[ccp4bb] Two postdoc positions in Ubiquitin Signalling, WEHI, Melbourne, Australia

[Bernhard Lechtenberg]

Dear colleagues,

The Ubiquitin Signalling Division at The Walter and Eliza Hall Institute (WEHI) 
is looking to recruit two Research Officers (Postdocs) to join our teams in the 
Lechtenberg and Komander labs.

WEHI in Melbourne is Australia’s pre-eminent medical research institute. The 
Ubiquitin Signalling Division at WEHI was established in 2018, and offers a 
vibrant, multi-disciplinary and highly collaborative environment at the 
forefront of ubiquitin research with access to state-of-the art facilities in 
proteomics, structural biology (Titan Krios G4, Australian Synchrotron), 
biophysics (SEC-MALS, ITC, SPR, mass photometry, MST), cell manipulation 
(CRISPR) and imaging facilities, and drug discovery through the National Drug 
Discovery Centre (NDDC). The division features a continuous stream of excellent 
publications, exciting preliminary data, and opportunities to establish 
research ideas in a multidisciplinary environment.

The Lechtenberg lab seeks to recruit a postdoc with structural biology 
(crystallography or cryo-EM) and biochemistry expertise to study large RBR E3 
ligase complexes. Preliminary data include an established mammalian expression 
system.
Details and applications: 
https://wehi.wd3.myworkdayjobs.com/en-US/WEHI/job/Research-Officer---Structural-Biology-of-E3-Ubiquitin-Ligases_JR1578-3
Contact: Dr. Bernhard Lechtenberg, 
lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>

The Komander lab seeks to recruit a postdoc with structural biology and protein 
biochemistry background to embark on an early-stage drug discovery project in 
the area of mitophagy and Parkinson’s disease, with excellent academic and 
translational / entrepreneurial outputs expected.
Details and applications: 
https://wehi.wd3.myworkdayjobs.com/en-US/WEHI/job/Parkville-Victoria-Australia/Research-Officer_JR1823-1
Contact: Prof. David Komander, d...@wehi.edu.au<mailto:d...@wehi.edu.au>

We offer three-year contracts with highly competitive salaries and benefits. 
Pre-application enquiries are encouraged via the contact details provided.

Please share this information within your networks.

Kind regards,

Bernhard

Bernhard C. Lechtenberg PhD
NHMRC Emerging Leadership Fellow
Laboratory Head
Ubiquitin Signalling Division​​
E lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>
T +61 3 9345 2217


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1G Royal Parade Parkville Victoria 3052 Australia

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Re: [ccp4bb] To Trim or Not to To Trim

[Bernhard Lechtenberg]

I found the poll I wrote about earlier. This actually is way older than I had 
expected (2011). You can see the poll results (which was run by Ed Pozharski) 
and discussion at the time here in the CCP4BB archive:
https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20268.html

In brief, the results of 240 respondents were:
Delete the atoms 43%
Let refinement take care of it by inflating B-factors41%
Set occupancy to zero12%
Other 4%


Bernhard

From: CCP4 bulletin board  on behalf of Debanu Das 

Date: Friday, 10 March 2023 at 2:56 pm
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] To Trim or Not to To Trim
We dealt with this in-depth during structural genomics days when we deposited 
over 1500 novel, high-quality, experimentally-phased structures into the PDB. 
Think it’s prudent to trim/truncate side chains without reliable density.

Non-structural biologists using PDB structures without expert help can err in 
any of these scenarios: misinterpreting most common/random rotamer, zero 
occupancy atoms, B-factors, etc.

What is the value of populating the PDB, which is a structural model 
repository, with such information that is not there, i.e., reliable structural 
model?

Any trained crystallographer/structural biologist can easily add in side chain 
information if needed for modeling/computational chemistry reasons.

Best regards,
Debanu

On Thu, Mar 9, 2023 at 6:50 PM Jurgen Bosch 
mailto:jxb...@case.edu>> wrote:
I’d say no trimming to side chains for the following reason: There are 
non-structural biologists using PDB files and if atoms are missing they don’t 
know what to do. A better approach is where no side chain density allows 
support of placement, pick the most common rotamer and set the occupancy to 
zero for those atoms lacking density support. More work for you but more 
accurate in my opinion.

Jürgen


___
Jürgen Bosch, PhD, MBA
Center for Global Health & Diseases
Case Western Reserve University
Cleveland, OH 44106
https://www.linkedin.com/in/jubosch/


CEO & Co-Founder at InterRayBio, LLC




On Mar 9, 2023, at 9:45 PM, Bernhard Lechtenberg 
<968307750321-dmarc-requ...@jiscmail.ac.uk<mailto:968307750321-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Hi Rhys,

I am also all for leaving side chains and letting the B-factors deal with the 
weak/absent density.

I don’t think there is a consensus, but I kind of remember that somebody did a 
poll a few years ago and if I remember correctly the main approaches were the 
one described above, or trimming the side-chains.

Bernhard

Bernhard C. Lechtenberg PhD
NHMRC Emerging Leadership Fellow
Laboratory Head
Ubiquitin Signalling Division​​
E lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>
T +61 3 9345 2217


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Rhys Grinter 
<22087c81e8c6-dmarc-requ...@jiscmail.ac.uk<mailto:22087c81e8c6-dmarc-requ...@jiscmail.ac.uk>>
Date: Friday, 10 March 2023 at 12:26 pm
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] To Trim or Not to To Trim
Hi All,

I'm trying to crowdsource an opinion on how people deal with modelling side 
chains with poorly resolved electron or cryoEM density.

My preference is to model the sidechain and allow the B-factors to go high in 
refinement to represent that the side chain is flexible. However, I'm aware 
that some people truncate sidechains if density is not present to justify 
modelling. I've also seen models where the sidechain is modelled but with zero 
occupancy if density isn't present.

Is there a consensus and justifying arguments for why one approach is better?

Cheers,

Rhys




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Re: [ccp4bb] To Trim or Not to To Trim

[Bernhard Lechtenberg]

Hi Rhys,

I am also all for leaving side chains and letting the B-factors deal with the 
weak/absent density.

I don’t think there is a consensus, but I kind of remember that somebody did a 
poll a few years ago and if I remember correctly the main approaches were the 
one described above, or trimming the side-chains.

Bernhard

Bernhard C. Lechtenberg PhD
NHMRC Emerging Leadership Fellow
Laboratory Head
Ubiquitin Signalling Division​​
E lechtenber...@wehi.edu.au
T +61 3 9345 2217


From: CCP4 bulletin board  on behalf of Rhys Grinter 
<22087c81e8c6-dmarc-requ...@jiscmail.ac.uk>
Date: Friday, 10 March 2023 at 12:26 pm
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] To Trim or Not to To Trim
Hi All,

I'm trying to crowdsource an opinion on how people deal with modelling side 
chains with poorly resolved electron or cryoEM density.

My preference is to model the sidechain and allow the B-factors to go high in 
refinement to represent that the side chain is flexible. However, I'm aware 
that some people truncate sidechains if density is not present to justify 
modelling. I've also seen models where the sidechain is modelled but with zero 
occupancy if density isn't present.

Is there a consensus and justifying arguments for why one approach is better?

Cheers,

Rhys




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Re: [ccp4bb] Postdoc opportunity at WEHI, Melbourne, Australia

[Bernhard Lechtenberg]

There are still a few days to apply for this postdoc position in my lab at WEHI 
in Melbourne. Application deadline is 11 March 2023 (AEDT; closer to 10 March 
for those in the US).

For questions please email me at 
lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>

Bernhard

From: Bernhard Lechtenberg 
Date: Thursday, 9 February 2023 at 11:04 am
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Postdoc opportunity at WEHI, Melbourne, Australia
Dear colleagues,

My lab is looking for a Research Officer (Postdoc) to strengthen our team in 
the Ubiquitin Signalling Division at WEHI in Melbourne, Australia.

We aim to understand the structure and molecular mechanisms of E3 ubiquitin 
ligases, their basic biology, and roles in human diseases such as cancer, 
inflammatory conditions, and neurodegeneration. In the past years, our lab has 
gained deep insights into the mechanisms and structures of several E3 ubiquitin 
ligase complexes using X-ray crystallography (Lechtenberg et al., Nature 2016; 
Cotton et al., Mol. Cell 2022; Wang, Cotton et al., Nat. Comms. 2023; Cotton & 
Lechtenberg, Biochem. Soc. Trans., 2020). We now want to expand on these 
efforts, further integrating cryo-electron microscopy.

We offer a vibrant, multi-disciplinary and highly collaborative environment at 
the forefront of ubiquitin research and structural biology with ready and 
regular access to state-of-the-art biophysics (SEC-MALS, ITC, SPR) and 
structural biology equipment incl. cryo-EM (Titan Krios G4 with Falcon 4 direct 
detector at the Bio21/WEHI Ian Holmes Imaging Centre), automated 
crystallisation, synchrotron (Australian Synchrotron) and NMR facilities.

Applicants should possess a PhD in a relevant discipline, a demonstrated track 
record of 1st author publications in peer reviewed journals in structural 
biology and strong practical experience in protein biochemistry and structural 
biology (cryo-EM or crystallography).

More information and application details here:
https://wehi.wd3.myworkdayjobs.com/en-US/WEHI/job/Research-Officer---Structural-Biology-of-E3-Ubiquitin-Ligases_JR1578-3


Application closing date:  27 Feb 2023 (AEST)

The start date is flexible. Pre-application enquiries are welcome and 
encouraged. Please email me at 
lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>.

Best wishes,
Bernhard
Bernhard C. Lechtenberg PhD
NHMRC Emerging Leadership Fellow
Laboratory Head
Ubiquitin Signalling Division​​
E lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>
T +61 3 9345 2217




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[ccp4bb] Postdoc opportunity at WEHI, Melbourne, Australia

[Bernhard Lechtenberg]

Dear colleagues,

My lab is looking for a Research Officer (Postdoc) to strengthen our team in 
the Ubiquitin Signalling Division at WEHI in Melbourne, Australia.

We aim to understand the structure and molecular mechanisms of E3 ubiquitin 
ligases, their basic biology, and roles in human diseases such as cancer, 
inflammatory conditions, and neurodegeneration. In the past years, our lab has 
gained deep insights into the mechanisms and structures of several E3 ubiquitin 
ligase complexes using X-ray crystallography (Lechtenberg et al., Nature 2016; 
Cotton et al., Mol. Cell 2022; Wang, Cotton et al., Nat. Comms. 2023; Cotton & 
Lechtenberg, Biochem. Soc. Trans., 2020). We now want to expand on these 
efforts, further integrating cryo-electron microscopy.

We offer a vibrant, multi-disciplinary and highly collaborative environment at 
the forefront of ubiquitin research and structural biology with ready and 
regular access to state-of-the-art biophysics (SEC-MALS, ITC, SPR) and 
structural biology equipment incl. cryo-EM (Titan Krios G4 with Falcon 4 direct 
detector at the Bio21/WEHI Ian Holmes Imaging Centre), automated 
crystallisation, synchrotron (Australian Synchrotron) and NMR facilities.

Applicants should possess a PhD in a relevant discipline, a demonstrated track 
record of 1st author publications in peer reviewed journals in structural 
biology and strong practical experience in protein biochemistry and structural 
biology (cryo-EM or crystallography).

More information and application details here:
https://wehi.wd3.myworkdayjobs.com/en-US/WEHI/job/Research-Officer---Structural-Biology-of-E3-Ubiquitin-Ligases_JR1578-3

Application closing date:  27 Feb 2023 (AEST)

The start date is flexible. Pre-application enquiries are welcome and 
encouraged. Please email me at 
lechtenber...@wehi.edu.au.

Best wishes,
Bernhard
Bernhard C. Lechtenberg PhD
NHMRC Emerging Leadership Fellow
Laboratory Head
Ubiquitin Signalling Division​​
E lechtenber...@wehi.edu.au
T +61 3 9345 2217


[WEHI Logo]


Walter and Eliza Hall Institute of Medical Research
1G Royal Parade Parkville Victoria 3052 Australia

www.wehi.edu.au

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WEHI acknowledges the Wurundjeri people of the Kulin Nation as the traditional 
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Re: [ccp4bb] A Problem of my Research Protein

[Bernhard Lechtenberg]

Hi Guohui,

Are you sure this is caused by disulfide bond formation? Do you have any 
reducing agent in your buffer? 5 Cys residues in 30 amino acids looks very much 
like a zinc finger to me. Is there any evidence for that? Are there any other 
zinc binding residues, i.e. His in this region?

Good luck,
Bernhard

From: CCP4 bulletin board  on behalf of Guohui SHANG 

Reply to: Guohui SHANG 
Date: Tuesday, 15 September 2020 at 4:22 pm
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] A Problem of my Research Protein

Hi Everyone，
  Well,My Research Protein is easily Dimerzation caused by Disulfide bond 
for many Cys.And it leads to the SEC peak too wide about 5 mL. Then I Cut N-ter 
30 Amino Acids in which 5 spaced Cys,but the SEC Peak is still wide and hard to 
Crystallize（as the protein is very pure and clean）.Besides, I have tried 
different Buffer pH ＜ pI or pH ＞ pI，but the SEC peak Do not Work at all.
  Anyone could offer your kindly ideas,I would thank you very much！


发自我的iPhone



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Re: [ccp4bb] Cell disruption

[Bernhard Lechtenberg]

Dear colleagues,

The CCP4BB community is amazing, as always. I received many comments by happy 
users of the Avestin Emulsiflex C3 (and C5); not so much on the Microfluidics 
LM20. Thanks for that, it will be a great help to decide which instrument to 
purchase.

Best,
Bernhard


From: CCP4 bulletin board  on behalf of Adam Middleton 

Reply to: Adam Middleton 
Date: Tuesday, 18 August 2020 at 2:56 pm
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Cell disruption

Hi Bernhard,

The Department has an Avestin emusiflex here, and it is kept in the cold room. 
Works great as long as your cells are properly resuspended and in a big enough 
volume. Highly recommended.

Adam



On Sun, Aug 16, 2020 at 4:48 PM Pascal Egea 
<4aa44fc90f38-dmarc-requ...@jiscmail.ac.uk<mailto:4aa44fc90f38-dmarc-requ...@jiscmail.ac.uk>>
 wrote:
Dear Bernhard ,
I would recommend the emulsified from Avestin. It is great . Depending on your 
budget you can ask for stainless  steel (probe  to slow tear ) or ceramic 
(longer lasting but a bit more expensive).
We have the ceramic but I have worked with the stainless steel one and it is 
really good with E. coli cells. Not the best for
Yeast despite pressurizing more. Yeast are better dealt with bead beaters or 
cryo-mill grinders.
3 passes are usually sufficient to process up to 250 ml of extract in a decent 
time (15-20) minutes .

We operate ours at room temperature just cool the serpentine tubing coming out 
of the chamber in ice . There is an option to add a cooling plate but we have 
never considered it. Overheating comes from overpressurizing but for E Coli at 
15000 psi max  3 passes will do the trick without overhea tu bf the sample. I 
have processEs successfully many membrane proteins And protein complexes with 
it.

I have never seen one in a cold room to be honest but it should be fine 
although I don’t think it would be necessary . I would Ask avestin about that .
Maintenance and cleaning is simple . Once in a while I will replace the seal, 
it s not very difficult to do. The whole assembly is metal so you can sterilize 
the whole thing After complete disassembly in extreme cases . This is a bit 
more involved but not overwhelming.
The people at Avestin are super nice and responsive ( Canadians) so they will 
guide you if necessary . And we have had their engineers show up sometimes to 
just check the instrument for free . I have had this one for 10 years in my lab 
now. And before that was using one for 7 years during my post doc.  I would not 
lyse bacteria by any other method now.

I hope this helps.
All the best.
Pascal

On Sat, Aug 15, 2020 at 9:08 PM Bernhard Lechtenberg 
mailto:lechtenber...@wehi.edu.au>> wrote:
Dear colleagues,

We are currently looking to purchase a cell disruptor/homogeniser mainly for 
routinely processing a few 100 mls of E. coli suspensions. With the current 
COVID-19 restrictions it is very difficult for us to test any equipment. I thus 
hope that some of you can share their experiences with the different models. I 
found a similar thread on the CCP4BB from 2013 but wondered if anybody had had 
some more up to date information.

We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the 
Microfluidics LM20 Microfluidizer. In particular we are interested to know more 
about ease of use, maintenance, reliability and if anybody operates these in a 
cold room (4°C).

Thanks in advance,

Bernhard


--
Bernhard C. Lechtenberg, Ph.D.
Laboratory Head
Ubiquitin Signalling Division
The Walter and Eliza Hall Institute
1G Royal 
Parade<https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g>
Parkville VIC 
3052<https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g>
Australia<https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g>
Phone: +61 3 9345 2217
Email: lechtenber...@wehi.edu.au<mailto:lechtenber...@wehi.edu.au>


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The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
Kulin
Nation as the traditional owners of the land where our campuses are located and
the continuing connection to country and community.
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--
Pascal F. Egea, PhD
Associate Project Scientist
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email

[ccp4bb] Cell disruption

[Bernhard Lechtenberg]

Dear colleagues,

We are currently looking to purchase a cell disruptor/homogeniser mainly for 
routinely processing a few 100 mls of E. coli suspensions. With the current 
COVID-19 restrictions it is very difficult for us to test any equipment. I thus 
hope that some of you can share their experiences with the different models. I 
found a similar thread on the CCP4BB from 2013 but wondered if anybody had had 
some more up to date information.

We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the 
Microfluidics LM20 Microfluidizer. In particular we are interested to know more 
about ease of use, maintenance, reliability and if anybody operates these in a 
cold room (4°C).

Thanks in advance,

Bernhard


--
Bernhard C. Lechtenberg, Ph.D.
Laboratory Head
Ubiquitin Signalling Division
The Walter and Eliza Hall Institute
1G Royal Parade
Parkville VIC 3052
Australia
Phone: +61 3 9345 2217
Email: lechtenber...@wehi.edu.au


___

The information in this email is confidential and intended solely for the 
addressee.
You must not disclose, forward, print or use it without the permission of the 
sender.

The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
Kulin
Nation as the traditional owners of the land where our campuses are located and
the continuing connection to country and community.
___



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Re: [ccp4bb] Coot "Fix nomenclature error" window

[Bernhard Lechtenberg]

Dear Evgenii,

The following from the Coot manual should help:
https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/docs/coot.html#set_002dnomenclature_002derrors_002don_002dread

add “set-nomenclature-errors-on-read ignore” to your Coot startup file. In case 
you are using they python file, e.g. “.coot.py”, I think you have to change 
this to “set_nomenclature_errors_on_read ignore”.

HTH,
Bernhard



--
Bernhard C. Lechtenberg, Ph.D.
Laboratory Head
Ubiquitin Signalling Division
The Walter and Eliza Hall Institute
1G Royal Parade
Parkville VIC 3052
Australia
Phone: +61 3 9345 2217
Email: lechtenber...@wehi.edu.au


From: CCP4 bulletin board  on behalf of Eugene Osipov 

Reply to: Eugene Osipov 
Date: Thursday, 14 May 2020 at 7:09 am
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Coot "Fix nomenclature error" window

Dear CCP4bb members,
how could I disable  checks for nomenclature errors and get rid of "Fix 
Nomenclature Errors" window at every Coot startup?

[cid:image001.png@01D629C5.C961E320]

Thanks in advance,
--
Evgenii Osipov
Laboratory for Biocrystallography,
Department of Pharmaceutical Sciences,
KU Leuven O



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[ccp4bb] Job posting - CryoEM Research Group Leader in Australia

[Bernhard Lechtenberg]

Dear colleagues,

We would like to bring to your attention a job opening for a Research Group 
Leader specialising in CryoEM at the The Walter and Eliza Hall Institute of 
Medical Research in Melbourne Australia.

For details see - https://www.wehi.edu.au/cryoem-laboratory-head

This is a 7-year appointment in the first instance with opportunities for 
further renewal. WEHI is a highly collaborative world-class institution that 
has been in operation for more than 100 years and was recently ranked in 
Nature’s Top 20 non-profit/non-governmental biomedical research institutes 
globally. The link above contains all necessary information for enquiries and 
applications.

Many thanks and kind regards,
Bernhard

--
Bernhard C. Lechtenberg, Ph.D.
Laboratory Head
Ubiquitin Signalling Division
The Walter and Eliza Hall Institute
1G Royal Parade
Parkville VIC 3052
Australia
Phone: +61 3 9345 2217
Email: lechtenber...@wehi.edu.au


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Re: [ccp4bb] covalent link to aa

[Bernhard Lechtenberg]

Hi Markus,

Did you load the .cif file into Coot before real space refinement? File -> 
Import CIF dictionary …
When you do that, Coot should be able to refine the link and that might then 
also help for the refinement in Refmac.

Bernhard
Bernhard C. Lechtenberg, PhD
Postdoctoral Associate
Riedl Lab
Cancer Metabolism and Signaling Networks Program
NCI-Designated Cancer Center

[cid:24A04CE0-5418-4257-A8D1-8084CA766BC9@burnham.org]

10901 N. Torrey Pines Road, La Jolla, CA 92037
T  858.646.3100 ext. 4216  E 
blechtenb...@sbpdiscovery.org
Science Benefiting Patients®

On Jan 15, 2019, at 12:49 PM, Markus Heckmann 
mailto:markus.21...@gmail.com>> wrote:

Dear all,
I have a structure where CYS is bound to OCA (octanoic acid). I
started with Jligand and created a CYS-OCA covalent link. It then
output a CIF file (verified looks OK). Add the LINK record to PDB
file:

LINK SG  CYS A 161 C1  OCA A 902CYS-OCA

Place OCA to the model using Coot. When I now use RealSpaceRefinement,
the OCA is placed on top of CYS itself. Coot does not recognise that
OCA-CYS link but why? therefore, I just place OCA at Place it in
*reasonable* position without any overlaps.


All files are located at
https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgist.github.com%2Fort163%2Fe6b8411f45e5224f7d46fc36c3324e6fdata=02%7C01%7Cblechtenberg%40SBPDISCOVERY.ORG%7Cddcea39782f54aa79e0008d67b2b4211%7C0b162723004547deb0699f1a7aa955a1%7C0%7C0%7C636831823062844471sdata=YuhoO%2B6giCrJwZ9R4Njw2UBpkp6c85PZHA%2FSYiBwZnw%3Dreserved=0

Now in REFMAC, input MTZ,PDB and the CIF file (from jligand). After
refinement, I find that the oxygen in the OCA is pointing wrongly. The
thioester bond is also NOT ideal distance (though density is good).

Is there anything wrong with my procedure? *Shouldnt* the refinement
optimise everything in OCA-CYS link?

Best
Markus



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Re: [ccp4bb] Na-Binding Protein?

[Bernhard Lechtenberg]

Dear Jacob,

The protease thrombin is another example. Thrombin is activated by Na+ (but not 
Li+ or K+). We have shown using NMR that Na+ binding allosterically stabilizes 
active conformations of thrombin. Additionally, numerous crystal structures of 
Na+-free (“slow” thrombin) and Na+-bound (“fast” thrombin) have been published.

These publications should give you a good overview:
https://www.ncbi.nlm.nih.gov/pubmed/20660315
https://www.ncbi.nlm.nih.gov/pubmed/22944689
http://www.pnas.org/content/100/24/13785/T1.expansion.html

Best,
Bernhard
Bernhard C. Lechtenberg, PhD
Postdoctoral Associate
Riedl Lab
Cancer Metabolism and Signaling Networks Program
NCI-Designated Cancer Center

[cid:24A04CE0-5418-4257-A8D1-8084CA766BC9@burnham.org]

10901 N. Torrey Pines Road, La Jolla, CA 92037
T  858.646.3100 ext. 4216  E 
blechtenb...@sbpdiscovery.org
Science Benefiting Patients®

On Jan 9, 2018, at 6:42 AM, Keller, Jacob 
> wrote:

Dear Crystallographers,

Is anyone aware of a soluble protein which changes large-scale conformation +/- 
Na+, and is specific for Na+ per se, or at least ignores K+ and Ca++? E.g., Rb+ 
or Li+ might be okay. Structural info would be a plus, but not a sine qua non.

Similarly, what about with K+ or Cl- specificities, but oblivious to similar 
common ions?

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
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Re: [ccp4bb] C-terminal amide

[Bernhard Lechtenberg]

HI Abhisek,

I had the same problem a few years back. Here is the solution I came up with 
thanks to help from the CCP4bb:

1) Add pointer atom in Coot NH2 and create link to C-terminal residue (Coot: 
Extensions -> Modeling -> Make link)
2) create cif file with correct link description (see below)
3) modify LINK record in PDB file to correct residues/ link name (TYR-NH2 if 
you use the file below with modifications for your case)
4) refine in Refmac with cif as LIB in

Augie Pioszak sent me the cif file copied below. you will have to change PHE to 
TYR for your particular case.

Hope that helps.

Best,
Bernhard

---

Bernhard,
You need a link record in the pdb file to link the NH2 amino group to your last 
peptide residue.  When I did this a few years back I had to include a .cif 
library file describing the link for use with Refmac.  See pdb entry 3c4m and 
below for the lib description I used.  I still use O, so not sure how coot 
handles it, but I would guess it will recognize the NH2 group just fine.  Hope 
this helps.
Regards,
Augie Pioszak

# ---   LIST OF LINKS ---
#
data_link_list
loop_
_chem_link.id
_chem_link.comp_id_1
_chem_link.mod_id_1
_chem_link.group_comp_1
_chem_link.comp_id_2
_chem_link.mod_id_2
_chem_link.group_comp_2
_chem_link.name
PHE-NH2  PHE  ..NH2  ..
bond_PHE-C_=_NH2-N
# --
#
# --- DESCRIPTION OF LINKS ---
#
data_link_PHE-NH2
#
loop_
_chem_link_bond.link_id
_chem_link_bond.atom_1_comp_id
_chem_link_bond.atom_id_1
_chem_link_bond.atom_2_comp_id
_chem_link_bond.atom_id_2
_chem_link_bond.type
_chem_link_bond.value_dist
_chem_link_bond.value_dist_esd
PHE-NH2  1 C   2 N single  1.3290.020
loop_
_chem_link_angle.link_id
_chem_link_angle.atom_1_comp_id
_chem_link_angle.atom_id_1
_chem_link_angle.atom_2_comp_id
_chem_link_angle.atom_id_2
_chem_link_angle.atom_3_comp_id
_chem_link_angle.atom_id_3
_chem_link_angle.value_angle
_chem_link_angle.value_angle_esd
PHE-NH2  1 O   1 C   2 N   122.0001.600
PHE-NH2  1 CA  1 C   2 N   118.2002.000
loop_
_chem_link_plane.link_id
_chem_link_plane.plane_id
_chem_link_plane.atom_comp_id
_chem_link_plane.atom_id
_chem_link_plane.dist_esd
PHE-NH2plane11 CA0.020
PHE-NH2plane11 C 0.020
PHE-NH2plane11 O 0.020
PHE-NH2plane12 N 0.020
# --

-
> On Oct 12, 2017, at 2:53 PM, Abhishek Anan  wrote:
> 
> Dear all,
> 
> I am trying to solve the structure of a peptide with C-terminal amide. In 
> order to add the amide group to the c-terminal TYR, I substituted TYR with 
> TYC (tyrosinamide) and created a link between the preceding GLY and TYC. When 
> refined in refmac, the pdb inserts a TER card between GLY and TYC and no 
> covalent bond is created between them. How do I fix this? I have also tried 
> to add NH2 pointer atom to TYR in coot and create a link between them but in 
> vain. I also imported the NH2.cif file into coot and tried to make a link but 
> of no use. I also tried to import NH2.cif into Jligand so I could get a link 
> record but it refuses to open the NH2.cif file. 
> 
> I would greatly appreciate any help!
> 
> Best regards,
> Abhishek

Re: [ccp4bb] Database for peptides in pdb format

[Bernhard Lechtenberg]

Hi,

if you just want a dipeptide, the easiest is probably to build one in PyMOL: 
Build - Residue - Leucine then Proline. You can save it in PDB format using 
File - Save Molecule…

You can probably do the same in Coot or JLigand if you want to use CCP4 
programs.

Hope that helps,
Bernhard

Bernhard C. Lechtenberg, PhD
Postdoctoral Fellow
Riedl Lab
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
Phone: 858.646.3100 x 4216
Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org




On Sep 3, 2014, at 11:17 AM, Kgosisejo, Oarabile 
o.kgosis...@usask.camailto:o.kgosis...@usask.ca wrote:

Hello all,

Is there a database for peptides in a pdb format? I am investigating the 
structure-function of a dipeptidase and I would like to dock a dipeptide, 
substrate (e.g. Leucine-Proline), on the active site but I have no idea where I 
can get the dipeptides for that. I searched in the PDB but I could not find any.

Your help is appreciated

Best Regards,

Oarabile M. Kgosisejo
o.kgosis...@usask.camailto:o.kgosis...@usask.ca

Re: [ccp4bb] off topic - UV absorbance of phosphotyrosine

[Bernhard Lechtenberg]

Hi Will,

I previously used an extinction coefficient of 600M-1cm-1 for phosphotyrosine 
at 280nm estimated from figure 2 of this publication:

http://www.ncbi.nlm.nih.gov/pubmed/2418612

Hope that helps,
Bernhard


Bernhard C. Lechtenberg, PhD
Postdoctoral Fellow
Riedl Lab
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
Phone: 858.646.3100 x 4216
Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org




On May 21, 2014, at 10:41 AM, Will Stanley 
will.stanley2...@gmail.commailto:will.stanley2...@gmail.com
 wrote:


Hello folks,

I would like to measure the concentrations of proteins containing 
phosphotyrosines using absorbance at 280nm.

I'm wondering about calculating extinction coefficients by the Edelhoch method 
- as used in ProtParam for example:

http://web.expasy.org/protparam/protparam-doc.html

Given that phosphotyrosine absorbs less strongly at 280nm than tyrosine, has 
anyone developed a modified extinction coefficient calculation that treats 
tyrosine and phosphotyrosine separately and has the same kind of accuracy that 
Edelhof achieved using amino acid analogues (e.g. comparing 
glycyl-L-tyrosylglycine to the phophorylated equivalent)?

Cheers,
Will.

[ccp4bb] Fwd: [ccp4bb] Add C-terminal amide - solved

[Bernhard Lechtenberg]

Thanks for the help, the issue is solved. Here a short summary in case anybody 
else has the same problem:

1) Add pointer atom in Coot NH2 and create link to C-terminal residue
2) create cif file with correct link description
3) modify LINK record in PDB to correct residues/ link name
4) refine in Refmac with cif as LIB in

Thanks especially to Maike and Augen

Bernhard


Begin forwarded message:

From: Bernhard Lechtenberg 
blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org
Subject: [ccp4bb] Add C-terminal amide
Date: October 29, 2013 6:32:37 PM PDT
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Reply-To: Bernhard Lechtenberg 
blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org

Hello experts,

I am currently working on a structure of a protein-peptide complex. The peptide 
was synthesized with a C-terminal amide group. What is the best way to add this 
in Coot and refine in Refmac? I did a search on the web but only found a 
protocol for CNS not for Coot/Refmac.

Thanks for the help.

Bernhard

Bernhard C. Lechtenberg, PhD
Postdoctoral Fellow
Riedl Lab
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
Phone: 858.646.3100 x 4216
Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org

[ccp4bb] Add C-terminal amide

[Bernhard Lechtenberg]

Hello experts,

I am currently working on a structure of a protein-peptide complex. The peptide 
was synthesized with a C-terminal amide group. What is the best way to add this 
in Coot and refine in Refmac? I did a search on the web but only found a 
protocol for CNS not for Coot/Refmac.

Thanks for the help.

Bernhard

Bernhard C. Lechtenberg, PhD
Postdoctoral Fellow
Riedl Lab
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
Phone: 858.646.3100 x 4216
Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org

[ccp4bb] Problems with key bindings in coot0.7.2 CCP4 install

[Bernhard Lechtenberg]

Hi all,

I recently installed CCP4 6.4 under Mac OS X 10.8.5 and now have problems using 
key bindings in the Coot version (0.7.2) that comes with CCP4. Although most of 
the standard key bindings still work, some of them (e.g. 'o' for other NCS 
chain) and most of the user-defined key bindings do not work. The key bindings 
file seems to be correctly loaded by Coot and the correct keys are listed in 
'Extensions - Settings - Key bindings' and the linked functions work when I 
click on the buttons in that list. The terminal shows the following message 
when I hit one of the keys that are not working:

(skip-to-next-ncs-chain 'forward)
((safe_scheme_command) Error in proc: key:  unbound-variable  args:  (#f 
Unbound variable: ~S (skip-to-next-ncs-chain) #f))

Everything works correctly in a separately installed Coot v0.7.1 with the same 
.coot.py file.

Any suggestions on what the problem might be?

Thanks for the help

Bernhard



Bernhard C. Lechtenberg, PhD
Postdoctoral Fellow
Riedl Lab
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road
La Jolla, CA 92037, USA
Phone: 858.646.3100 x 4216
Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org

Re: [ccp4bb] Win-coot: replace residue

[Bernhard Lechtenberg]

Hi Uma,

when I do the same procedure (in OSX, but the problem seems to be the same), 
the new residue is stored in a new chain with residue number 1. So you would 
need to renumber the residue and change the chain ID back to the original chain 
ID. It's annoyingly complicated, but it works for me.

Hope that helps,
Bernhard



On Sep 11, 2013, at 12:56 PM, Uma Ratu 
rosiso2...@gmail.commailto:rosiso2...@gmail.com wrote:

Hello,

I try to replace one of cysteine residue to CSX using Win-coot.

Here is how I did:
Extensions  Modelling  Replace Residue... and enter the three letter code.

The program place the CSX residue, but with break bond. The new residue is no 
longer linked with other residue and not in the chain.

I then try to replace with other residues, such as ALA. The results are the 
same: the new residues all with break bond.
How could I put the replaced residue back to the main-chain?

I use Win-coot-0.7.

Thank you

Uma

Re: [ccp4bb] Coot chain ID

[Bernhard Lechtenberg]

In Coot you can use

Extensions - Modelling - Reorder Chains

Bernahrd
-- 
Bernhard Lechtenberg
PhD student
Huntington lab
University of Cambridge
Department of Haematology
Cambridge Institute for Medical Research
Wellcome Trust/MRC Building, Hills Road
Cambridge, CB2 0XY
United Kingdom


-Original Message-
From: Dipankar Manna dipanka...@aurigene.com
Reply-to: Dipankar Manna dipanka...@aurigene.com
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Coot chain ID
Date: Sat, 14 Apr 2012 06:55:10 +

Dear All,

 

I fitted a ligand into a structure along with SO4 and waters (COOT).
Then I did the “Merge Molecules” and did the refinement. In output PDB
file the chain ID sequence is showing B, A, C( ligand, protein and SO4
respectively) and the ATOM numbering is starting from ligand (Chain B
instead of Chain A). How can I make the sequence A, B and C (Protein,
ligand and SO4).

 

Best wishes

 

Dipankar






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