Re: [ccp4bb] biomolecular NMR for IDPs

[Georg Mlynek]

Dear Harry, you can run alphafold via 
https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb#scrollTo=woIxeCPygt7K


*Cited*

*"Differences to AlphaFold v2.0*

In comparison to AlphaFold v2.0, this Colab notebook uses*no templates 
(homologous structures)*and a selected portion of theBFD database 
<https://bfd.mmseqs.com/>. We have validated these changes on several 
thousand recent PDB structures. While accuracy will be near-identical to 
the full AlphaFold system on many targets, a small fraction have a large 
drop in accuracy due to the smaller MSA and lack of templates. For best 
reliability, we recommend instead using thefull open source AlphaFold 
<https://github.com/deepmind/alphafold/>, or theAlphaFold Protein 
Structure Database <https://alphafold.ebi.ac.uk/>."



Br, Georg.





Am 17.08.2021 um 18:12 schrieb Harry Powell - CCP4BB:

Hi

Forgive me if I’ve missed something, but I can’t find a way to run AlphaFold 
without installing it locally - in which case I need a reasonable GPU.

On the other hand, RoseTTAFold does pretty much the same thing and is available 
via David Baker’s web server - upload your sequence and sit back and wait. The 
overall models that come out are very similar to those from AlphaFold (not 
_quite_ as good…) but are generally available wihtout having to install the 
software and maybe invest in a good GPU (DeepMind doesn’t have to worry about 
the cost or hassle of such things).


https://www.bakerlab.org/index.php/2021/07/15/accurate-protein-structure-prediction-accessible/
 (see the link at the bottom of the page just next to Minkyung Baek’s photo)

On the third hand, both AlphaFold and RoseTTAFold are very good at finding 
models for proteins that have good order at the secondary structure level. I 
don’t know much about IDPs, but I thought that they were intrinsically 
disordered because they lack 3D structure. Secondary structure is most 
certainly three dimensional…

Just another two ha’porth.

Harry


On 17 Aug 2021, at 16:59, George Sheldrick  wrote:




As Joel has suggested before, alphafold on an IDP would be interesting and 
would seem like a zero-cost starting point - perhaps one you have tried already.


Sent from ProtonMail mobile



 Original Message 
On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:

Hi Sorin,

I hate to say it, but this is a really tough and expensive one. Solving a true 
conformational ensemble of one IDP of decent size (~>70 residues) at something 
like decent resolution is hard, and not that many labs actually do it (it's 
usually a different set of NMR techniques than solving folding proteins, and that 
knowledge is even somewhat specialized even within the NMR community). Solving a 
co-structural ensemble of two IDPs that bind is even harder, and I'm hard pressed 
to remember a single case right now where it's been done (probably has, but very 
rarely). Assuming they express really well and produce decent spectra, it is in 
theory doable, but I'd assume multiple years of work by a very good student or 
postdoc from a lab that specializes in this and many thousands of dollars (I'd 
very roughly assume ~$10k in materials costs alone) would be required for that 
co-structure.

The SAXS route is certainly less expensive and faster if it works and gets you 
the info you need, but it certainly will be low-res. I'm not as familiar with 
it, but if you can differentially label the proteins, the neutron equivalent of 
SAXS might also help with the co-structural ensemble to differentiate which 
protein is where in the resulting blob.

Scott

Scott Horowitz, Ph.D.
Assistant Professor
Department of Chemistry & Biochemistry
Knoebel Institute for Healthy Aging
University of Denver
  
ECS Building

2155 E. Wesley Ave
Denver, CO 80208
Phone: 303-871-4326
Fax: 303-871-7915
Zoom Room: https://udenver.zoom.us/my/scotthorowitz
Email: scott.horow...@du.edu
Office: Room 561   Lab: Room 505

From: CCP4 bulletin board  on behalf of Roopa Thapar 
<070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
Sent: Sunday, August 15, 2021 8:20 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
  
[External Email From]: owner-ccp...@jiscmail.ac.uk



Hello Sorin,


1. The cost of getting NMR data on the IDPs you propose depends upon the 
expression levels of the protein/s as you will need to label with 15N and 13C - 
and depending upon your overall yields per liter of E.coli culture, this can 
add up.  In addition you will need to run triple resonance experiments - so you 
should look into the hourly charge to access the NMR spectrometers where you 
are located.  Moreover, you need to account for time required for optimization 
of solution conditions to collect the NMR data as the sample needs to be 
homogenous (as in no aggregation) at millimolar or hundreds of micromolar 
concentration.   As Ethan Me

Re: [ccp4bb] biomolecular NMR for IDPs

[CCP4BB]

lDDT = Local Difference Distance Test, for reference...

Harry
--
Dr Harry Powell

> On 17 Aug 2021, at 20:21, Randy John Read  wrote:
> 
> What the DeepMind people say about this is that when part of a predicted 
> structure has a low predicted lDDT (a CASP measure of model quality), this is 
> a good indicator that it may be intrinsically disordered. 
> 
> Best wishes,
> 
> Randy Read
> 
>> On 17 Aug 2021, at 17:13, Sorin Draga  wrote:
>> 
>> Dear all, 
>> 
>> Regarding the AlphaFold2: while its results are impressive for properly 
>> folded proteins, I would caution against using it for IDPs: they do not have 
>> a stable 3D structure that can be predicted,  instead they occupy an 
>> ensemble of structures, with a high degree of heterogeneity. While you will 
>> find models of IDPs predicted by AlphaFold, they only represent (at best) 
>> one of many local minima that these proteins occupy.
>> 
>> Again, many thanks for all your excellent contributions! 
>> 
>> Kind regards, 
>> 
>> Sorin 
>> 
>> On Tue, Aug 17, 2021 at 11:59 AM George Sheldrick 
>>  wrote:
>> 
>> 
>> 
>> 
>> 
>> As Joel has suggested before, alphafold on an IDP would be interesting and 
>> would seem like a zero-cost starting point - perhaps one you have tried 
>> already.
>> 
>> 
>> 
>> Sent from ProtonMail mobile
>> 
>> 
>> 
>>  Original Message 
>> On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:
>> 
>> Hi Sorin,
>> 
>> I hate to say it, but this is a really tough and expensive one. Solving a 
>> true conformational ensemble of one IDP of decent size (~>70 residues) at 
>> something like decent resolution is hard, and not that many labs actually do 
>> it (it's usually a different set of NMR techniques than solving folding 
>> proteins, and that knowledge is even somewhat specialized even within the 
>> NMR community). Solving a co-structural ensemble of two IDPs that bind is 
>> even harder, and I'm hard pressed to remember a single case right now where 
>> it's been done (probably has, but very rarely). Assuming they express really 
>> well and produce decent spectra, it is in theory doable, but I'd assume 
>> multiple years of work by a very good student or postdoc from a lab that 
>> specializes in this and many thousands of dollars (I'd very roughly assume 
>> ~$10k in materials costs alone) would be required for that co-structure.
>> 
>> The SAXS route is certainly less expensive and faster if it works and gets 
>> you the info you need, but it certainly will be low-res. I'm not as familiar 
>> with it, but if you can differentially label the proteins, the neutron 
>> equivalent of SAXS might also help with the co-structural ensemble to 
>> differentiate which protein is where in the resulting blob.
>> 
>> Scott
>> 
>> Scott Horowitz, Ph.D.
>> Assistant Professor
>> Department of Chemistry & Biochemistry
>> Knoebel Institute for Healthy Aging
>> University of Denver
>> 
>> ECS Building
>> 2155 E. Wesley Ave
>> Denver, CO 80208
>> Phone: 303-871-4326
>> Fax: 303-871-7915
>> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
>> Email: scott.horow...@du.edu
>> Office: Room 561   Lab: Room 505
>> 
>> From: CCP4 bulletin board  on behalf of Roopa Thapar 
>> <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
>> Sent: Sunday, August 15, 2021 8:20 AM
>> To: CCP4BB@JISCMAIL.AC.UK 
>> Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>> 
>> [External Email From]: owner-ccp...@jiscmail.ac.uk
>> 
>> 
>> Hello Sorin,
>> 
>> 
>> 1. The cost of getting NMR data on the IDPs you propose depends upon the 
>> expression levels of the protein/s as you will need to label with 15N and 
>> 13C - and depending upon your overall yields per liter of E.coli culture, 
>> this can add up.  In addition you will need to run triple resonance 
>> experiments - so you should look into the hourly charge to access the NMR 
>> spectrometers where you are located.  Moreover, you need to account for time 
>> required for optimization of solution conditions to collect the NMR data as 
>> the sample needs to be homogenous (as in no aggregation) at millimolar or 
>> hundreds of micromolar concentration.   As Ethan Merritt suggested, it would 
>> be a good idea to use SAXS first as it requires very little sample, no 
>> isotope labeling, and you can try to narrow down the solution conditions 
&g

Re: [ccp4bb] biomolecular NMR for IDPs

[Randy John Read]

What the DeepMind people say about this is that when part of a predicted 
structure has a low predicted lDDT (a CASP measure of model quality), this is a 
good indicator that it may be intrinsically disordered. 

Best wishes,

Randy Read

> On 17 Aug 2021, at 17:13, Sorin Draga  wrote:
> 
> Dear all, 
> 
> Regarding the AlphaFold2: while its results are impressive for properly 
> folded proteins, I would caution against using it for IDPs: they do not have 
> a stable 3D structure that can be predicted,  instead they occupy an ensemble 
> of structures, with a high degree of heterogeneity. While you will find 
> models of IDPs predicted by AlphaFold, they only represent (at best) one of 
> many local minima that these proteins occupy.
> 
> Again, many thanks for all your excellent contributions! 
> 
> Kind regards, 
> 
> Sorin 
> 
> On Tue, Aug 17, 2021 at 11:59 AM George Sheldrick  
> wrote:
> 
> 
> 
> 
> 
> As Joel has suggested before, alphafold on an IDP would be interesting and 
> would seem like a zero-cost starting point - perhaps one you have tried 
> already.
> 
> 
> 
> Sent from ProtonMail mobile
> 
> 
> 
>  Original Message 
> On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:
> 
> Hi Sorin,
> 
> I hate to say it, but this is a really tough and expensive one. Solving a 
> true conformational ensemble of one IDP of decent size (~>70 residues) at 
> something like decent resolution is hard, and not that many labs actually do 
> it (it's usually a different set of NMR techniques than solving folding 
> proteins, and that knowledge is even somewhat specialized even within the NMR 
> community). Solving a co-structural ensemble of two IDPs that bind is even 
> harder, and I'm hard pressed to remember a single case right now where it's 
> been done (probably has, but very rarely). Assuming they express really well 
> and produce decent spectra, it is in theory doable, but I'd assume multiple 
> years of work by a very good student or postdoc from a lab that specializes 
> in this and many thousands of dollars (I'd very roughly assume ~$10k in 
> materials costs alone) would be required for that co-structure.
> 
> The SAXS route is certainly less expensive and faster if it works and gets 
> you the info you need, but it certainly will be low-res. I'm not as familiar 
> with it, but if you can differentially label the proteins, the neutron 
> equivalent of SAXS might also help with the co-structural ensemble to 
> differentiate which protein is where in the resulting blob.
> 
> Scott
> 
> Scott Horowitz, Ph.D.
> Assistant Professor
> Department of Chemistry & Biochemistry
> Knoebel Institute for Healthy Aging
> University of Denver
>  
> ECS Building
> 2155 E. Wesley Ave
> Denver, CO 80208
> Phone: 303-871-4326
> Fax: 303-871-7915
> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
> Email: scott.horow...@du.edu
> Office: Room 561   Lab: Room 505
> 
> From: CCP4 bulletin board  on behalf of Roopa Thapar 
> <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> Sent: Sunday, August 15, 2021 8:20 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>  
> [External Email From]: owner-ccp...@jiscmail.ac.uk
> 
> 
> Hello Sorin,
> 
> 
> 1. The cost of getting NMR data on the IDPs you propose depends upon the 
> expression levels of the protein/s as you will need to label with 15N and 13C 
> - and depending upon your overall yields per liter of E.coli culture, this 
> can add up.  In addition you will need to run triple resonance experiments - 
> so you should look into the hourly charge to access the NMR spectrometers 
> where you are located.  Moreover, you need to account for time required for 
> optimization of solution conditions to collect the NMR data as the sample 
> needs to be homogenous (as in no aggregation) at millimolar or hundreds of 
> micromolar concentration.   As Ethan Merritt suggested, it would be a good 
> idea to use SAXS first as it requires very little sample, no isotope 
> labeling, and you can try to narrow down the solution conditions that would 
> be best suited for NMR.  The Kratky plots, Rg values under different solution 
> conditions can give very useful information about conformational states and 
> ensembles populated by IDPs.  However, although NMR tends to be more 
> expensive than other techniques but is perfect for IDPs as you point out you 
> can get residue specific information.  A combined NMR/SAXS approach has 
> proven to be very useful to validate computational models.
> 
> 2. In general, CROs are much more expensive particularly for generating 
> isotopical

Re: [ccp4bb] biomolecular NMR for IDPs

[Krieger, James M]

Dear Harry,

Thanks very much for the info about RoseTTAFold. As for AlphaFold, there are 
ways to run it through Google Colab e.g. https://t.co/kxvjLVFwQx

I’d also advise against using these for IDPs.

Best wishes
James

On Aug 17, 2021, at 17:13, Harry Powell - CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:

Hi

Forgive me if I’ve missed something, but I can’t find a way to run AlphaFold 
without installing it locally - in which case I need a reasonable GPU.

On the other hand, RoseTTAFold does pretty much the same thing and is available 
via David Baker’s web server - upload your sequence and sit back and wait. The 
overall models that come out are very similar to those from AlphaFold (not 
_quite_ as good…) but are generally available wihtout having to install the 
software and maybe invest in a good GPU (DeepMind doesn’t have to worry about 
the cost or hassle of such things).

  
https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.bakerlab.org%2Findex.php%2F2021%2F07%2F15%2Faccurate-protein-structure-prediction-accessible%2Fdata=04%7C01%7Ckriegerj%40PITT.EDU%7Cf9f072cb89204d071c5108d96199e97e%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C637648135958426818%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=kDqYYceh876jRrCLDQxN3%2FhQaMk34Scgog4RRnTHAso%3Dreserved=0
 (see the link at the bottom of the page just next to Minkyung Baek’s photo)

On the third hand, both AlphaFold and RoseTTAFold are very good at finding 
models for proteins that have good order at the secondary structure level. I 
don’t know much about IDPs, but I thought that they were intrinsically 
disordered because they lack 3D structure. Secondary structure is most 
certainly three dimensional…

Just another two ha’porth.

Harry

On 17 Aug 2021, at 16:59, George Sheldrick  wrote:




As Joel has suggested before, alphafold on an IDP would be interesting and 
would seem like a zero-cost starting point - perhaps one you have tried already.


Sent from ProtonMail mobile



 Original Message 
On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:

Hi Sorin,

I hate to say it, but this is a really tough and expensive one. Solving a true 
conformational ensemble of one IDP of decent size (~>70 residues) at something 
like decent resolution is hard, and not that many labs actually do it (it's 
usually a different set of NMR techniques than solving folding proteins, and 
that knowledge is even somewhat specialized even within the NMR community). 
Solving a co-structural ensemble of two IDPs that bind is even harder, and I'm 
hard pressed to remember a single case right now where it's been done (probably 
has, but very rarely). Assuming they express really well and produce decent 
spectra, it is in theory doable, but I'd assume multiple years of work by a 
very good student or postdoc from a lab that specializes in this and many 
thousands of dollars (I'd very roughly assume ~$10k in materials costs alone) 
would be required for that co-structure.

The SAXS route is certainly less expensive and faster if it works and gets you 
the info you need, but it certainly will be low-res. I'm not as familiar with 
it, but if you can differentially label the proteins, the neutron equivalent of 
SAXS might also help with the co-structural ensemble to differentiate which 
protein is where in the resulting blob.

Scott

Scott Horowitz, Ph.D.
Assistant Professor
Department of Chemistry & Biochemistry
Knoebel Institute for Healthy Aging
University of Denver

ECS Building
2155 E. Wesley Ave
Denver, CO 80208
Phone: 303-871-4326
Fax: 303-871-7915
Zoom Room: 
https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fudenver.zoom.us%2Fmy%2Fscotthorowitzdata=04%7C01%7Ckriegerj%40PITT.EDU%7Cf9f072cb89204d071c5108d96199e97e%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C637648135958426818%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000sdata=HUuXJBDq0iobMIA1xAtvEEyhmcpM1xxE6W4bGjX3oVQ%3Dreserved=0
Email: scott.horow...@du.edu
Office: Room 561   Lab: Room 505

From: CCP4 bulletin board  on behalf of Roopa Thapar 
<070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
Sent: Sunday, August 15, 2021 8:20 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs

[External Email From]: owner-ccp...@jiscmail.ac.uk


Hello Sorin,


1. The cost of getting NMR data on the IDPs you propose depends upon the 
expression levels of the protein/s as you will need to label with 15N and 13C - 
and depending upon your overall yields per liter of E.coli culture, this can 
add up.  In addition you will need to run triple resonance experiments - so you 
should look into the hourly charge to access the NMR spectrometers where you 
are located.  Moreover, you need to account for time required for optimization 
of solution conditions to collect the NMR data as the sampl

Re: [ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Dear all,

Regarding the AlphaFold2: while its results are impressive for properly
folded proteins, I would caution against using it for IDPs: they do not
have a stable 3D structure that can be predicted,  instead they occupy an
ensemble of structures, with a high degree of heterogeneity. While you will
find models of IDPs predicted by AlphaFold, they only represent (at best)
one of many local minima that these proteins occupy.

Again, many thanks for all your excellent contributions!

Kind regards,

Sorin

On Tue, Aug 17, 2021 at 11:59 AM George Sheldrick 
wrote:

>
>
>
> As Joel has suggested before, alphafold on an IDP would be interesting and
> would seem like a zero-cost starting point - perhaps one you have tried
> already.
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:
>
>
> Hi Sorin,
>
> I hate to say it, but this is a really tough and expensive one. Solving a
> true conformational ensemble of one IDP of decent size (~>70 residues) at
> something like decent resolution is hard, and not that many labs actually
> do it (it's usually a different set of NMR techniques than solving folding
> proteins, and that knowledge is even somewhat specialized even within the
> NMR community). Solving a co-structural ensemble of two IDPs that bind is
> even harder, and I'm hard pressed to remember a single case right now where
> it's been done (probably has, but very rarely). Assuming they express
> really well and produce decent spectra, it is in theory doable, but I'd
> assume multiple years of work by a very good student or postdoc from a lab
> that specializes in this and many thousands of dollars (I'd very roughly
> assume ~$10k in materials costs alone) would be required for that
> co-structure.
>
> The SAXS route is certainly less expensive and faster if it works and gets
> you the info you need, but it certainly will be low-res. I'm not as
> familiar with it, but if you can differentially label the proteins, the
> neutron equivalent of SAXS might also help with the co-structural ensemble
> to differentiate which protein is where in the resulting blob.
>
> Scott
>
> Scott Horowitz, Ph.D.
>
> Assistant Professor
>
> Department of Chemistry & Biochemistry
>
> Knoebel Institute for Healthy Aging
>
> University of Denver
>
>
>
> ECS Building
>
> 2155 E. Wesley Ave
>
> Denver, CO 80208
>
> Phone: 303-871-4326
>
> Fax: 303-871-7915
>
> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
>
> Email: scott.horow...@du.edu
>
> Office: Room 561   Lab: Room 505
>
> --
> *From:* CCP4 bulletin board 
>  on behalf of Roopa Thapar
> <0000070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* Sunday, August 15, 2021 8:20 AM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> 
> *Subject:* [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>
> [External Email From]: *owner-ccp...@jiscmail.ac.uk*
>
> Hello Sorin,
>
>
> 1. The cost of getting NMR data on the IDPs you propose depends upon the
> expression levels of the protein/s as you will need to label with 15N and
> 13C - and depending upon your overall yields per liter of E.coli culture,
> this can add up.  In addition you will need to run triple resonance
> experiments - so you should look into the hourly charge to access the NMR
> spectrometers where you are located.  Moreover, you need to account for
> time required for optimization of solution conditions to collect the NMR
> data as the sample needs to be homogenous (as in no aggregation) at
> millimolar or hundreds of micromolar concentration.   As Ethan Merritt
> suggested, it would be a good idea to use SAXS first as it requires very
> little sample, no isotope labeling, and you can try to narrow down the
> solution conditions that would be best suited for NMR.  The Kratky plots,
> Rg values under different solution conditions can give very useful
> information about conformational states and ensembles populated by IDPs.
> However, although NMR tends to be more expensive than other techniques
> but is perfect for IDPs as you point out you can get residue specific
> information.  A combined NMR/SAXS approach has proven to be very useful to
> validate computational models.
>
> 2. In general, CROs are much more expensive particularly for generating
> isotopically labeled samples - it is cost-prohibitive for academic labs.
> Genscript is one CRO that will express proteins, but I am not sure if they
> will make isotopically labeled proteins for NMR.
>
> 3. The amount of protein needed depends upon the size of the molecule

Re: [ccp4bb] biomolecular NMR for IDPs

[Harry Powell - CCP4BB]

Hi

Forgive me if I’ve missed something, but I can’t find a way to run AlphaFold 
without installing it locally - in which case I need a reasonable GPU.

On the other hand, RoseTTAFold does pretty much the same thing and is available 
via David Baker’s web server - upload your sequence and sit back and wait. The 
overall models that come out are very similar to those from AlphaFold (not 
_quite_ as good…) but are generally available wihtout having to install the 
software and maybe invest in a good GPU (DeepMind doesn’t have to worry about 
the cost or hassle of such things).


https://www.bakerlab.org/index.php/2021/07/15/accurate-protein-structure-prediction-accessible/
 (see the link at the bottom of the page just next to Minkyung Baek’s photo)

On the third hand, both AlphaFold and RoseTTAFold are very good at finding 
models for proteins that have good order at the secondary structure level. I 
don’t know much about IDPs, but I thought that they were intrinsically 
disordered because they lack 3D structure. Secondary structure is most 
certainly three dimensional…

Just another two ha’porth.

Harry

> On 17 Aug 2021, at 16:59, George Sheldrick  wrote:
> 
> 
> 
> 
> As Joel has suggested before, alphafold on an IDP would be interesting and 
> would seem like a zero-cost starting point - perhaps one you have tried 
> already.
> 
> 
> Sent from ProtonMail mobile
> 
> 
> 
>  Original Message 
> On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:
> 
> Hi Sorin,
> 
> I hate to say it, but this is a really tough and expensive one. Solving a 
> true conformational ensemble of one IDP of decent size (~>70 residues) at 
> something like decent resolution is hard, and not that many labs actually do 
> it (it's usually a different set of NMR techniques than solving folding 
> proteins, and that knowledge is even somewhat specialized even within the NMR 
> community). Solving a co-structural ensemble of two IDPs that bind is even 
> harder, and I'm hard pressed to remember a single case right now where it's 
> been done (probably has, but very rarely). Assuming they express really well 
> and produce decent spectra, it is in theory doable, but I'd assume multiple 
> years of work by a very good student or postdoc from a lab that specializes 
> in this and many thousands of dollars (I'd very roughly assume ~$10k in 
> materials costs alone) would be required for that co-structure.
> 
> The SAXS route is certainly less expensive and faster if it works and gets 
> you the info you need, but it certainly will be low-res. I'm not as familiar 
> with it, but if you can differentially label the proteins, the neutron 
> equivalent of SAXS might also help with the co-structural ensemble to 
> differentiate which protein is where in the resulting blob.
> 
> Scott
> 
> Scott Horowitz, Ph.D.
> Assistant Professor
> Department of Chemistry & Biochemistry
> Knoebel Institute for Healthy Aging
> University of Denver
>  
> ECS Building
> 2155 E. Wesley Ave
> Denver, CO 80208
> Phone: 303-871-4326
> Fax: 303-871-7915
> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
> Email: scott.horow...@du.edu
> Office: Room 561   Lab: Room 505
> 
> From: CCP4 bulletin board  on behalf of Roopa Thapar 
> <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> Sent: Sunday, August 15, 2021 8:20 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>  
> [External Email From]: owner-ccp...@jiscmail.ac.uk
> 
> 
> Hello Sorin,
> 
> 
> 1. The cost of getting NMR data on the IDPs you propose depends upon the 
> expression levels of the protein/s as you will need to label with 15N and 13C 
> - and depending upon your overall yields per liter of E.coli culture, this 
> can add up.  In addition you will need to run triple resonance experiments - 
> so you should look into the hourly charge to access the NMR spectrometers 
> where you are located.  Moreover, you need to account for time required for 
> optimization of solution conditions to collect the NMR data as the sample 
> needs to be homogenous (as in no aggregation) at millimolar or hundreds of 
> micromolar concentration.   As Ethan Merritt suggested, it would be a good 
> idea to use SAXS first as it requires very little sample, no isotope 
> labeling, and you can try to narrow down the solution conditions that would 
> be best suited for NMR.  The Kratky plots, Rg values under different solution 
> conditions can give very useful information about conformational states and 
> ensembles populated by IDPs.  However, although NMR tends to be more 
> expensive than other techniques but is perfect for IDPs as you point out you 
> can get residue s

Re: [ccp4bb] biomolecular NMR for IDPs

[George Sheldrick]

As Joel has suggested before, alphafold on an IDP would be interesting 
and would seem like a zero-cost starting point - perhaps one you have 
tried already.




Sent from ProtonMail mobile



 Original Message 
On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:


   Hi Sorin,

   I hate to say it, but this is a really tough and expensive one.
   Solving a true conformational ensemble of one IDP of decent size
   (~>70 residues) at something like decent resolution is hard, and not
   that many labs actually do it (it's usually a different set of NMR
   techniques than solving folding proteins, and that knowledge is even
   somewhat specialized even within the NMR community). Solving a
   co-structural ensemble of two IDPs that bind is even harder, and I'm
   hard pressed to remember a single case right now where it's been
   done (probably has, but very rarely). Assuming they express really
   well and produce decent spectra, it is in theory doable, but I'd
   assume multiple years of work by a very good student or postdoc from
   a lab that specializes in this and many thousands of dollars (I'd
   very roughly assume ~$10k in materials costs alone) would be
   required for that co-structure.

   The SAXS route is certainly less expensive and faster if it works
   and gets you the info you need, but it certainly will be low-res.
   I'm not as familiar with it, but if you can differentially label the
   proteins, the neutron equivalent of SAXS might also help with the
   co-structural ensemble to differentiate which protein is where in
   the resulting blob.

   Scott

   Scott Horowitz, Ph.D.

   Assistant Professor

   Department of Chemistry & Biochemistry

   Knoebel Institute for Healthy Aging

   University of Denver

   ECS Building

   2155 E. Wesley Ave

   Denver, CO 80208

   Phone: 303-871-4326

   Fax: 303-871-7915

   Zoom Room: https://udenver.zoom.us/my/scotthorowitz
   <https://udenver.zoom.us/my/scotthorowitz>**

   Email: scott.horow...@du.edu

   Office: Room 561   Lab: Room 505


   
   *From:* CCP4 bulletin board  on behalf of
   Roopa Thapar <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
   *Sent:* Sunday, August 15, 2021 8:20 AM
   *To:* CCP4BB@JISCMAIL.AC.UK 
   *Subject:* [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
   [External Email From]: *owner-ccp...@jiscmail.ac.uk*



   Hello Sorin,


   1. The cost of getting NMR data on the IDPs you propose depends upon
   the expression levels of the protein/s as you will need to label
   with 15N and 13C - and depending upon your overall yields per liter
   of E.coli culture, this can add up.  In addition you will need to
   run triple resonance experiments - so you should look into the
   hourly charge to access the NMR spectrometers where you are
   located.  Moreover, you need to account for time required for
   optimization of solution conditions to collect the NMR data as the
   sample needs to be homogenous (as in no aggregation) at millimolar
   or hundreds of micromolar concentration.   As Ethan Merritt
   suggested, it would be a good idea to use SAXS first as it requires
   very little sample, no isotope labeling, and you can try to narrow
   down the solution conditions that would be best suited for NMR. The
   Kratky plots, Rg values under different solution conditions can give
   very useful information about conformational states and ensembles
   populated by IDPs. However, although NMR tends to be more expensive
   than other techniques but is perfect for IDPs as you point out you
   can get residue specific information.  A combined NMR/SAXS approach
   has proven to be very useful to validate computational models.

   2. In general, CROs are much more expensive particularly for
   generating isotopically labeled samples - it is cost-prohibitive for
   academic labs.  Genscript is one CRO that will express proteins, but
   I am not sure if they will make isotopically labeled proteins for NMR.

   3. The amount of protein needed depends upon the size of the
   molecule.  You will need at least 2-3 samples that are
   differentially labeled with 15N, 13C (also since you want data on
   the free and bound forms of the complex) at 0.5 - 1 mM depending
   upon the size of the molecule which relates to the complexity of the
   NMR spectrum due to number of resonances and the relaxation times. 
   The total volume required for each sample is between 280 ul - 600
   ul, depending upon which type of instrumentation and NMR probes you
   have access to.

   Hope this helps!

   Best regards,
   Roopa

   On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga
wrote:


   Hello everyone,

   I do realize that this is not a NMR focused group, but I do hope
   that there are a few spectroscopists lurking around that could
   possibly answer a few questions (I am more of a
  

Re: [ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Thank you all for the very helpful information and suggestions!

Kind regards,

Sorin

On Sun, Aug 15, 2021 at 12:48 PM Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> As Joel has suggested before, alphafold on an IDP would be interesting and
> would seem like a zero-cost starting point - perhaps one you have tried
> already.
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote:
>
>
> Hi Sorin,
>
> I hate to say it, but this is a really tough and expensive one. Solving a
> true conformational ensemble of one IDP of decent size (~>70 residues) at
> something like decent resolution is hard, and not that many labs actually
> do it (it's usually a different set of NMR techniques than solving folding
> proteins, and that knowledge is even somewhat specialized even within the
> NMR community). Solving a co-structural ensemble of two IDPs that bind is
> even harder, and I'm hard pressed to remember a single case right now where
> it's been done (probably has, but very rarely). Assuming they express
> really well and produce decent spectra, it is in theory doable, but I'd
> assume multiple years of work by a very good student or postdoc from a lab
> that specializes in this and many thousands of dollars (I'd very roughly
> assume ~$10k in materials costs alone) would be required for that
> co-structure.
>
> The SAXS route is certainly less expensive and faster if it works and gets
> you the info you need, but it certainly will be low-res. I'm not as
> familiar with it, but if you can differentially label the proteins, the
> neutron equivalent of SAXS might also help with the co-structural ensemble
> to differentiate which protein is where in the resulting blob.
>
> Scott
>
> Scott Horowitz, Ph.D.
>
> Assistant Professor
>
> Department of Chemistry & Biochemistry
>
> Knoebel Institute for Healthy Aging
>
> University of Denver
>
>
>
> ECS Building
>
> 2155 E. Wesley Ave
>
> Denver, CO 80208
>
> Phone: 303-871-4326
>
> Fax: 303-871-7915
>
> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
>
> Email: scott.horow...@du.edu
>
> Office: Room 561   Lab: Room 505
>
> ------
> *From:* CCP4 bulletin board  on behalf of Roopa
> Thapar <070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* Sunday, August 15, 2021 8:20 AM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>
> [External Email From]: *owner-ccp...@jiscmail.ac.uk*
>
> Hello Sorin,
>
>
> 1. The cost of getting NMR data on the IDPs you propose depends upon the
> expression levels of the protein/s as you will need to label with 15N and
> 13C - and depending upon your overall yields per liter of E.coli culture,
> this can add up.  In addition you will need to run triple resonance
> experiments - so you should look into the hourly charge to access the NMR
> spectrometers where you are located.  Moreover, you need to account for
> time required for optimization of solution conditions to collect the NMR
> data as the sample needs to be homogenous (as in no aggregation) at
> millimolar or hundreds of micromolar concentration.   As Ethan Merritt
> suggested, it would be a good idea to use SAXS first as it requires very
> little sample, no isotope labeling, and you can try to narrow down the
> solution conditions that would be best suited for NMR.  The Kratky plots,
> Rg values under different solution conditions can give very useful
> information about conformational states and ensembles populated by IDPs.
> However, although NMR tends to be more expensive than other techniques
> but is perfect for IDPs as you point out you can get residue specific
> information.  A combined NMR/SAXS approach has proven to be very useful to
> validate computational models.
>
> 2. In general, CROs are much more expensive particularly for generating
> isotopically labeled samples - it is cost-prohibitive for academic labs.
> Genscript is one CRO that will express proteins, but I am not sure if they
> will make isotopically labeled proteins for NMR.
>
> 3. The amount of protein needed depends upon the size of the molecule.
> You will need at least 2-3 samples that are differentially labeled with
> 15N, 13C (also since you want data on the free and bound forms of the
> complex) at 0.5 - 1 mM depending upon the size of the molecule which
> relates to the complexity of the NMR spectrum due to number of resonances
> and the relaxation times.  The total volume required for each sample is
> between 280 ul - 600 ul, depending upon which type of instrumentation a

Re: [ccp4bb] biomolecular NMR for IDPs

[Jon Cooper]

As Joel has suggested before, alphafold on an IDP would be interesting and 
would seem like a zero-cost starting point - perhaps one you have tried already.

Sent from ProtonMail mobile

 Original Message 
On 15 Aug 2021, 15:53, Scott Horowitz wrote:

> Hi Sorin,
>
> I hate to say it, but this is a really tough and expensive one. Solving a 
> true conformational ensemble of one IDP of decent size (~>70 residues) at 
> something like decent resolution is hard, and not that many labs actually do 
> it (it's usually a different set of NMR techniques than solving folding 
> proteins, and that knowledge is even somewhat specialized even within the NMR 
> community). Solving a co-structural ensemble of two IDPs that bind is even 
> harder, and I'm hard pressed to remember a single case right now where it's 
> been done (probably has, but very rarely). Assuming they express really well 
> and produce decent spectra, it is in theory doable, but I'd assume multiple 
> years of work by a very good student or postdoc from a lab that specializes 
> in this and many thousands of dollars (I'd very roughly assume ~$10k in 
> materials costs alone) would be required for that co-structure.
>
> The SAXS route is certainly less expensive and faster if it works and gets 
> you the info you need, but it certainly will be low-res. I'm not as familiar 
> with it, but if you can differentially label the proteins, the neutron 
> equivalent of SAXS might also help with the co-structural ensemble to 
> differentiate which protein is where in the resulting blob.
>
> Scott
>
> [Scott Horowitz, Ph.D.]
>
> Assistant Professor
>
> Department of Chemistry & Biochemistry
>
> Knoebel Institute for Healthy Aging
>
> University of Denver
>
> ECS Building
>
> 2155 E. Wesley Ave
>
> Denver, CO 80208
>
> Phone: 303-871-4326
>
> Fax: 303-871-7915
>
> Zoom Room: https://udenver.zoom.us/my/scotthorowitz
>
> Email: scott.horow...@du.edu
>
> Office: Room 561 Lab: Room 505
>
> ---
>
> From: CCP4 bulletin board  on behalf of Roopa Thapar 
> <0000070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
> Sent: Sunday, August 15, 2021 8:20 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs
>
> [External Email From]: owner-ccp...@jiscmail.ac.uk
>
> Hello Sorin,
>
> 1. The cost of getting NMR data on the IDPs you propose depends upon the 
> expression levels of the protein/s as you will need to label with 15N and 13C 
> - and depending upon your overall yields per liter of E.coli culture, this 
> can add up. In addition you will need to run triple resonance experiments - 
> so you should look into the hourly charge to access the NMR spectrometers 
> where you are located. Moreover, you need to account for time required for 
> optimization of solution conditions to collect the NMR data as the sample 
> needs to be homogenous (as in no aggregation) at millimolar or hundreds of 
> micromolar concentration. As Ethan Merritt suggested, it would be a good idea 
> to use SAXS first as it requires very little sample, no isotope labeling, and 
> you can try to narrow down the solution conditions that would be best suited 
> for NMR. The Kratky plots, Rg values under different solution conditions can 
> give very useful information about conformational states and ensembles 
> populated by IDPs. However, although NMR tends to be more expensive than 
> other techniques but is perfect for IDPs as you point out you can get residue 
> specific information. A combined NMR/SAXS approach has proven to be very 
> useful to validate computational models.
>
> 2. In general, CROs are much more expensive particularly for generating 
> isotopically labeled samples - it is cost-prohibitive for academic labs. 
> Genscript is one CRO that will express proteins, but I am not sure if they 
> will make isotopically labeled proteins for NMR.
>
> 3. The amount of protein needed depends upon the size of the molecule. You 
> will need at least 2-3 samples that are differentially labeled with 15N, 13C 
> (also since you want data on the free and bound forms of the complex) at 0.5 
> - 1 mM depending upon the size of the molecule which relates to the 
> complexity of the NMR spectrum due to number of resonances and the relaxation 
> times. The total volume required for each sample is between 280 ul - 600 ul, 
> depending upon which type of instrumentation and NMR probes you have access 
> to.
>
> Hope this helps!
>
> Best regards,
> Roopa
>
> On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga 
>  wrote:
>
> Hello everyone,
>
> I do realize that this is not a N

Re: [ccp4bb] biomolecular NMR for IDPs

[Scott Horowitz]

Hi Sorin,

I hate to say it, but this is a really tough and expensive one. Solving a true 
conformational ensemble of one IDP of decent size (~>70 residues) at something 
like decent resolution is hard, and not that many labs actually do it (it's 
usually a different set of NMR techniques than solving folding proteins, and 
that knowledge is even somewhat specialized even within the NMR community). 
Solving a co-structural ensemble of two IDPs that bind is even harder, and I'm 
hard pressed to remember a single case right now where it's been done (probably 
has, but very rarely). Assuming they express really well and produce decent 
spectra, it is in theory doable, but I'd assume multiple years of work by a 
very good student or postdoc from a lab that specializes in this and many 
thousands of dollars (I'd very roughly assume ~$10k in materials costs alone) 
would be required for that co-structure.

The SAXS route is certainly less expensive and faster if it works and gets you 
the info you need, but it certainly will be low-res. I'm not as familiar with 
it, but if you can differentially label the proteins, the neutron equivalent of 
SAXS might also help with the co-structural ensemble to differentiate which 
protein is where in the resulting blob.

Scott

Scott Horowitz, Ph.D.
Assistant Professor
Department of Chemistry & Biochemistry
Knoebel Institute for Healthy Aging
University of Denver

ECS Building
2155 E. Wesley Ave
Denver, CO 80208
Phone: 303-871-4326
Fax: 303-871-7915
Zoom Room: https://udenver.zoom.us/my/scotthorowitz
Email: scott.horow...@du.edu
Office: Room 561   Lab: Room 505



From: CCP4 bulletin board  on behalf of Roopa Thapar 
<070a21fba45f-dmarc-requ...@jiscmail.ac.uk>
Sent: Sunday, August 15, 2021 8:20 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs

[External Email From]: owner-ccp...@jiscmail.ac.uk


Hello Sorin,


1. The cost of getting NMR data on the IDPs you propose depends upon the 
expression levels of the protein/s as you will need to label with 15N and 13C - 
and depending upon your overall yields per liter of E.coli culture, this can 
add up.  In addition you will need to run triple resonance experiments - so you 
should look into the hourly charge to access the NMR spectrometers where you 
are located.  Moreover, you need to account for time required for optimization 
of solution conditions to collect the NMR data as the sample needs to be 
homogenous (as in no aggregation) at millimolar or hundreds of micromolar 
concentration.   As Ethan Merritt suggested, it would be a good idea to use 
SAXS first as it requires very little sample, no isotope labeling, and you can 
try to narrow down the solution conditions that would be best suited for NMR.  
The Kratky plots, Rg values under different solution conditions can give very 
useful information about conformational states and ensembles populated by IDPs. 
 However, although NMR tends to be more expensive than other techniques but is 
perfect for IDPs as you point out you can get residue specific information.  A 
combined NMR/SAXS approach has proven to be very useful to validate 
computational models.

2. In general, CROs are much more expensive particularly for generating 
isotopically labeled samples - it is cost-prohibitive for academic labs.  
Genscript is one CRO that will express proteins, but I am not sure if they will 
make isotopically labeled proteins for NMR.

3. The amount of protein needed depends upon the size of the molecule.  You 
will need at least 2-3 samples that are differentially labeled with 15N, 13C 
(also since you want data on the free and bound forms of the complex) at 0.5 - 
1 mM depending upon the size of the molecule which relates to the complexity of 
the NMR spectrum due to number of resonances and the relaxation times.  The 
total volume required for each sample is between 280 ul - 600 ul, depending 
upon which type of instrumentation and NMR probes you have access to.

Hope this helps!

Best regards,
Roopa

On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga 
 wrote:


Hello everyone,

I do realize that this is not a NMR focused group, but I do hope that there are 
a few spectroscopists lurking around that could possibly answer a few questions 
(I am more of a modeler/computationalist):

The problem: I have two intrinsically disordered proteins that are known to 
interact (let's call them 1 and 2). I would like to get structural information 
(a conformational ensemble) for 1 and for the "complex" (1+2). Further down the 
line (depending on whether this is possible) I would also like to evaluate 
potential small molecule inhibitors for the said complex. Both 1 and 2 are <200 
aminoacids long.

The questions:

1. Could the cost of determining the "structure" for 1 and 1+2 be estimated? To 
be more precise, I am looking for a ball-park figure on how much a NMR 
measureme

Re: [ccp4bb] biomolecular NMR for IDPs

[Harry Powell - CCP4BB]

Hi

Just my two ha’porth.

I’m currently involved in a project where my collaborators are investigating 
the interactions between protein pairs (both hetero and homo) - they 
specifically asked me _not_ to give them any models from ensembles (actually, 
they said “no NMR structures because they are ensembles") - because (unless 
they try their “docking” with every member of the ensemble) they have no idea 
which member of the ensemble they should use for their studies. If you don't 
actually know how the different parts of a protein are oriented with respect to 
itself, how can you know how they will be disposed to another protein?

Which is not to say you shouldn’t try (or even that I’m right) - what it does 
indicate is that if you include ensembles (from any source) you are going to 
make your life harder and also possibly cast doubt on the results.

Harry


> On 15 Aug 2021, at 14:57, Krieger, James M  wrote:
> 
> It is possible to get an ensemble for an intrinsically disordered segment 
> from NMR. We did this in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008819/
> 
> Best wishes 
> James 
> 
>> On Aug 15, 2021, at 14:48, Jon Cooper 
>> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
>> 
>> Hello, my numpty-level understanding is that being intrinsically disorder 
>> and giving high-resolution structural data are mutually exclusive. I will 
>> re-read your e-mails. Hope this helps. Cheers, Jon.C.
>> 
>> 
>> Sent from ProtonMail mobile
>> 
>> 
>> 
>>  Original Message 
>> On 15 Aug 2021, 09:16, Sorin Draga < sor.dr...@gmail.com> wrote:
>> 
>> Hello Ethan, 
>> 
>> Thank you for the suggestions. I should have mentioned in my initial post 
>> that my intention is to first conduct a high throughput virtual screening on 
>> these proteins, thus I would need high "resolution" of the structures which 
>> SAXS could not provide, as far as I understand.
>> SAXS/SAS might become useful at a later stage, when I have a small number of 
>> potential inhibitors identified.
>> 
>> Kind regards,
>> 
>> Sorin
>> 
>> On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt  wrote:
>> It is possible that you could address some of your questions
>> more quickly and much more cheaply by small-angle scattering,
>> either light (SAS) or X-ray (SAXS).
>> 
>> I would suggest looking into those avenues first.
>> 
>> If you have well behaved (i.e. non-aggregating) purified proein
>> and access to synchrotron beam time (easily requested),
>> a series of SAXS experiments could probably be conducted in one day.
>> I don't want to oversell SAXS, I'm not really an enthusiast.
>> But this case, categorizing the interaction of two poorly ordered proteins
>> in solution and in particular the facilitation or disruption of this
>> interaction by small molecules, should be well within its scope.
>> 
>> best
>> 
>> Ethan
>> 
>> On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
>> > Hello everyone,
>> > 
>> > I do realize that this is not a NMR focused group, but I do hope that there
>> > are a few spectroscopists lurking around that could possibly answer a few
>> > questions (I am more of a modeler/computationalist):
>> > 
>> > The problem: I have two intrinsically disordered proteins that are known to
>> > interact (let's call them 1 and 2). I would like to get structural
>> > information (a conformational ensemble) for 1 and for the "complex" (1+2).
>> > Further down the line (depending on whether this is possible) I would also
>> > like to evaluate potential small molecule inhibitors for the said complex.
>> > Both 1 and 2 are <200 aminoacids long.
>> > 
>> > The questions:
>> > 
>> > 1. Could the cost of determining the "structure" for 1 and 1+2 be
>> > estimated? To be more precise, I am looking for a ball-park figure on how
>> > much a NMR measurement would cost in this case.
>> > 2. Could anyone recommend a good group/CRO that could provide such a
>> > service and not have an astronomical cost?
>> > 3. Any other suggestions/thoughts that you think might be worth mentioning
>> > (minimum quantity of protein necessary, purity, type of NMR etc)
>> > 
>> > Many thanks for your help and time!
>> > 
>> > Cheers!
>> > 
>> > Sorin
>> > 
>> > 
>> > 
>> > To unsubscribe from the CCP4BB list, click the following link:
>> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> > 
>> > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 
>> > list hosted by www.jiscmail.ac.uk, terms & conditions are available at 
>> > https://www.jiscmail.ac.uk/policyandsecurity/
>> > 
>> 
>> 
>> -- 
>> Ethan A Merritt
>> Biomolecular Structure Center,  K-428 Health Sciences Bldg
>> MS 357742,   University of Washington, Seattle 98195-7742
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>> 
>> To unsubscribe 

Re: [ccp4bb] biomolecular NMR for IDPs

[Roopa Thapar]

 Hello Sorin,

1. The cost of getting NMR data on the IDPs you propose depends upon the 
expression levels of the protein/s as you will need to label with 15N and 13C - 
and depending upon your overall yields per liter of E.coli culture, this can 
add up.  In addition you will need to run triple resonance experiments - so you 
should look into the hourly charge to access the NMR spectrometers where you 
are located.  Moreover, you need to account for time required for optimization 
of solution conditions to collect the NMR data as the sample needs to be 
homogenous (as in no aggregation) at millimolar or hundreds of micromolar 
concentration.   As Ethan Merritt suggested, it would be a good idea to use 
SAXS first as it requires very little sample, no isotope labeling, and you can 
try to narrow down the solution conditions that would be best suited for NMR.  
The Kratky plots, Rg values under different solution conditions can give very 
useful information about conformational states and ensembles populated by IDPs. 
 However, although NMR tends to be more expensive than other techniques but is 
perfect for IDPs as you point out you can get residue specific information.  A 
combined NMR/SAXS approach has proven to be very useful to validate 
computational models.
2. In general, CROs are much more expensive particularly for generating 
isotopically labeled samples - it is cost-prohibitive for academic labs.  
Genscript is one CRO that will express proteins, but I am not sure if they will 
make isotopically labeled proteins for NMR.
3. The amount of protein needed depends upon the size of the molecule.  You 
will need at least 2-3 samples that are differentially labeled with 15N, 13C 
(also since you want data on the free and bound forms of the complex) at 0.5 - 
1 mM depending upon the size of the molecule which relates to the complexity of 
the NMR spectrum due to number of resonances and the relaxation times.  The 
total volume required for each sample is between 280 ul - 600 ul, depending 
upon which type of instrumentation and NMR probes you have access to.
Hope this helps!
Best regards,Roopa
On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga 
 wrote:  
 
 Hello everyone, 

I do realize that this is not a NMR focused group, but I do hope that there are 
a few spectroscopists lurking around that could possibly answer a few questions 
(I am more of a modeler/computationalist):

The problem: I have two intrinsically disordered proteins that are known to 
interact (let's call them 1 and 2). I would like to get structural information 
(a conformational ensemble) for 1 and for the "complex" (1+2). Further down the 
line (depending on whether this is possible) I would also like to evaluate 
potential small molecule inhibitors for the said complex. Both 1 and 2 are <200 
aminoacids long. 

The questions:
1. Could the cost of determining the "structure" for 1 and 1+2 be estimated? To 
be more precise, I am looking for a ball-park figure on how much a NMR 
measurement would cost in this case. 2. Could anyone recommend a good group/CRO 
that could provide such a service and not have an astronomical cost?3. Any 
other suggestions/thoughts that you think might be worth mentioning (minimum 
quantity of protein necessary, purity, type of NMR etc)

Many thanks for your help and time!

Cheers! 

Sorin


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
  



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Re: [ccp4bb] biomolecular NMR for IDPs

[Krieger, James M]

I'm not really sure. I did this many years ago during my masters and applied it 
to a much shorter segment (~20 amino acids), which we could chemically 
synthesise and use for natural abundance HSQC experiments. The computational 
cost was also much less as a consequence of the system size I imagine. I 
suggest you get in touch with someone who actually does NMR. I just wanted to 
let you know that this is potentially possible.

Best wishes
James

From: Sorin Draga 
Sent: Sunday, August 15, 2021 3:03 PM
To: Krieger, James M 
Cc: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] biomolecular NMR for IDPs

Hi James,

Could you shed some light on the costs involved? This has been done also in the 
case of the 2 proteins that I am interested in - however the data is not 
publically available and the authors of the articles are reluctant to share the 
data, which I could understand.

Many thanks!

On Sun, Aug 15, 2021 at 9:58 AM Krieger, James M 
mailto:krieg...@pitt.edu>> wrote:
It is possible to get an ensemble for an intrinsically disordered segment from 
NMR. We did this in 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008819/<https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC4008819%2F=04%7C01%7CKRIEGERJ%40pitt.edu%7Ca4967022b2ba4240136a08d95ff58230%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C637646330321934147%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000=Yu9h%2FE4MhX4rHtXBHDYtA5RHjtALvIo%2B%2BfB3%2BoX1JcE%3D=0>

Best wishes
James

On Aug 15, 2021, at 14:48, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk<mailto:488a26d62010-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Hello, my numpty-level understanding is that being intrinsically disorder and 
giving high-resolution structural data are mutually exclusive. I will re-read 
your e-mails. Hope this helps. Cheers, Jon.C.


Sent from ProtonMail mobile



 Original Message 
On 15 Aug 2021, 09:16, Sorin Draga < 
sor.dr...@gmail.com<mailto:sor.dr...@gmail.com>> wrote:

Hello Ethan,

Thank you for the suggestions. I should have mentioned in my initial post that 
my intention is to first conduct a high throughput virtual screening on these 
proteins, thus I would need high "resolution" of the structures which SAXS 
could not provide, as far as I understand.
SAXS/SAS might become useful at a later stage, when I have a small number of 
potential inhibitors identified.

Kind regards,

Sorin

On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt 
mailto:merr...@uw.edu>> wrote:
It is possible that you could address some of your questions
more quickly and much more cheaply by small-angle scattering,
either light (SAS) or X-ray (SAXS).

I would suggest looking into those avenues first.

If you have well behaved (i.e. non-aggregating) purified proein
and access to synchrotron beam time (easily requested),
a series of SAXS experiments could probably be conducted in one day.
I don't want to oversell SAXS, I'm not really an enthusiast.
But this case, categorizing the interaction of two poorly ordered proteins
in solution and in particular the facilitation or disruption of this
interaction by small molecules, should be well within its scope.

best

Ethan

On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
> Hello everyone,
>
> I do realize that this is not a NMR focused group, but I do hope that there
> are a few spectroscopists lurking around that could possibly answer a few
> questions (I am more of a modeler/computationalist):
>
> The problem: I have two intrinsically disordered proteins that are known to
> interact (let's call them 1 and 2). I would like to get structural
> information (a conformational ensemble) for 1 and for the "complex" (1+2).
> Further down the line (depending on whether this is possible) I would also
> like to evaluate potential small molecule inhibitors for the said complex.
> Both 1 and 2 are <200 aminoacids long.
>
> The questions:
>
> 1. Could the cost of determining the "structure" for 1 and 1+2 be
> estimated? To be more precise, I am looking for a ball-park figure on how
> much a NMR measurement would cost in this case.
> 2. Could anyone recommend a good group/CRO that could provide such a
> service and not have an astronomical cost?
> 3. Any other suggestions/thoughts that you think might be worth mentioning
> (minimum quantity of protein necessary, purity, type of NMR etc)
>
> Many thanks for your help and time!
>
> Cheers!
>
> Sorin
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://nam12.safelink

Re: [ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Hi James,

Could you shed some light on the costs involved? This has been done also in
the case of the 2 proteins that I am interested in - however the data is
not publically available and the authors of the articles are reluctant to
share the data, which I could understand.

Many thanks!

On Sun, Aug 15, 2021 at 9:58 AM Krieger, James M  wrote:

> It is possible to get an ensemble for an intrinsically disordered segment
> from NMR. We did this in
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008819/
>
> Best wishes
> James
>
> On Aug 15, 2021, at 14:48, Jon Cooper <
> 488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> Hello, my numpty-level understanding is that being intrinsically disorder
> and giving high-resolution structural data are mutually exclusive. I will
> re-read your e-mails. Hope this helps. Cheers, Jon.C.
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 15 Aug 2021, 09:16, Sorin Draga < sor.dr...@gmail.com> wrote:
>
>
> Hello Ethan,
>
> Thank you for the suggestions. I should have mentioned in my initial post
> that my intention is to first conduct a high throughput virtual screening
> on these proteins, thus I would need high "resolution" of the structures
> which SAXS could not provide, as far as I understand.
> SAXS/SAS might become useful at a later stage, when I have a small number
> of potential inhibitors identified.
>
> Kind regards,
>
> Sorin
>
> On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt  wrote:
>
>> It is possible that you could address some of your questions
>> more quickly and much more cheaply by small-angle scattering,
>> either light (SAS) or X-ray (SAXS).
>>
>> I would suggest looking into those avenues first.
>>
>> If you have well behaved (i.e. non-aggregating) purified proein
>> and access to synchrotron beam time (easily requested),
>> a series of SAXS experiments could probably be conducted in one day.
>> I don't want to oversell SAXS, I'm not really an enthusiast.
>> But this case, categorizing the interaction of two poorly ordered proteins
>> in solution and in particular the facilitation or disruption of this
>> interaction by small molecules, should be well within its scope.
>>
>> best
>>
>> Ethan
>>
>> On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
>> > Hello everyone,
>> >
>> > I do realize that this is not a NMR focused group, but I do hope that
>> there
>> > are a few spectroscopists lurking around that could possibly answer a
>> few
>> > questions (I am more of a modeler/computationalist):
>> >
>> > The problem: I have two intrinsically disordered proteins that are
>> known to
>> > interact (let's call them 1 and 2). I would like to get structural
>> > information (a conformational ensemble) for 1 and for the "complex"
>> (1+2).
>> > Further down the line (depending on whether this is possible) I would
>> also
>> > like to evaluate potential small molecule inhibitors for the said
>> complex.
>> > Both 1 and 2 are <200 aminoacids long.
>> >
>> > The questions:
>> >
>> > 1. Could the cost of determining the "structure" for 1 and 1+2 be
>> > estimated? To be more precise, I am looking for a ball-park figure on
>> how
>> > much a NMR measurement would cost in this case.
>> > 2. Could anyone recommend a good group/CRO that could provide such a
>> > service and not have an astronomical cost?
>> > 3. Any other suggestions/thoughts that you think might be worth
>> mentioning
>> > (minimum quantity of protein necessary, purity, type of NMR etc)
>> >
>> > Many thanks for your help and time!
>> >
>> > Cheers!
>> >
>> > Sorin
>> >
>> > 
>> >
>> > To unsubscribe from the CCP4BB list, click the following link:
>> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>> >
>> > This message was issued to members of www.jiscmail.ac.uk/CCP4BB
>> ,
>> a mailing list hosted by www.jiscmail.ac.uk
>> 

Re: [ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Hello Jon,

Indeed - in the case of NMR, as far as I understand, we can't talk about
resolution (that is why I used quotes). What I need here is a structure (or
ensemble of structures) to which I could hope to dock ligands with some
level of accuracy - something that SAXS could not provide.



On Sun, Aug 15, 2021 at 9:48 AM Jon Cooper 
wrote:

> Hello, my numpty-level understanding is that being intrinsically disorder
> and giving high-resolution structural data are mutually exclusive. I will
> re-read your e-mails. Hope this helps. Cheers, Jon.C.
>
>
> Sent from ProtonMail mobile
>
>
>
>  Original Message 
> On 15 Aug 2021, 09:16, Sorin Draga < sor.dr...@gmail.com> wrote:
>
>
> Hello Ethan,
>
> Thank you for the suggestions. I should have mentioned in my initial post
> that my intention is to first conduct a high throughput virtual screening
> on these proteins, thus I would need high "resolution" of the structures
> which SAXS could not provide, as far as I understand.
> SAXS/SAS might become useful at a later stage, when I have a small number
> of potential inhibitors identified.
>
> Kind regards,
>
> Sorin
>
> On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt  wrote:
>
>> It is possible that you could address some of your questions
>> more quickly and much more cheaply by small-angle scattering,
>> either light (SAS) or X-ray (SAXS).
>>
>> I would suggest looking into those avenues first.
>>
>> If you have well behaved (i.e. non-aggregating) purified proein
>> and access to synchrotron beam time (easily requested),
>> a series of SAXS experiments could probably be conducted in one day.
>> I don't want to oversell SAXS, I'm not really an enthusiast.
>> But this case, categorizing the interaction of two poorly ordered proteins
>> in solution and in particular the facilitation or disruption of this
>> interaction by small molecules, should be well within its scope.
>>
>> best
>>
>> Ethan
>>
>> On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
>> > Hello everyone,
>> >
>> > I do realize that this is not a NMR focused group, but I do hope that
>> there
>> > are a few spectroscopists lurking around that could possibly answer a
>> few
>> > questions (I am more of a modeler/computationalist):
>> >
>> > The problem: I have two intrinsically disordered proteins that are
>> known to
>> > interact (let's call them 1 and 2). I would like to get structural
>> > information (a conformational ensemble) for 1 and for the "complex"
>> (1+2).
>> > Further down the line (depending on whether this is possible) I would
>> also
>> > like to evaluate potential small molecule inhibitors for the said
>> complex.
>> > Both 1 and 2 are <200 aminoacids long.
>> >
>> > The questions:
>> >
>> > 1. Could the cost of determining the "structure" for 1 and 1+2 be
>> > estimated? To be more precise, I am looking for a ball-park figure on
>> how
>> > much a NMR measurement would cost in this case.
>> > 2. Could anyone recommend a good group/CRO that could provide such a
>> > service and not have an astronomical cost?
>> > 3. Any other suggestions/thoughts that you think might be worth
>> mentioning
>> > (minimum quantity of protein necessary, purity, type of NMR etc)
>> >
>> > Many thanks for your help and time!
>> >
>> > Cheers!
>> >
>> > Sorin
>> >
>> > 
>> >
>> > To unsubscribe from the CCP4BB list, click the following link:
>> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> >
>> > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
>> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
>> available at https://www.jiscmail.ac.uk/policyandsecurity/
>> >
>>
>>
>> --
>> Ethan A Merritt
>> Biomolecular Structure Center,  K-428 Health Sciences Bldg
>> MS 357742,   University of Washington, Seattle 98195-7742
>>
>>
>>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>



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Re: [ccp4bb] biomolecular NMR for IDPs

[Krieger, James M]

It is possible to get an ensemble for an intrinsically disordered segment from 
NMR. We did this in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008819/

Best wishes
James

On Aug 15, 2021, at 14:48, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

Hello, my numpty-level understanding is that being intrinsically disorder and 
giving high-resolution structural data are mutually exclusive. I will re-read 
your e-mails. Hope this helps. Cheers, Jon.C.


Sent from ProtonMail mobile



 Original Message 
On 15 Aug 2021, 09:16, Sorin Draga < sor.dr...@gmail.com> wrote:

Hello Ethan,

Thank you for the suggestions. I should have mentioned in my initial post that 
my intention is to first conduct a high throughput virtual screening on these 
proteins, thus I would need high "resolution" of the structures which SAXS 
could not provide, as far as I understand.
SAXS/SAS might become useful at a later stage, when I have a small number of 
potential inhibitors identified.

Kind regards,

Sorin

On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt 
mailto:merr...@uw.edu>> wrote:
It is possible that you could address some of your questions
more quickly and much more cheaply by small-angle scattering,
either light (SAS) or X-ray (SAXS).

I would suggest looking into those avenues first.

If you have well behaved (i.e. non-aggregating) purified proein
and access to synchrotron beam time (easily requested),
a series of SAXS experiments could probably be conducted in one day.
I don't want to oversell SAXS, I'm not really an enthusiast.
But this case, categorizing the interaction of two poorly ordered proteins
in solution and in particular the facilitation or disruption of this
interaction by small molecules, should be well within its scope.

best

Ethan

On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
> Hello everyone,
>
> I do realize that this is not a NMR focused group, but I do hope that there
> are a few spectroscopists lurking around that could possibly answer a few
> questions (I am more of a modeler/computationalist):
>
> The problem: I have two intrinsically disordered proteins that are known to
> interact (let's call them 1 and 2). I would like to get structural
> information (a conformational ensemble) for 1 and for the "complex" (1+2).
> Further down the line (depending on whether this is possible) I would also
> like to evaluate potential small molecule inhibitors for the said complex.
> Both 1 and 2 are <200 aminoacids long.
>
> The questions:
>
> 1. Could the cost of determining the "structure" for 1 and 1+2 be
> estimated? To be more precise, I am looking for a ball-park figure on how
> much a NMR measurement would cost in this case.
> 2. Could anyone recommend a good group/CRO that could provide such a
> service and not have an astronomical cost?
> 3. Any other suggestions/thoughts that you think might be worth mentioning
> (minimum quantity of protein necessary, purity, type of NMR etc)
>
> Many thanks for your help and time!
>
> Cheers!
>
> Sorin
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of 
> www.jiscmail.ac.uk/CCP4BB,
>  a mailing list hosted by 
> www.jiscmail.ac.uk,
>  terms & conditions are available at 
> 

Re: [ccp4bb] biomolecular NMR for IDPs

[Jon Cooper]

Hello, my numpty-level understanding is that being intrinsically disorder and 
giving high-resolution structural data are mutually exclusive. I will re-read 
your e-mails. Hope this helps. Cheers, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 15 Aug 2021, 09:16, Sorin Draga wrote:

> Hello Ethan,
>
> Thank you for the suggestions. I should have mentioned in my initial post 
> that my intention is to first conduct a high throughput virtual screening on 
> these proteins, thus I would need high "resolution" of the structures which 
> SAXS could not provide, as far as I understand.
> SAXS/SAS might become useful at a later stage, when I have a small number of 
> potential inhibitors identified.
> Kind regards,
> Sorin
>
> On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt  wrote:
>
>> It is possible that you could address some of your questions
>> more quickly and much more cheaply by small-angle scattering,
>> either light (SAS) or X-ray (SAXS).
>>
>> I would suggest looking into those avenues first.
>>
>> If you have well behaved (i.e. non-aggregating) purified proein
>> and access to synchrotron beam time (easily requested),
>> a series of SAXS experiments could probably be conducted in one day.
>> I don't want to oversell SAXS, I'm not really an enthusiast.
>> But this case, categorizing the interaction of two poorly ordered proteins
>> in solution and in particular the facilitation or disruption of this
>> interaction by small molecules, should be well within its scope.
>>
>> best
>>
>> Ethan
>>
>> On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
>>> Hello everyone,
>>>
>>> I do realize that this is not a NMR focused group, but I do hope that there
>>> are a few spectroscopists lurking around that could possibly answer a few
>>> questions (I am more of a modeler/computationalist):
>>>
>>> The problem: I have two intrinsically disordered proteins that are known to
>>> interact (let's call them 1 and 2). I would like to get structural
>>> information (a conformational ensemble) for 1 and for the "complex" (1+2).
>>> Further down the line (depending on whether this is possible) I would also
>>> like to evaluate potential small molecule inhibitors for the said complex.
>>> Both 1 and 2 are <200 aminoacids long.
>>>
>>> The questions:
>>>
>>> 1. Could the cost of determining the "structure" for 1 and 1+2 be
>>> estimated? To be more precise, I am looking for a ball-park figure on how
>>> much a NMR measurement would cost in this case.
>>> 2. Could anyone recommend a good group/CRO that could provide such a
>>> service and not have an astronomical cost?
>>> 3. Any other suggestions/thoughts that you think might be worth mentioning
>>> (minimum quantity of protein necessary, purity, type of NMR etc)
>>>
>>> Many thanks for your help and time!
>>>
>>> Cheers!
>>>
>>> Sorin
>>>
>>> 
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>>
>>> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 
>>> list hosted by www.jiscmail.ac.uk, terms & conditions are available at 
>>> https://www.jiscmail.ac.uk/policyandsecurity/
>>>
>>
>> --
>> Ethan A Merritt
>> Biomolecular Structure Center, K-428 Health Sciences Bldg
>> MS 357742, University of Washington, Seattle 98195-7742
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

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hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Hello Ethan,

Thank you for the suggestions. I should have mentioned in my initial post
that my intention is to first conduct a high throughput virtual screening
on these proteins, thus I would need high "resolution" of the structures
which SAXS could not provide, as far as I understand.
SAXS/SAS might become useful at a later stage, when I have a small number
of potential inhibitors identified.

Kind regards,

Sorin

On Sat, Aug 14, 2021 at 5:55 PM Ethan A Merritt  wrote:

> It is possible that you could address some of your questions
> more quickly and much more cheaply by small-angle scattering,
> either light (SAS) or X-ray (SAXS).
>
> I would suggest looking into those avenues first.
>
> If you have well behaved (i.e. non-aggregating) purified proein
> and access to synchrotron beam time (easily requested),
> a series of SAXS experiments could probably be conducted in one day.
> I don't want to oversell SAXS, I'm not really an enthusiast.
> But this case, categorizing the interaction of two poorly ordered proteins
> in solution and in particular the facilitation or disruption of this
> interaction by small molecules, should be well within its scope.
>
> best
>
> Ethan
>
> On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
> > Hello everyone,
> >
> > I do realize that this is not a NMR focused group, but I do hope that
> there
> > are a few spectroscopists lurking around that could possibly answer a few
> > questions (I am more of a modeler/computationalist):
> >
> > The problem: I have two intrinsically disordered proteins that are known
> to
> > interact (let's call them 1 and 2). I would like to get structural
> > information (a conformational ensemble) for 1 and for the "complex"
> (1+2).
> > Further down the line (depending on whether this is possible) I would
> also
> > like to evaluate potential small molecule inhibitors for the said
> complex.
> > Both 1 and 2 are <200 aminoacids long.
> >
> > The questions:
> >
> > 1. Could the cost of determining the "structure" for 1 and 1+2 be
> > estimated? To be more precise, I am looking for a ball-park figure on how
> > much a NMR measurement would cost in this case.
> > 2. Could anyone recommend a good group/CRO that could provide such a
> > service and not have an astronomical cost?
> > 3. Any other suggestions/thoughts that you think might be worth
> mentioning
> > (minimum quantity of protein necessary, purity, type of NMR etc)
> >
> > Many thanks for your help and time!
> >
> > Cheers!
> >
> > Sorin
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
> > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/
> >
>
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> MS 357742,   University of Washington, Seattle 98195-7742
>
>
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] biomolecular NMR for IDPs

[Ethan A Merritt]

It is possible that you could address some of your questions
more quickly and much more cheaply by small-angle scattering,
either light (SAS) or X-ray (SAXS).

I would suggest looking into those avenues first.

If you have well behaved (i.e. non-aggregating) purified proein
and access to synchrotron beam time (easily requested),
a series of SAXS experiments could probably be conducted in one day.
I don't want to oversell SAXS, I'm not really an enthusiast.
But this case, categorizing the interaction of two poorly ordered proteins
in solution and in particular the facilitation or disruption of this
interaction by small molecules, should be well within its scope.

best

Ethan

On Saturday, 14 August 2021 14:12:40 PDT Sorin Draga wrote:
> Hello everyone,
> 
> I do realize that this is not a NMR focused group, but I do hope that there
> are a few spectroscopists lurking around that could possibly answer a few
> questions (I am more of a modeler/computationalist):
> 
> The problem: I have two intrinsically disordered proteins that are known to
> interact (let's call them 1 and 2). I would like to get structural
> information (a conformational ensemble) for 1 and for the "complex" (1+2).
> Further down the line (depending on whether this is possible) I would also
> like to evaluate potential small molecule inhibitors for the said complex.
> Both 1 and 2 are <200 aminoacids long.
> 
> The questions:
> 
> 1. Could the cost of determining the "structure" for 1 and 1+2 be
> estimated? To be more precise, I am looking for a ball-park figure on how
> much a NMR measurement would cost in this case.
> 2. Could anyone recommend a good group/CRO that could provide such a
> service and not have an astronomical cost?
> 3. Any other suggestions/thoughts that you think might be worth mentioning
> (minimum quantity of protein necessary, purity, type of NMR etc)
> 
> Many thanks for your help and time!
> 
> Cheers!
> 
> Sorin
> 
> 
> 
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-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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[ccp4bb] biomolecular NMR for IDPs

[Sorin Draga]

Hello everyone,

I do realize that this is not a NMR focused group, but I do hope that there
are a few spectroscopists lurking around that could possibly answer a few
questions (I am more of a modeler/computationalist):

The problem: I have two intrinsically disordered proteins that are known to
interact (let's call them 1 and 2). I would like to get structural
information (a conformational ensemble) for 1 and for the "complex" (1+2).
Further down the line (depending on whether this is possible) I would also
like to evaluate potential small molecule inhibitors for the said complex.
Both 1 and 2 are <200 aminoacids long.

The questions:

1. Could the cost of determining the "structure" for 1 and 1+2 be
estimated? To be more precise, I am looking for a ball-park figure on how
much a NMR measurement would cost in this case.
2. Could anyone recommend a good group/CRO that could provide such a
service and not have an astronomical cost?
3. Any other suggestions/thoughts that you think might be worth mentioning
(minimum quantity of protein necessary, purity, type of NMR etc)

Many thanks for your help and time!

Cheers!

Sorin



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