Re: [ccp4bb] N-terminal PCA as artifact of crystallization?

[Jared Sampson]

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 <
488a26d62010-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  on behalf of Jared
> Sampson 
> *Sent:* 21 April 2021 16:15
> *To:* 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
> 
> and find out how our cutting-edge measurement science has a positive impact
> in the real world
> --
>
> 
>  Keep
> in touch  
>  
> --
> NPL 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.

[ccp4bb] Future Planning: Entries with extended PDB and CCD ID codes will be distributed in PDBx/mmCIF format only

[Jasmine Young]

http://www.wwpdb.org/news/news?year=2021#607760112786e73a79c76f9d

wwPDB, in collaboration with the PDBx/mmCIF Working Group 
, has set plans to extend the length of 
ID codes for PDB and Chemical Component Dictionary (CCD) ID entries in 
the future. Entries issued with these extended IDs will not be supported 
by the legacy PDB file format.


CCD entries are currently identified by unique three-character 
alphanumeric codes. At current growth rates, we anticipate running out 
of available new codes in the next three to four years. At this point, 
the wwPDB will issue four-character alphanumeric codes for CCD IDs in 
the OneDep system. Due to constraints of the legacy PDB file format, 
entries containing these new, four character ID codes will only be 
distributed in PDBx/mmCIF format. The wwPDB will begin implementation of 
extended CCD ID codes in 2022.


In addition, wwPDB also plans extended PDB ID length to eight characters 
prefixed by ‘PDB’, e.g., pdb_1abc. Each PDB ID has a corresponding 
Digital Object Identifier (DOI), often required for manuscript 
submission to journals and described in publications by the structure 
authors. Both extended PDB IDs and corresponding PDB DOIs, along with 
existing four character PDB IDs, will be included in the PDBx/mmCIF 
formatted files for all new entries by Fall 2021.


For example, PDB entry 1ABC will also have the extended PDB ID 
(pdb_1abc) and the corresponding PDB DOI (10.2210/pdb1abc/pdb) 
listed in the _database_2 PDBx/mmCIF category.


loop_
_database_2.database_id
_database_2.database_code
_database_2.pdbx_database_accession
_database_2.pdbx_DOI
PDB   1abc pdb_1abc 10.2210/pdb1abc/pdb
WWPDB   D_1x     ?       ?

Once four-character PDB IDs are all consumed, newly-deposited PDB 
entries will only be issued extended PDB ID codes, and entries will only 
be distributed in PDBx/mmCIF format.


wwPDB is asking PDB users and related software developers to review code 
and begin to remove such limitations for the future.


--
Regards,

Jasmine

===
Jasmine Young, Ph.D.
Biocuration Team Lead
RCSB Protein Data Bank
Research Professor
Institute for Quantitative Biomedicine
Rutgers, The State University of New Jersey
174 Frelinghuysen Rd
Piscataway, NJ 08854-8087

Email: jasm...@rcsb.rutgers.edu
Phone: (848)445-0103 ext 4920
Fax: (732)445-4320
===



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/

Re: [ccp4bb] N-terminal PCA as artifact of crystallization?

[Jon Cooper]

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 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  on behalf of Jared Sampson 
> 
> Sent: 21 April 2021 16:15
> To: 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/](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

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] N-terminal PCA as artifact of crystallization?

[Isabel Moraes]

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  on behalf of Jared Sampson 

Sent: 21 April 2021 16:15
To: 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
 and find out how our cutting-edge measurement science has a positive impact in 
the real world


[http://resource.npl.co.uk/images/npl-logo.jpg]
Keep in touch 
[http://resource.npl.co.uk/images/twitter.jpg]   
[http://resource.npl.co.uk/images/facebook.jpg] 
  
[http://resource.npl.co.uk/images/youtube.jpg] 


NPL 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

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/

[ccp4bb] Job posting: Scientist-I (Structural biology) at Frederick National Laboratory for Cancer Research

[Dhirendra K Simanshu]

Hello everyone,

The Frederick National Laboratory for Cancer Research is looking for a
Scientist with experience in protein chemistry and structural biology
(crystallography). The candidate will join the structural biology group
within the NCI RAS Initiative program and work on the protein-protein and
protein-small molecule complexes.

For detailed information and to apply for this position, please use this
link:
https://leidosbiomed.csod.com/ats/careersite/jobdetails.aspx?site=4&c=leidosbiomed&id=1845

Best regards
Dhirendra Simanshu


--

Dhirendra Simanshu

Principal Scientist, Team Lead - RAS Structural Biology

NCI RAS Initiative

Frederick National Laboratory for Cancer Research

8560 Progress Drive, C1012, Frederick, MD 21701

dhirendra.siman...@fnlcr.nih.gov 



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/

[ccp4bb] N-terminal PCA as artifact of crystallization?

[Jared Sampson]

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

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/

[ccp4bb] CASE PhD project available on DNA helicases - University of York and Oxford Nanopore Technologies.

[Michael Plevin]

Dear All

Can I request your assistance in finding a PhD student?

We have a 4-year fully-funded CASE PhD project available for an October 2021 
start. The project is a collaboration between myself, Professor James Chong 
(York) and Oxford Nanopore Technologies. The project will include structural 
studies of DNA helicases using X-ray crystallography and cryo-EM. 

Project and application details can be found via the link below.

https://www.findaphd.com/phds/project/wr-dtp-icase-project-with-ont-enhancing-motor-protein-performance-for-nanopore-dna-sequencing-via-directed-and-darwinian-evolutionary-approaches/?p125515
 


Funding restrictions mean that this position is only fully-funded for UK 
residents. Non UK residents would have to pay additional fees. More information 
on residence conditions can be found here 
.

Application deadline: 29 April 2021

Many thanks in advance

Michael






Dr Michael J Plevin
Senior Lecturer in Molecular Biophysics
York Biomedical Research Institute
York Structural Biology Laboratory
Department of Biology
University of York
Wentworth Way
York, YO10 5DD
United Kingdom

Office: +44 1904 328682
Mobile: +44 7796 303001
Twitter: @mjplevin 
ORCID: orcid.org/-0003-2057-8291 

Web: www.york.ac.uk/biology/research/biochemistry-biophysics/michael-plevin/ 


EMAIL DISCLAIMER: http://www.york.ac.uk/docs/disclaimer/email.htm 






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/

Re: [ccp4bb] ccp4i2 crashes on MacOSX 10.13.6 - High Sierra

[Harry Powell - CCP4BB]

Blimey, I’m surprised I found this about 15 minutes after posting, having spent 
all this week trying to locate it.

It *seems* that I had libs from an openssl installed via Homebrew that the 
openssl supplied with CCP4 found before its own. Deleting those files 
(/usr/local/lib/libssl.1.1.dylib and its link /usr/local/lib/libssl.dylib) 
looks like it’s solved my problem.

Talk about needles in haystacks…

Harry

> On 21 Apr 2021, at 10:42, Harry Powell - CCP4BB  
> wrote:
> 
> Hi folks
> 
> I have an old-ish Mac (Mac Pro, mid-2010, 3.46 6-core Intel Xeon, 32GB RAM) 
> running High Sierra (OSX 10.13.6) - the box will not support newer versions 
> of OSX or MacOS, so I can’t “upgrade” any further on the OS front (well, I 
> could if I really wanted to hack beyond Apple’s recommendations). 
> 
> I’ve been in touch with CCP4 central, but they have been unable to reproduce 
> my issue (over the course of several days), so I though I would ask here…
> 
> Please don’t say “buy a new Mac”...
> 
> It runs the CCP4 programs as per spec if I avoid CCP4I2, e.g. runs DIALS, 
> runs iMosflm, runs QTMG, runs Coot, etc etc etc.
> 
> BUT, if I run ccp4i2 problems soon follow - 
> 
> (1) start iMosflm - I get the “running man” and that’s all. If I try to stop 
> the job with any of the four options, I2 crashes.
> 
> (2) if I have any jobs in the Job list and left click in that window, I2 
> crashes.
> 
> (3) If I run xia2, the job starts and appears to run, but when I leave the 
> xia2 task, I2 crashes.
> 
> (4) Try to run Coot from I2, it never starts.
> 
> I get a whole bunch of errors if I launch a console that indicate there’s an 
> issue with QT and SSL (I won’t bore everyone here with the details) so it’s 
> likely there’s something wrong there.
> 
> Any (useful) ideas?
> 
> Harry
> 
> 



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/

[ccp4bb] ccp4i2 crashes on MacOSX 10.13.6 - High Sierra

[Harry Powell - CCP4BB]

Hi folks

I have an old-ish Mac (Mac Pro, mid-2010, 3.46 6-core Intel Xeon, 32GB RAM) 
running High Sierra (OSX 10.13.6) - the box will not support newer versions of 
OSX or MacOS, so I can’t “upgrade” any further on the OS front (well, I could 
if I really wanted to hack beyond Apple’s recommendations). 

I’ve been in touch with CCP4 central, but they have been unable to reproduce my 
issue (over the course of several days), so I though I would ask here…

Please don’t say “buy a new Mac”...

It runs the CCP4 programs as per spec if I avoid CCP4I2, e.g. runs DIALS, runs 
iMosflm, runs QTMG, runs Coot, etc etc etc.

BUT, if I run ccp4i2 problems soon follow - 

(1) start iMosflm - I get the “running man” and that’s all. If I try to stop 
the job with any of the four options, I2 crashes.

(2) if I have any jobs in the Job list and left click in that window, I2 
crashes.

(3) If I run xia2, the job starts and appears to run, but when I leave the xia2 
task, I2 crashes.

(4) Try to run Coot from I2, it never starts.

I get a whole bunch of errors if I launch a console that indicate there’s an 
issue with QT and SSL (I won’t bore everyone here with the details) so it’s 
likely there’s something wrong there.

Any (useful) ideas?

Harry



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/