Print

Print
Well - that more or less proves the residue is a CYS - there is a peak in the PHAN map right on the S.
And that the extra density does not contain an anomalous scatterer so is probably not a metal or a sulphate or...

But that still doesnt explain WHAT it is . Sorry not to be more help..

Eleanor

On Wed, 10 Jul 2019 at 11:32, Lumbini Yadav <[log in to unmask]> wrote:
No I am using ccp4i. I tried doing SAD refinement in refmac and the output image is attached below .
I do not seen density near cysteine that was visible in Fo-Fc map

On Wed, Jul 10, 2019 at 3:40 PM Eleanor Dodson <[log in to unmask]> wrote:
The key word for refmac is ANOM MAPONLY
Are you using GUI2?
Eleanor

On Wed, 10 Jul 2019 at 09:32, Lumbini Yadav <[log in to unmask]> wrote:
I have soaked my crystals in  sodium dithionite a reducing agent. I have not done mass spec but sequence is confirmed

On Tue, Jul 9, 2019 at 9:26 PM Bonsor, Daniel <[log in to unmask]> wrote:

Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification?

What about S-Acetyl-cysteine (3-letter code: SCY).

 

Best,

 

Dan

 

Daniel A Bonsor PhD.

Sundberg Lab

Institute of Human Virology

University of Maryland, Baltimore

725 W Lombard Street N370

Baltimore

Maryland

MD 21201

Tel: (410) 706-7457

 

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Lumbini Yadav
Sent: Tuesday, July 09, 2019 5:22 AM
To: [log in to unmask]
Subject: [ccp4bb] Fo-Fc density close to cysteine residue

 

Dear all,

 

We have found a huge Fo-Fc density close to cysteine residue (see attached image) in the structure with resolution of 1.2A. In the crystallization condition, we have PEG 3350, Potassium phosphate monobasic, glycerol and protein was in Tris and NaCl. Before freezing the crystals were soaked in mother liquor containing sodium dithionite.

 

I have tried different modified cysteine (CSX, CSO, OCS, CME, CSS, SNC, CSD, CXM, SCH, CSU) from the library and also SO3, SO2 and peroxide. But in all the screenings we do see some part of Fo-Fc density unaddressed at 3 sigma.

 

Does anyone have an idea about what this density could be? Covalent modification?

 

Thanks.

 



Kind regards,

Lumbini

 

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


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


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