I-PINE selective labelling

[Chris Waudby]

Hi Woonghee,

Quick question about submitting selective labelling information to I-PINE: I know some methyl spin system types in my CH spectrum, and have stereospecific Leu/Val information - how do I input this information to submit to I-PINE?

Best,

Chris

[Lee, Woonghee]

Hi Chris,

 

I-PINE accepts N15 selective labeling information from N-HSQC. You can easily do that by using “user label” (ctrl+l) and check that option from PINE-SPARKY.2 plugin. https://nmrfam.wisc.edu/pine-sparky2/

 

Methyl labeling information can be used if you have assigned already, and by pre-assignment option. It improves side-chain assignment quality, however, it does not go upstream and helps backbone assignment automatically. Instead, you can limit the choices by finding correlated N-HSQC peaks and put user-labels like described above. Then, it will affect the backbone, too.

 

Cheers,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

woongh...@ucdenver.edu

https://sites.google.com/view/wlee-group 

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5^th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA

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[Chris Waudby]

Hi Woonghee,

OK - perhaps this reflects my ignorance of how I-PINE is working. So rather than a simultaneous analysis, the backbone is assigned first of all using HNCA/HNCO type experiments, and only then are side-chains assigned using NOESY/COSY/TOCSY data?

Perhaps it would help to be more explicit about my particular case: I'm looking at a 45 kDa heterodimer of known structure, although we can't separately label the two subunits, so for all intents and purposes I'm treating it as a single chain. I've got HNCO/HNCACO/HNCA/HNCOCA/CCONH data plus HCH/CCH COSY and 15N/13C NOESY experiments. I also have a 4D HCCH TOCSY - though as this doesn't appear to be supported I can incorporate at least some of the information in this by projecting down to a pair of 3Ds. Lastly, I have a stereospecific ILV spectrum, so together with a CT-HSQC I can identify proR/proS leucine/valine methyls, as well as methionine methyls - but is there any way to incorporate this information into the analysis, as I'd assume it might help the NOESY-based assignments?

Best,

Chris

[Lee, Woonghee]

Hi Chris,

 

Experiments containing CB is very important and it looks you don’t have any experiments for that. Secondary chemical shifts are indicators of amino acid types and secondary structures and also you can have another connectivity between spin systems. Typically the CBCA(CO)NH and HNCACB combination is very powerful, however, HN(CA)CB can be used instead of HNCACB in your case while size is large. Giving CB connection between i-1 and i is the most effective thing for now than anything else.

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[Chris Waudby]

Completely agree! Unfortunately I’ve really struggled to get a good CB measurement, even with an HN(CA)CB - have tried several different pulse sequences now. I’ve got a fairly reasonable (H)CCONH TOCSY though, which at least gives CB shifts for the i-1. Is this experiment used by I-PINE for the backbone stage of the analysis?

[Lee, Woonghee]

Hi Chris,

 

(H)CCONH-TOCSY is used in sidechain assignments. Ideally, it would have been nice if we just extracted CA/CB and used that to build spin systems, however, it didn’t work robust like you wanted. Considering `mis’-extraction of atom types vs. getting more information, it isn’t really helpful for real-world users in general because inputs are noisy and lacking many cases ironically which can bias to the wrong spin system connection. However, if you can determine CA/CB from (H)CCONH-TOCSY and (H)CCH-TOCSY, you can make synthetic CBCA(CO)NH (from CCONH) and HNCACB (by combining two experiments) confidently, you may use as inputs and it will improve the results greatly.

 

Best,

Woonghee

 

--

Woonghee Lee, I.E.I.P., M.S., Ph.D.

 

Assistant Professor

Department of Chemistry

University of Colorado Denver

1151 Arapahoe St. (Science Bldg.) Rm 4128A

Denver, CO 80217-3364, USA

woongh...@ucdenver.edu

https://sites.google.com/view/wlee-group 

https://clas.ucdenver.edu/chemistry/woonghee-lee

 

Shipping/Mailing Address:

Woonghee Lee

1201 5^th St. UCD CHEM-194

P.O. Box 173364 (USPS)

Denver, CO 80204, USA