Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Difficult Molecular replacement

[Gong, Zhen]

Hello Marco,

I also feel that it might be due to the wrong space group because all the 
homologous models and alphafold model did not improve the R values. Make sure 
that you turn on “All possible in same pointgroup” when you run 
Phenix.Phaser-MR. I work with P212121 crystals a lot. Sometimes, if the 
diffraction quality was not good, the data will be indexed as P222, or P21212 
etc, and sometimes even C2221. Try all possible in same pointgroup and see what 
happens. Good luck!

Zhen

From: CCP4 bulletin board  on behalf of Marco Bravo 

Date: Monday, February 19, 2024 at 20:17
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [EXTERNAL] Re: [ccp4bb] Difficult Molecular replacement
[You don't often get email from mbrav...@ucr.edu. Learn why this is important 
at https://aka.ms/LearnAboutSenderIdentification ]

Hello Todd, I get the best solution for p22121 space group after MR with an LLG 
score of 640 from phaser. and the Rfree is .4748. However after MR refinement 
does not lower the Rfree and it appears to make the Rfree worse. The XDS 
software indicates that my best solution is P21 21 2. Often Phaser MR places 
the solution in P 21 21 2. The helicase is a superfamily 2 helicase and is only 
monomeric. Its a 543aa long protein with a MW of 62Kda. It should have two RecA 
like domains at the core but the protein I have crystallized has a previously 
uncharacterized n-terminal domain responsible for tight single stranded DNA 
binding.  I have tried different space groups manually but that resulted in 
clashing. I will be frank I do need to work on my crystallography background as 
crystal lattices, space group, and self-rotaion functions are limited. Thank 
you so far for your help , I will try further trimming down my search model 
into separate domain and trying that in the meantime.



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Re: [ccp4bb] Difficult Molecular replacement

[Marco Bravo]

Hello Todd, I get the best solution for p22121 space group after MR with an LLG 
score of 640 from phaser. and the Rfree is .4748. However after MR refinement 
does not lower the Rfree and it appears to make the Rfree worse. The XDS 
software indicates that my best solution is P21 21 2. Often Phaser MR places 
the solution in P 21 21 2. The helicase is a superfamily 2 helicase and is only 
monomeric. Its a 543aa long protein with a MW of 62Kda. It should have two RecA 
like domains at the core but the protein I have crystallized has a previously 
uncharacterized n-terminal domain responsible for tight single stranded DNA 
binding.  I have tried different space groups manually but that resulted in 
clashing. I will be frank I do need to work on my crystallography background as 
crystal lattices, space group, and self-rotaion functions are limited. Thank 
you so far for your help , I will try further trimming down my search model 
into separate domain and trying that in the meantime.



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Re: [ccp4bb] Difficult Molecular replacement

[Green, Todd Jason]

Hello Marc-

My first thought is do you have the correct space group? Are you searching for 
other space groups in the point group? Second, helicases are multimers, but the 
orientation of the monomers that make up the multimers may deviate 
significantly. If you didn't use a monomer, it's worth doing so. Likewise, 
using the hexamer might be better. Is searching with a complete multimer 
sensible, ie would crystallographic symmetry cause overlaps due to coincidence 
of a non-crystallographic axis with a crystallographic axis? If you are 
searching with a multimer, even if you get a solution, you may very likely need 
to rigid body refine all "domains/subdomains" of the solution to proceed toward 
better r-factors. Lastly, trim as much as you can from the search model to get 
to the core, give that a shot.

Can you calculate the self-rotation function? What does it tell you about 
potential symmetry and orientation of symmetry vs. your best solution?

What program are you using, phaser? what does the LLG score look like?

I'm happy to discuss further if you like.

Best-
Todd

From: CCP4 bulletin board  on behalf of Marco Bravo 

Sent: Monday, February 19, 2024 4:44 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Difficult Molecular replacement

Hello all,
I recently collected data on some plate crystals for a previously 
uncharacterized protein at the ALS light source. The XDS auto-data processing 
log output indicates that my resolution is 2.8 angstroms. The protein is a 
helicase with homologs already in the protein data bank making it a suitable 
target for molecular replacement which I thought initially. However after 
trying molecular replacements with all known homologs in the protein data bank 
the R values remain high after MR >0.5. After an initial round of Rigid body or 
restrained refinement. The R values still remain very high at around >.5. I 
have tried MR with Rosetta and alphaphold models but the problem of high R 
values persists. The best solution I get is from the CCP4 cloud automatic 
molecular replacement and model building pipeline which gives me a free R value 
of 0.46.. However the solution is only for residues ~100-326 out of a 543 amino 
acid long protein. And even then the model still has a lot of missing residues 
and truncated sidechains and overall fits the map quite poorly. Does anyone 
have any suggestions about how I can solve my structure if at all possible at 
this point? I ran the crystals and pre-crystalized samples on a gel and it 
appears that the protein remains stable during crystallization as the molecular 
weight did not change or any degradation does not appear.



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Re: [ccp4bb] Difficult Molecular replacement

[Artem Evdokimov]

Hello Marco,

First: compare your unit cell parameters with RCSB (there is an option for
that in the search, very helpful). Give it say 5% error margin.

Worst case scenario - cry a little, having found that your protein is
something else...

Next, check in depth for space group oddities, twinning, or general data
misbehavior.

Next, split your helicase into two lobes (if this is the right kind of
helicase) and do MR with both halves, like it was a complex of two proteins
(requires a bit of skill with MR - but nothing terrifyingly hard tbh). If
there are more domains - split into more (RQC, HRDC, etc.)

Hope this helps.

Artem




On Mon, Feb 19, 2024, 5:54 PM Marco Bravo  wrote:

> Hello all,
> I recently collected data on some plate crystals for a previously
> uncharacterized protein at the ALS light source. The XDS auto-data
> processing log output indicates that my resolution is 2.8 angstroms. The
> protein is a helicase with homologs already in the protein data bank making
> it a suitable target for molecular replacement which I thought initially.
> However after trying molecular replacements with all known homologs in the
> protein data bank the R values remain high after MR >0.5. After an initial
> round of Rigid body or restrained refinement. The R values still remain
> very high at around >.5. I have tried MR with Rosetta and alphaphold models
> but the problem of high R values persists. The best solution I get is from
> the CCP4 cloud automatic molecular replacement and model building pipeline
> which gives me a free R value of 0.46.. However the solution is only for
> residues ~100-326 out of a 543 amino acid long protein. And even then the
> model still has a lot of missing residues and truncated sidechains and
> overall fits the map quite poorly. Does anyone have any suggestions about
> how I can solve my structure if at all possible at this point? I ran the
> crystals and pre-crystalized samples on a gel and it appears that the
> protein remains stable during crystallization as the molecular weight did
> not change or any degradation does not appear.
>
> 
>
> 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
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Re: [ccp4bb] Difficult Molecular replacement

[Tom Peat]

Hello Marco,

This may seem a little obvious, but did you check the sequence of your protein 
from the gel/ crystals?
I've found mass spec to be very useful in this regard as we've had at least two 
instances of having a protein that looked to be the right size/ correct 
protein, but wasn't after mass spec analysis.
Good luck, tom

From: CCP4 bulletin board  on behalf of Marco Bravo 

Sent: Tuesday, February 20, 2024 9:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Difficult Molecular replacement

[You don't often get email from mbrav...@ucr.edu. Learn why this is important 
at https://aka.ms/LearnAboutSenderIdentification ]

Hello all,
I recently collected data on some plate crystals for a previously 
uncharacterized protein at the ALS light source. The XDS auto-data processing 
log output indicates that my resolution is 2.8 angstroms. The protein is a 
helicase with homologs already in the protein data bank making it a suitable 
target for molecular replacement which I thought initially. However after 
trying molecular replacements with all known homologs in the protein data bank 
the R values remain high after MR >0.5. After an initial round of Rigid body or 
restrained refinement. The R values still remain very high at around >.5. I 
have tried MR with Rosetta and alphaphold models but the problem of high R 
values persists. The best solution I get is from the CCP4 cloud automatic 
molecular replacement and model building pipeline which gives me a free R value 
of 0.46.. However the solution is only for residues ~100-326 out of a 543 amino 
acid long protein. And even then the model still has a lot of missing residues 
and truncated sidechains and overall fits the map quite poorly. Does anyone 
have any suggestions about how I can solve my structure if at all possible at 
this point? I ran the crystals and pre-crystalized samples on a gel and it 
appears that the protein remains stable during crystallization as the molecular 
weight did not change or any degradation does not appear.



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[ccp4bb] Difficult Molecular replacement

[Marco Bravo]

Hello all,
I recently collected data on some plate crystals for a previously 
uncharacterized protein at the ALS light source. The XDS auto-data processing 
log output indicates that my resolution is 2.8 angstroms. The protein is a 
helicase with homologs already in the protein data bank making it a suitable 
target for molecular replacement which I thought initially. However after 
trying molecular replacements with all known homologs in the protein data bank 
the R values remain high after MR >0.5. After an initial round of Rigid body or 
restrained refinement. The R values still remain very high at around >.5. I 
have tried MR with Rosetta and alphaphold models but the problem of high R 
values persists. The best solution I get is from the CCP4 cloud automatic 
molecular replacement and model building pipeline which gives me a free R value 
of 0.46.. However the solution is only for residues ~100-326 out of a 543 amino 
acid long protein. And even then the model still has a lot of missing residues 
and truncated sidechains and overall fits the map quite poorly. Does anyone 
have any suggestions about how I can solve my structure if at all possible at 
this point? I ran the crystals and pre-crystalized samples on a gel and it 
appears that the protein remains stable during crystallization as the molecular 
weight did not change or any degradation does not appear. 



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[ccp4bb] Computational Instrument Scientist - Biology

[O'Neill, Hugh]

We are seeking a Computational Instrument Scientist (CIS) for small-angle 
neutron scattering (SANS). This position resides in the Neutron Scattering 
Division (NSD) at Oak Ridge National Laboratory (ORNL). Applications are sought 
from highly creative and motivated individuals with demonstrated 
skills/experience in computational method development who will join the 
interdisciplinary team of instrument scientists that support the SANS 
instrument suite at the Spallation Neutron Source (SNS) and High Flux Isotope 
Reactor (HFIR) at ORNL.
The CIS is the liaison between the experimental and computational aspects of 
the neutron scattering science program and coordinates with instrument 
scientists and software developers in the development of sustainable products 
for the user community.  In this position, you will emphasize developing 
software analysis tools for biological research, and improving automation for 
translating measurements into knowledge, that are applicable across the 
instruments in the SANS suite. As part of your career, you will have 
opportunities to use other neutron scattering instruments at SNS and HFIR to 
pursue your scientific interests.

A link to the position advertisement can be found here:

https://jobs.ornl.gov/job/Oak-Ridge-Computational-Instrument-Scientist-Biology-TN-37830/1117249200/

From: O'Neill, Hugh
Sent: Monday, November 6, 2023 9:36 PM
To: ccp4bb@jiscmail.ac.uk
Subject: Biochemist post-doctoral associate position

Biochemist Position at Oak Ridge National Laboratory


We are seeking a Postdoctoral Research Associate with expertise in biochemistry 
and molecular biology to join an interdisciplinary team in the Neutron 
Scattering Division (NSD) at Oak Ridge National Laboratory. You will contribute 
to a project that aims to develop an integrated platform for rapid production 
and characterization of viral proteins to aid in the design of therapeutics. 
You will be responsible for developing and executing approaches to produce 
deuterium-labeled proteins for small-angle neutron scattering measurements to 
provide insights into the structural properties of these macromolecular 
complexes. Taking this position, you will have the opportunity to work with a 
team comprising of specialists in structural biology, assay development, X-ray 
and neutron diffraction and scattering, and computation. As part of our 
research team, you will be affiliated with the ORNL Center for Structural 
Molecular Biology, and you will have access 
to a tool suite that includes leading small-angle scattering and neutron 
diffraction facilities, bio-fermentation laboratories, and biophysical 
characterization laboratories, in addition to in-house small angle X-ray 
scattering and X-ray diffraction instrumentation. The position represents an 
excellent opportunity for you to develop your career and interact with leading 
scientists from around the world.

A link to the position advertisement can be found here:

https://jobs.ornl.gov/job/Oak-Ridge-Postdoctoral-Research-Associate-Biochemist-TN-37830/1081996500/






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[ccp4bb] Use a Python Package to Access the RCSB PDB Search API

[Dennis Piehl]

RCSB PDB now maintains a Python package that can be used for accessing our 
Search API, rcsbsearchapi 
(https://rcsbsearchapi.readthedocs.io/en/latest/index.html).

The package can be installed from PyPI or by downloading the source code on 
GitHub (https://github.com/rcsb/py-rcsbsearchapi). This repository will serve 
as the central hub for all future development, bug fixes, and discussions 
related to the RCSB PDB Search API package.

With this package, users can perform all the same types of queries they are 
familiar with from the Advanced Search builder on RCSB.org, all through a 
Pythonic interface. These capabilities include:

• Structure and chemical attribute search
• Sequence search
• Sequence motif search
• Structure similarity search
• Structure motif search
• Chemical similarity search
• Option to include computed structure models (CSMs) in search

A full overview of these features with examples is available 
(https://rcsbsearchapi.readthedocs.io/en/latest/quickstart.html). Users are 
also encouraged to review the examples on GitHub in the README file and Jupyter 
notebooks (https://github.com/rcsb/py-rcsbsearchapi).

This Python wrapper was developed by undergraduates Rusham Bhatt (University of 
Maryland Baltimore County) and Santiago Blaumann (Cornell University) under the 
direction of Dennis Piehl and Brinda Vallat (RCSB PDB) as part of the Rutgers 
RISE (Research Intensive Summer Experience, https://www.rise.rutgers.edu/).

RCSB PDB appreciates all RCSB PDB API contributors, with special thanks Spencer 
Bliven for providing the original groundwork for the package, and Rutgers 
undergraduate Pratyoy Biswas for expanding the work performed during the RISE 
at Rutgers program.

The Search API and Data API are the two main APIs that power RCSB.org. The 
Search API retrieves PDB IDs that match given search conditions, while the Data 
API serves to retrieve data for a given PDB ID. Detailed documentation on RCSB 
PDB Web Service APIs is available 
(https://www.rcsb.org/docs/programmatic-access/web-services-overview). 

Stay up-to-date with API developments by viewing (or subscribing) to the RCSB 
PDB API announcements Google group 
(https://groups.google.com/a/rcsb.org/g/api/about).

---
Dennis Piehl
RCSB Protein Data Bank
Institute for Quantitative Biomedicine
Rutgers, The State University of New Jersey



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[ccp4bb] CBMS Lecture Series - Olga Rechkoblit - February 21st, 13:30 (EST)

[Stojanoff, Vivian]

You are cordially invited to join  the Center for Biomolecular Structure 
Lecture Series ………..





Olga  Rechkoblit

Mount Sinai School of Medicine



WEDNESDAY, February 21, 13:30 (EST)



"Activation of bacterial immune system by cyclic nucleotides"



Register in advance for this meeting:


https://bnl.zoomgov.com/meeting/register/vJIsdOGprDojEjg7-CJFcbqdzjJEXsKmbYk



   Time conversion Link: 
https://www.worldtimebuddy.com/



Abstract: The bacterial CBASS system is similar to the cGAS-STING system in 
humans, containing an enzyme that synthesizes a cyclic nucleotide upon viral 
infection and an effector that senses the second messenger for the anti-viral 
response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease 
domain, is the most abundant CBASS effector, yet the mechanism by which it 
becomes activated for cell killing remains unknown. We present here 
high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) 
with second messengers. The key to PsCap5 activation is a dimer-to-tetramer 
transition, whereby the binding of second messenger to dimer triggers an 
open-to-closed transformation of the SAVED domains, furnishing a surface for 
assembly of the tetramer. This movement propagates to the HNH domains, 
juxtaposing and converting two HNH domains into states for DNA destruction. 
These results show how Cap5 effects bacterial cell suicide and we provide 
proof-in-principle data that the CBASS can be extrinsically activated to limit 
bacterial infections.



==



Vivian Stojanoff, PhD

Education, Training, Outreach

User Program

p 1(631) 344 8375

e lifescie...@bnl.gov

w https://www.bnl.gov/ps/lifesciences/



Address:

Center for Biomolecular Structure

National Synchrotron Light Source II

Building 745

Brookhaven National Laboratory

Upton NY 11973





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Re: [ccp4bb] Which version of ccp4 introduced i2run and/or where does it live?

[Klureza, Maggie]

Hi Kay,

I did find
/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/site-packages/ccp4i2/bin/i2run
on my Mac, and was able to use that to successfully bring up the
import_merged help documentation, so I think that's working - thanks!

For general reference, thanks to an individual reply I got: Evidently i2run
is located in 7.0/7.1 installations at ccp4-7.1/share/ccp4i2/bin/i2run (and
I was able to produce the same import_merged help documentation via that
approach as well).

Thanks!
Maggie

On Mon, Feb 19, 2024 at 3:37 AM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Hi Maggie,
>
> I have CCP4 version 8.0 on my Mac and it does have the file
>
> /Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/bin/i2run
>
> If I run it without arguments, it says
> CCP4 /Applications/ccp4-8.0
> ccp4i2 version 1.1.0
> ccp4i2 source revision 6539
> Failed with exception  list index out of range
> Traceback (most recent call last):
>   File
> "/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
> line 721, in 
> theRunner = CI2Runner(sys.argv)
>   File
> "/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
> line 34, in __init__
> self.add_arguments(theParser, cmdLineArgs)
>   File
> "/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
> line 381, in add_arguments
> taskName = cmdLineArgs[1]
> IndexError: list index out of range
>
> so it seems to work in principle. However, the directory where i2run lives
> is not in the $PATH so one might want to
> sudo ln -s
> /Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/bin/i2run
> /Applications/ccp4-8.0/bin
>
> in order to run it with just "i2run".
> There is documentation in
>
> /Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/docs/sphinx/build/html/_sources/i2run/i2run.rst.txt
>
> Hope this helps,
> Kay
>
>
> On Sat, 17 Feb 2024 19:21:21 -0500, Klureza, Maggie <
> mklur...@g.harvard.edu> wrote:
>
> >Hi all,
> >
> >I would very much like to make use of i2run's capacity for scripting,
> >rather than pressing the same sequence of GUI buttons a few dozen times,
> >but I'm having trouble actually calling it.
> >
> >The documentation  I
> >found gives example code snippets structured as "i2run [function]
> >[options]", but even immediately after sourcing ccp4, entering e.g. "i2run
> >import_merged --help" returns an error saying "bash: i2run: command not
> >found".
> >
> >I found only one reference to i2run on the ccp4bb archive
> >
> >(actually from just 2 days ago!), which included a script that called
> i2run
> >using the full path of
> >"/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/bin/i2run crank2". However,
> I
> >was not able to find any equivalent path in either of the 2 ccp4
> >installations I have access to.
> >
> >Ideally I'd like to use our lab's installation of ccp4 on our university
> >computational cluster, which is currently ccp4-7.1. Attempting to follow
> >the analogous path took me to "ccp4-7.1/lib/python2.7/site-packages",
> which
> >did not contain a ccp4i2 directory. I'm wondering if perhaps i2run wasn't
> >introduced until ccp4-8.0, and we would need an updated installation?
> >Except, I also have ccp4 downloaded on my laptop (a Mac, if that's
> >relevant), and going to "ccp4-8.0/lib/python3.7/site-packages" there still
> >did not produce a ccp4i2 directory.
> >
> >Therefore, I'm wondering: Does ccp4-7.1 contain i2run, or would I need to
> >update to 8.0 no matter what? And, almost regardless of the answer to that
> >first question: What is the proper way to call i2run/where does it live
> >within a ccp4 installation? I've been searching through the online
> >documentation, but all the information I could find seemed to assume that
> >i2run was readily accessible given the existence of a ccp4 installation,
> so
> >I'm a bit stumped.
> >
> >Many thanks,
> >Maggie
> >
> >
> >
> >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/
> >
>
>
>



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This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 

Re: [ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

[Nicholas Clark]

Hi Javier,

For a few years (during an industry job) we regularly cloned directly into
BL21 (we made our own high competency cells) and confirmed via expression
before plasmid sequencing. Only after sequencing did we transform into a
cloning strain for miniprep and “long term storage”.

We didn’t see significant issues with RecA but more so over time we lost
the DNA to nuclease activity (endA). However, this took months to see any
appreciable loss.

If your goal is only plasmid recovery, as Jon stated, your current plan
should work perfectly. Be sure to use the “optional wash” in your miniprep
kit (e.g., Buffer PB in the Qiagen kit) and transform into Top10 within a
month and you shouldn’t have significant issues.

Best,

Nick Clark

Nicholas D. Clark (He/Him)
PhD Candidate
Malkowski Lab
University at Buffalo
Department of Structural Biology
Jacob's School of Medicine & Biomedical Sciences
955 Main Street, RM 5130
Buffalo, NY 14203

Cell: 716-830-1908


On Sun, Feb 18, 2024 at 7:50 PM Javier Gonzalez  wrote:

> Dear all,
> I'm sure this issue comes up very often, but for the first time in our lab
> we need to recover a pET-type expression plasmid from a BL21-like E. coli
> strain (NEB's T7 Express).
> I know a RecA+ strain is not suitable for plasmid production, but the
> basic plan is to grow and mini-prep the cells to recover the plasmid and
> later transform another E coli strain (Top10) to make frozen stocks and for
> plasmid production.
> Is this a regular practice or is there any known protocol we should follow?
> Any advice will be greatly appreciated.
>
> Best wishes,
> Javier
>
> --
> Dr. Javier M. González
> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
> Universidad Nacional de Santiago del Estero (UNSE)
> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
> Santiago del Estero. Argentina
> Tel: +54-(0385)-4238352
> Email  Twitter
> 
>
>
> --
>
> 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] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Flavio Di Pisa]

Dear all,
thank you for your valuable suggestions.

Have a good day,
Flavio.

Il 16/02/24 15:36, Karla J. F. Satchell ha scritto:


Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide 
info on one type recommended by another. Others may have further 
suggestions for Flavio.


*From: *CCP4 bulletin board  on behalf of Karla 
J. F. Satchell 

*Date: *Friday, February 16, 2024 at 7:13 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi everyone—

Thought I might chime in.

This technology was developed in my lab based on our studies of toxins 
and we have methods papers and two patent.


The CPD-tag works great for production of protein with minimal 
residual residues on the protein. Our studies indicate that it can 
help solubilize protein during expression. Protein is purified with 
the C-terminal CPD tag, and the entire CPD and 6xHis-tag are released 
by addition of InsP6 (phytic acid) which is very inexpensive.


The only restriction of the recognition sequence is small-Leu-small so 
a small residue plus Leucine does remain on your protein. There does 
also need to be a little flexibility at the end of your protein for 
access to the protease so our vectors add extra Gly-Ala so the 
residual is GAAL. CPD is particularly useful for small proteins if the 
additional residues do not interfere with your assays.


https://pubmed.ncbi.nlm.nih.gov/28056928/ 



https://pubmed.ncbi.nlm.nih.gov/31773580/ 



https://patents.google.com/patent/US8257946B2/en 



https://patents.google.com/patent/US8383400B2/en 



Here is a link to the original paper on mechanism of action of CPD

https://pubmed.ncbi.nlm.nih.gov/19620709/ 



These are not commercially available as despite multiple marketing 
attempts, there was little interest mid-2010s from biotech for 
licensing new cloning technologies and we abandoned advanced development


If this technology is interesting to you to add to your commercial 
biotech portfolio, please feel free to reach out to me.


Flavio, we can share our vector, but my institution does require an 
MTA as technology is patented. Most people just use synthetic DNA to 
generate the exact clone they want so we rarely get requests but happy 
to share sequences if you need for your synthetic design.


Karla Satchell

*From: *CCP4 bulletin board  on behalf of 
Srivastava, Dhiraj 

*Date: *Friday, February 16, 2024 at 6:42 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi Flavio

      While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease 
cleavage site of your choice. However this strategy will results in 
quite a few extra residues from protease site.


An alternative, which is not commercial but available through addgene, 
is cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in 
solubility and expression.


https://www.addgene.org/38251/ 



There are other variants of this vector available on addgene that you 
can choose from. Depending on your c terminal sequence, it may leave 
either no extra residues or only one or two residues.


Dhiraj



*From:*CCP4 bulletin board  on behalf of Flavio 
Di Pisa 

*Sent:* Friday, February 16, 2024 3:33 AM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the 

Re: [ccp4bb] Which version of ccp4 introduced i2run and/or where does it live?

[Kay Diederichs]

Hi Maggie,

I have CCP4 version 8.0 on my Mac and it does have the file
/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/bin/i2run

If I run it without arguments, it says
CCP4 /Applications/ccp4-8.0
ccp4i2 version 1.1.0
ccp4i2 source revision 6539
Failed with exception  list index out of range
Traceback (most recent call last):
  File 
"/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
 line 721, in 
theRunner = CI2Runner(sys.argv)
  File 
"/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
 line 34, in __init__
self.add_arguments(theParser, cmdLineArgs)
  File 
"/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/core/CCP4I2Runner.py",
 line 381, in add_arguments
taskName = cmdLineArgs[1]
IndexError: list index out of range

so it seems to work in principle. However, the directory where i2run lives is 
not in the $PATH so one might want to
sudo ln -s 
/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/bin/i2run
 /Applications/ccp4-8.0/bin

in order to run it with just "i2run". 
There is documentation in
/Applications/ccp4-8.0/Frameworks/Python.framework/Versions/3.7/lib/python3.7/site-packages/ccp4i2/docs/sphinx/build/html/_sources/i2run/i2run.rst.txt

Hope this helps,
Kay


On Sat, 17 Feb 2024 19:21:21 -0500, Klureza, Maggie  
wrote:

>Hi all,
>
>I would very much like to make use of i2run's capacity for scripting,
>rather than pressing the same sequence of GUI buttons a few dozen times,
>but I'm having trouble actually calling it.
>
>The documentation  I
>found gives example code snippets structured as "i2run [function]
>[options]", but even immediately after sourcing ccp4, entering e.g. "i2run
>import_merged --help" returns an error saying "bash: i2run: command not
>found".
>
>I found only one reference to i2run on the ccp4bb archive
>
>(actually from just 2 days ago!), which included a script that called i2run
>using the full path of
>"/ccp4-8.0/lib/python3.7/site-packages/ccp4i2/bin/i2run crank2". However, I
>was not able to find any equivalent path in either of the 2 ccp4
>installations I have access to.
>
>Ideally I'd like to use our lab's installation of ccp4 on our university
>computational cluster, which is currently ccp4-7.1. Attempting to follow
>the analogous path took me to "ccp4-7.1/lib/python2.7/site-packages", which
>did not contain a ccp4i2 directory. I'm wondering if perhaps i2run wasn't
>introduced until ccp4-8.0, and we would need an updated installation?
>Except, I also have ccp4 downloaded on my laptop (a Mac, if that's
>relevant), and going to "ccp4-8.0/lib/python3.7/site-packages" there still
>did not produce a ccp4i2 directory.
>
>Therefore, I'm wondering: Does ccp4-7.1 contain i2run, or would I need to
>update to 8.0 no matter what? And, almost regardless of the answer to that
>first question: What is the proper way to call i2run/where does it live
>within a ccp4 installation? I've been searching through the online
>documentation, but all the information I could find seemed to assume that
>i2run was readily accessible given the existence of a ccp4 installation, so
>I'm a bit stumped.
>
>Many thanks,
>Maggie
>
>
>
>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/
>



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[ccp4bb] Unable to access my old projects in new installed ccp4

[Lumbini Yadav]

Hi,

I have uninstalled ccp4 from my laptop and reinstalled a recent version. I
am unable to access my old run and also my project directory looks empty. I
am able to individually connect to each project.  Since I have multiple
projects is there a way to connect all my project at once



Thanking you



Regards

lumbini



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