[3dem] [EXTERNAL] correspondance between protein populations in solution and on the grid?

[Reza Khayat]

?Hi,


Zuben raises an important yet forgotten point, the distinction between precision and accuracy.


Best wishes,
Reza


Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Brown, Zuben <zb2218 at cumc.columbia.edu>
Sent: Tuesday, August 10, 2021 1:49 PM
To: vincent Chaptal; CCPEM at JISCMAIL.AC.UK; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] [EXTERNAL] correspondance between protein populations in solution and on the grid?

Hi Vincent,

Some work by Alex Noble et al. is a good place to start:

https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_34257&d=DwIFAw&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=sdjs5U1nHdzHSfPVxB_ytuv4ZgTPk-uA8ewbrEDLDhI&s=oT1n1B4xhjUS-RZgOtuPdr7qRFkTn-j0hiL2Ud02G1g&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__elifesciences.org_articles_34257&d=DwMFAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=wEohFrvUZAPTjYJdfYQ0TZZkRNs3OsEOIZnNDg6yKyE&s=wfuo7bv7lVIbDFcYfNlfjzv9BjBGrv_8-mXJOOzvvgY&e=>

Samples behave in a strange way on the grid, and even if you were to collect grids in triplicate how would you link your observations to what is happening in the test tube?

Perhaps component A has high affinity to the grid bars and always aggregates there.  Even multiple independent grids wouldn't show you that -- so making a claim about enzymatics from cryo-EM is not straightforward.

Other people certainly are more informed than me, but it seems like a big leap.

best wishes,
Zuben
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of vincent Chaptal <vincent.chaptal at ibcp.fr>
Sent: Tuesday, August 10, 2021 9:04 AM
To: CCPEM at JISCMAIL.AC.UK <CCPEM at JISCMAIL.AC.UK>; 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: [EXTERNAL] [3dem] correspondance between protein populations in solution and on the grid?

Hi,

when performing enzymatic assays, we look at triplicates and we can observe some degree of variation between experimental triplicates (also between biological triplicates but no exactly the topic is this question). Sometimes, I even wonder how homogeneous my purified protein is in the tube, and if I pipet a true homogeneous solution.

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 lipuid?
I was also wondering if conformation A and B would enter foilholes similarly or if some conformations enter holes more preferentially?

I couldn't find litterature on the matter, but I image it's been looked at; could you point me to the right refences?

This discussion on micro-domains in solution, or micro-concentration of particles in liquid, becomes more vivid when using pico-liters type of deposition on grids as we see coming (spotiton, etc...). Should we then collect 3 grids to link to structural enzymology?

Looking forward to your comments.
Best
Vincent

--

Vincent Chaptal, PhD

Director of GdR APPICOM

Drug Resistance and Membrane Proteins Lab


MMSB -UMR5086

7 passage du Vercors

69007 LYON

FRANCE

+33 4 37 65 29 01

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