber these reactions by thousands to 
one.  Colin Nave just described this in his posting, so I won't repeat 
it here, but the primary consequence of any ionizing radiation is to put 
a whole bunch of atoms into chemically excited states.  How this 
chemical energy dissipates depends on the structure of the material, and 
this makes it hard to predict if you don't know the structure.

-James


Bart Hazes wrote:
> Hi James,
>
> We used to talk about primary and secondary radiation damage. The 
> former operates at room temperature where free radicals were said to 
> be formed in solution and diffuse around to damage proteins. Under 
> cryoconditions this no longer happens, leading to greatly improved 
> crystal life time, but we still have primary radiation damage, with 
> the photons directly hitting the protein.
>
> It was my understanding that this was still considered to form a free 
> radical at the affected atom without there being any diffusion 
> involved. Sulfur atoms would be more sensitive as they have a larger 
> X-ray cross-section or because they may act as free-radical sinks 
> where free radicals generated nearby strip an electron from the 
> sulfur, thereby satisfying their own electronic configuration and 
> converting the sulfur into a radical state. For instance, in 
> ribonucleotide reductase a tyrosine free radical is formed 
> spontaneously (using oxygen and an dinuclear iron site) and "jumps" 
> over 20 Angstrom from one subunit to another to form a thiyl free 
> radical in the active site. It then "jumps" back to the tyrosine upon 
> completion of the catalytic cycle. Although we don't know how it 
> jumps, certainly not by diffusion, there is general agreement that it 
> does happen.
>
> People have also observed broken disulfides in cryocrystal structures 
> with the sulfurs at a distance that is too long for a disulfide but 
> too short for a normal non-bonded sulfur-sulfur interaction. I seem to 
> remember that this distance was suggested to indicate the presence of 
> a thiyl free radical. I'm no chemist of physicist so can't evaluate if 
> that claim is reasonable but if it is then that would be direct 
> evidence to support the involvement of a free radical state in 
> radiation damage.
>
> So I guess my questions/comments are
> - what are the great many good reasons to think that free radicals do 
> NOT play a role in radiation damage under cryo.
> - although diffusion does not happen below 130K, radicals do appear to 
> teleport, at least over short distances.
>
> Bart
>
> James Holton wrote:
>> I don't mean to single anyone out, but the assignment of "free 
>> radicals" as the species mediating radiation damage at cryo 
>> temperatures is a "pet peeve" of mine.  Free radicals have been shown 
>> to mediate damage at room temperature (and there is a VERY large body 
>> of literature on this), but there are a great many good reasons to 
>> think that free radicals do NOT play a role in radiation damage under 
>> cryo.
>>
>> This "assignment" of free radicals to damage is often made 
>> (flippantly) in the literature, but I feel a strong need to point out 
>> that there is NO EVIDENCE of a free radical diffusion mechanism for 
>> radiation damage below ~130K.  To the contrary there is a great deal 
>> of evidence that water, buffers and protein crystals below ~130 K are 
>> in a state of matter known as a "solid", and molecules (such as free 
>> radicals) do not diffuse through solids (except on geological 
>> timescales).  If you are worried that the x-ray beam is heating your 
>> crystal to >130 K, then have a look at Snell et. al. JSR 14 109-15 
>> (2007).  They showed quite convincingly that this just can't happen 
>> for anything but the most exotic situations.
>>
>> There is evidence, however, of energy transfer taking place between 
>> different regions of the crystal, but energy transfer does not 
>> require molecular diffusion or any other kind of mass transport.  In 
>> fact, solid-state chemistry is generally mediated by cascading 
>> neighbor-to-neighbor reactions that do not involve "diffusion" in the 
>> traditional sense.  Electricity is an example of this kind of 
>> chemistry, and these reactions are a LOT faster than diffusion.  The 
>> closest analogy to "diffusion" is that the propagating reaction can 
>> be seen as a "species" of sorts that is moving around inside the 
>> sample.  Entities like this are formally called quasiparticles.  Some 
>> quasiparticles are charged, but others are not.  If you don't know 
>> what a quasiparticle is, you can look them up in wikipedia.
>> Some have tried to rescue the "free radical" statements about 
>> radiation damage by claiming that individual electrons are 
>> "radicals".  I guess this must come from the "pressure" of such a 
>> large body of free-radical literature at room temperature.  However, 
>> IMHO this is about as useful as declaring that every chemical 
>> reaction is a "free radical" reaction (since they involve the 
>> movement of electrons).   I think it best that we try to call the 
>> chemistry what it is and try to stamp out rumors that mechanisms are 
>> known when in reality they are not.
>>
>> Just my little rant.
>>
>> -James Holton
>> MAD Scientist
>>
>>
>
>