Thank you for all the references and comments (as always). I understand now that in the context of the protein N-terminus, cyclization of Glu to PCA appears to happen spontaneously at low pH and therefore is also possible with non-eukaryotically expressed samples. I'll look more closely at the various relevant cases as I go forward with this analysis.
Much appreciated! Best, Jared On Wed, Apr 21, 2021 at 12:25 PM Jon Cooper < 0000488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello, there's another one at 1.1 A in 5JK4 which was a contaminant that > crystallised over 2 years. No mass-spec, though! Cheers, Jon.C. > > http://scripts.iucr.org/cgi-bin/paper?S2059798316010433 > > > Sent from ProtonMail mobile > > > > -------- Original Message -------- > On 21 Apr 2021, 17:01, Isabel Moraes < isabel.mor...@npl.co.uk> wrote: > > > Dear Jared, > > I advise you to have a look into our very recent Nat Comms paper (in > particular supplementary information) > https://doi.org/10.1038/s41467-020-20596-0 > > > > In our high-resolution crystal structures of the light-adapted (*6S6C*) > and dark-adapted (*6GUX*) state of Archaerhodopsin-3 (AR3), solved to 1.1 > Å and 1.3 Å respectively, the N-terminus residue Gln7 is modified to a > pyroglutamyl group (PCA). In our paper, we confirm this modification by > native mass spectroscopy. The AR3 protein was produce from its natural > source and any detergent was used during the purification or > crystallisation processes. Crystals were grown at pH5.5. > > I hope it helps > Isabel > > > ------------------------------------------------------------------ > Isabel Moraes, PhD > Principal Research Scientist - Structural Biology > National Physical Laboratory (NPL) > Hampton Rd | Teddington | Middlesex | TW11 0LW > ------------------------------------------------------------------- > > > ------------------------------ > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Jared > Sampson <jared.samp...@columbia.edu> > *Sent:* 21 April 2021 16:15 > *To:* CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> > *Subject:* [EXTERNAL] [ccp4bb] N-terminal PCA as artifact of > crystallization? > > > CAUTION: This email originated from outside of NPL. Do not click links or > open attachments unless you recognize the sender and know the content is > safe. > > Dear all, > > I'm looking at a crystal structure (1H4G) where the N-terminal Glu residue > has cyclized to pyroglutamic acid (PCA). The protein was expressed in and > secreted from bacteria (*Bacillus licheniformis*), and the > crystallization conditions for 3 ul hanging drops were 2 ul protein > solution (10 mg/ml in 100 mM sodium acetate pH 6.0) + 1 ul reservoir > solution (100 mM MES pH 6.5, 30% ammonium sulphate). > > As I wouldn't typically expect this kind of post-translational > modification to appear in bacteria (please correct me if I'm mistaken about > this), I suspect the presence of PCA here to be an artifact of > crystallization. > > Have others seen cyclization of N-terminal Glu or Gln to PCA under such > acidic crystallization conditions? I'd be interested in seeing any > relevant literature you might be able to suggest. > > Many thanks, > > Jared > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > Visit the NPL website > <https://www.npl.co.uk/?utm_source=Email%20Footer&utm_medium=email&utm_campaign=Homepage> > and find out how our cutting-edge measurement science has a positive impact > in the real world > ------------------------------ > > <https://www.npl.co.uk/?utm_source=Email%20Footer&utm_medium=email&utm_campaign=Homepage> > Keep > in touch <https://www.npl.co.uk/contact> <http://www.twitter.com/npl> > <http://www.facebook.com/npldigital> <http://www.youtube.com/npldigital> > ------------------------------ > NPL Privacy Policy <https://www.npl.co.uk/privacy-policy/> > > If you have received this message in error, please notify us and remove it > from your system. > NPL Management Ltd cannot guarantee that the e-mail or any attachments are > free from viruses. > > NPL Management Ltd is a company registered in England and Wales, number: > 2937881 > Registered office: National Physical Laboratory | Hampton Road | > Teddington, Middlesex | UK | TW11 0LW > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=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/
