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

Wed Aug 11 01:50:59 PDT 2021

Hi there,

Unfortunately no clear references to cite, but as I am facing similar problems, I find this an interesting topic.
It obviously goes case by case, but there is a good number of different techniques that give us insights on what is happening in solution, so we can compare with what we see under the microscope:
- if the structures have different Rh, size exclusion chromatography will tell proportions - I’d rather have this done with a SEC-MALS system, it not only measures molecular masses, but is also very good in detecting aggregation, even when it’s very dynamic;
- if you don’t have a lot of protein available, some characterization might be tried with DLS (although we don’t see it very often in recent publications, but we use a lot in the backstage);
- if the microscope suggests changes in secondary structure, good and old circular dichroism;
- if you have a lot of protein, NMR … rather thinking of identifying interactions between specific residues than solving structures in this case.
(… List far from being exhaustive)

Depending on how subtle the differences between conformations A and B are, comparison with structures from negative staining might show if the air-water interface is messing things up.

Anyways, I agree that making grids in triplicates will not tell how much interference is being added by the freezing procedure, so IMO it is important to build the argumentation with results from different techniques. Certainly if you know how to provoke changes in proportions A vs. B with ligands or different buffers or PTM, it will get even better.

The next level of difficulty, which probably makes a different topic, is to check if the different conformations are not artefacts from protein production or manipulation - see for instance the proteasomes that appear heterogeneous under the microscope when they are purified with an affinity column (a large proportion looses the cap), but are just fine when precipitated with ammonium sulfate. I apologize for the lack of reference and also for having forgotten who presented this nice work in a CryoEM colloquium years ago… even worse because the authors are probably in this mailing list, I hope they will forgive me.

---------------------------------------------------
Carlos KIKUTI, PhD
UMR144 - CNRS - Institut Curie
12 rue Lhomond 75005 Paris, France
+33 6 82 87 62 76
carlos.kikuti at curie.fr	

> 
> 
> Message: 1
> Date: Tue, 10 Aug 2021 17:49:05 +0000
> From: "Brown, Zuben" <zb2218 at cumc.columbia.edu>
> To: vincent Chaptal <vincent.chaptal at ibcp.fr>, "CCPEM at JISCMAIL.AC.UK"
> 	<CCPEM at JISCMAIL.AC.UK>, "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] [EXTERNAL] correspondance between protein
> 	populations in solution and on the grid?
> Message-ID:
> 	<BL0PR02MB4771F861CD149033BA5ADAE995F79 at BL0PR02MB4771.namprd02.prod.outlook.com>
> 	
> Content-Type: text/plain; charset="us-ascii"
> 
> 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=DwIFAg&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
> 
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.appicom.cnrs.fr&d=DwIFAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=wEohFrvUZAPTjYJdfYQ0TZZkRNs3OsEOIZnNDg6yKyE&s=LvCeO7hjiRmOCUsdQt7VrpNmwhBQ1OMYzNHQctiQ6yk&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.appicom.cnrs.fr&d=DwMDaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=qrt2reBNwFsAVKl8vw75LCHg-wJUzh2f0l8AbUbjYMU&s=D4e4wIyVRpIAoUnMB_OQnn1FTcxriRAj2xlohCaWC-I&e=>
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> ------------------------------
> 
> Message: 2
> Date: Wed, 11 Aug 2021 00:03:41 +0000
> From: Reza Khayat <rkhayat at ccny.cuny.edu>
> To: "CCPEM at JISCMAIL.AC.UK" <CCPEM at JISCMAIL.AC.UK>,
> 	"3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] [EXTERNAL] correspondance between protein
> 	populations in solution and on the grid?
> Message-ID: <1628640222405.24669 at ccny.cuny.edu>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> ?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
> 
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.appicom.cnrs.fr&d=DwIFAw&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=sdjs5U1nHdzHSfPVxB_ytuv4ZgTPk-uA8ewbrEDLDhI&s=QsS_flqC9i9NxkUwwDgoZJr1lVJDS0e6O166w-AxANo&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.appicom.cnrs.fr&d=DwMDaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=qrt2reBNwFsAVKl8vw75LCHg-wJUzh2f0l8AbUbjYMU&s=D4e4wIyVRpIAoUnMB_OQnn1FTcxriRAj2xlohCaWC-I&e=>
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