Vincent 

Thanks for your great question.  This is a very exciting and great question!   I also share in your thoughts and wonder what it is I can really claim with regards to conformational/compositional population heterogeneity.  I would like to echo your query and add a comment.  I am just a Ph.D. student but this mirrors another conversation I was having with some friends the other day and would love some further discussion if possible.

The comment and link by John Rubinstein does seem to suggest that an independent way of corroborating the observations of heterogeneity from cryoEM can be seen via NMR.  Roughly the same population regardless of ice and grids essentially.  Although, I do not see that group making any direct claim even with an alternative method producing very similar results. 

\\--  John Rubinstein, I see in that paper you relate conf. change to function. But outside of publication, I would be curious to know what you thought about your similar sample population ratios.  Do you speculate this has any "real" connection to the ratio population of conformations that might truly be in your sample equally applied to the CryoEM grid and solution via NMR?  

Zuben's link, made another solid point and that paper is very good at detailing the (almost) uncontrollable variations of particle positions within a foil hole.  

But I would like to echo vincent's thoughts:

>>>>When we observe the same solution under the microscope, is it the same population of particles in 2D/3D classes and in >>>>the test tube?
>>>>If we see 40% of conformation A, and 60% of conformation B in initial/crude 3D classification, can we make the claim that >>>>it represents the actual population as in liquid? 

And I would like to ask then, what is it we CAN claim about this type of very common observation of population conformations?

Fair enough linking what's observed in the microscope to enzymatic activity is tenuous at best.  It should have another independent source of corroboration  (like in John's link).  And, there are amazingly detailed observations to support that there are effects at the foil hole level affecting the sample and reconstructions (serious props to Nobel et, al., for that insane and wonderful paper.  I mean my god, I love those tilt movies with animated illustration figures ... GORGEOUS!). 

However, I do not feel convinced that the grid/ice effects are the complete cause of population conformation differences between conformations A and B either.  Especially in the case of added ligands or substrates to a sample before freezing/imaging i.e., induced conformations seen elsewhere.

So where does that leave us?  No one has measured the effects of ice/grid on conformation population ratios, and the absence of evidence is not proof of anything.  Despite the knowledge about what happens to single particles at the foil hole level under cryoEM conditions.  This is where I get unsure and lose my ground.  Does anyone, feel like rescuing a student who has put his own brain on, dare I say, thin ice ... (couldn't help myself )


Awesome comments, great question, and thanks!!! 

Kako

########################################################################

To unsubscribe from the CCPEM list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCPEM&A=1

This message was issued to members of www.jiscmail.ac.uk/CCPEM, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/