Re: [ccp4bb] preparing DL-Malic acid stock solution

[Janet Newman]

The way I have done it (and it was sort of fun in a mad scientist way) was to 
mix up solid DL-malic acid and sodium hydroxide in the right amounts and add 
water as needed.  This generates  of heat, but gets around the 
solubility minimum which is impossible to get out of:

I mixed 40.23 g of DL-malic acid powder (MW=134.1) with 24g of NaOH pellets 
(MW=40) in a large beaker with a stirrer bar.  Put the beaker in a larger 
container of ice, and put that on a stirrer in the cold room. Add 80 mL H20 to 
the beaker containing the chemicals, stand back - lots of heat evolved.
when everything has quietened down, make up to 100 mL with water (we filtered 
through a 0.22 um filter).

Cheers, Janet

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
Berry
Sent: Friday, 9 June 2017 2:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] preparing DL-Malic acid stock solution

(di)Sodium D,L-malate is available from Sigma-Aldrich (# CDS023113) and
probably dissolves to give a 3M solution which is slightly alkaline.
If pK2 is 5.1, then an insignificant amt of malic acid should bring the
pH down to 7 (If you have to add a significant amount, just add more
water to dilute to a final conc of 3M Malate + Malic acid)


On 06/08/2017 12:32 PM, Diana Tomchick wrote:
> As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
> malic acid salt is less soluble than the acid or the fully neutralized salt.
>
> The pKa2 of malic acid is 5.1.
>
> Contact the people at Hampton Research
>
> https://www.hamptonresearch.com/contact_us.aspx
>
> and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.
>
> It is possible that you need to fully neutralize it before it turns clear, or 
> that you may also need to gently heat it.
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu 
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato 
> > 
> wrote:
>
>
> Dear all,
> we’ve recently having trouble preparing a 3 M stock solution of DL-Malic 
> Acid, pH 7 (which we had, so it’s doable!).
> When we reach pH 3 - 4 the solution turns milky white and does not goes back 
> to a clear solution even when the pH is raised.
> Does anybody have any advice on how to get a clear solution? Has anyone gone 
> through the same?
> Thanks in advance,
> ciao,
> Sebastiano
>
>
> --
> *Sebastiano Pasqualato, PhD*
> Crystallography Unit
> Department of Experimental Oncology
> European Institute of Oncology
> IFOM-IEO Campus
> via Adamello, 16
> 20139 - Milano
> Italy
>
> tel +39 02 9437 5167
> fax +39 02 9437 5990
> web http://is.gd/IEOXtalUnit
>
>
>
> --
>
> UTSouthwestern
>
> Medical Center
>
> The future of medicine, today.
>

[ccp4bb] Off topic but of interest to SSRL users

[Edward Snell]

Dear All,

For those that have Facebook accounts the SSRL Users Organization has 
established a Facebook page at https://www.facebook.com/ssrluec/. This has 
details on the SSRL/LCLS users meeting and news stories coming from SSRL.

Recently you may or may not have heard that the US Department of Energy is 
proposing mothballing SSRL in a few months. With current administration 
priorities and the reduction in in person visits (with the success of remote 
data collection) SSRL has been targeted. This will have considerable impact on 
SSRL staff and users. The SSRL Users Organization will be providing guidelines 
on what the user community can do to educate Washington DC as to how important 
SSRL has been for career development, discovery, and the economy of the USA.  
In advance of that advice please visit the page and show your support.

Thanks,

Eddie Snell.

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
[cid:image001.png@01D2E06C.77B7F500]
Heisenberg was probably here!

Re: [ccp4bb] preparing DL-Malic acid stock solution

[Edward A. Berry]

(di)Sodium D,L-malate is available from Sigma-Aldrich (# CDS023113) and
probably dissolves to give a 3M solution which is slightly alkaline.
If pK2 is 5.1, then an insignificant amt of malic acid should bring the
pH down to 7 (If you have to add a significant amount, just add more
water to dilute to a final conc of 3M Malate + Malic acid)


On 06/08/2017 12:32 PM, Diana Tomchick wrote:

As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
malic acid salt is less soluble than the acid or the fully neutralized salt.

The pKa2 of malic acid is 5.1.

Contact the people at Hampton Research

https://www.hamptonresearch.com/contact_us.aspx

and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.

It is possible that you need to fully neutralize it before it turns clear, or 
that you may also need to gently heat it.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu 
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato > wrote:


Dear all,
we’ve recently having trouble preparing a 3 M stock solution of DL-Malic Acid, 
pH 7 (which we had, so it’s doable!).
When we reach pH 3 - 4 the solution turns milky white and does not goes back to 
a clear solution even when the pH is raised.
Does anybody have any advice on how to get a clear solution? Has anyone gone 
through the same?
Thanks in advance,
ciao,
Sebastiano


--
*Sebastiano Pasqualato, PhD*
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit



--

UTSouthwestern

Medical Center

The future of medicine, today.

Re: [ccp4bb] preparing DL-Malic acid stock solution

[Diana Tomchick]

As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
malic acid salt is less soluble than the acid or the fully neutralized salt.

The pKa2 of malic acid is 5.1.

Contact the people at Hampton Research

https://www.hamptonresearch.com/contact_us.aspx

and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.

It is possible that you need to fully neutralize it before it turns clear, or 
that you may also need to gently heat it.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato 
> wrote:


Dear all,
we’ve recently having trouble preparing a 3 M stock solution of DL-Malic Acid, 
pH 7 (which we had, so it’s doable!).
When we reach pH 3 - 4 the solution turns milky white and does not goes back to 
a clear solution even when the pH is raised.
Does anybody have any advice on how to get a clear solution? Has anyone gone 
through the same?
Thanks in advance,
ciao,
Sebastiano


--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit





UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] preparing DL-Malic acid stock solution

[Sebastiano Pasqualato]

Dear all,
we’ve recently having trouble preparing a 3 M stock solution of DL-Malic Acid, 
pH 7 (which we had, so it’s doable!).
When we reach pH 3 - 4 the solution turns milky white and does not goes back to 
a clear solution even when the pH is raised.
Does anybody have any advice on how to get a clear solution? Has anyone gone 
through the same?
Thanks in advance,
ciao,
Sebastiano


-- 
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit

[ccp4bb] Postdoctoral position in time-resolved EM at the University of Leeds

[Stephen Muench]

Dear All,
A BBSRC funded postdoctoral position is available to become 
part of an international team working on new methodologies for time resolved 
single particle cryo-EM allowing us to trap different conformational states of 
proteins and protein complexes in the µs-ms timeframe. The project will be led 
by the Muench group at the University of Leeds and will involve collaborations 
with Prof Howard White (Eastern Virginia Medical School) and Prof Martin 
Trebbin (Hamburg University centre for ultrafast imaging). This will bring 
together the world class EM facilities in the Leeds Astbury Biostructure 
facility, including two Titan Krios microscopes and the cutting-edge technology 
from the microfluidics field in Hamburg. The successful candidate will work 
between Leeds and Hamburg adapting the microfluidic technology developed in the 
Trebbin group and installing it within the current setup in Leeds. This project 
will build on the significant recent advances in the EM field and previous 
experience in developing time-resolved electron microscopy approaches to 
develop a novel system that can trap conformational states in the µs-ms 
timeframe. This would be an excellent opportunity for someone looking to pursue 
the latest developments in EM and someone who may wish to develop their own 
independent research utilizing this new technology. 

Closing Date: 23 June 2017.

For further details please contact s.p.mue...@leeds.ac.uk  and/or visit the 
following link http://jobs.leeds.ac.uk/FBSBM1065

Best wishes,

Ste

Re: [ccp4bb] AutoSol invalid MTZ column_types error

[Terwilliger, Thomas Charles]

?Hi Mintu,

I will answer you on the Phenix mailing list!

All the best,

Tom T



From: CCP4 bulletin board  on behalf of Mintu Chandra 

Sent: Wednesday, June 7, 2017 9:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AutoSol invalid MTZ column_types error


Dear all,



I am getting an error message "Invalid MTZ column_types for the given
miller_array." while I try to run AutoSol. The last line in the log is
"Adding array ['N(+)', 'N(-)'] to output file with type II".
Do you have any suggestion how to fix this error?

Thanks a lot in advance. Best,

Mintu

Re: [ccp4bb] Problems with an exonuclease

[Mohammad Khan]

Dear all,

I apologize that till now I was making the mistake of not sending the
replies in the common thread, but somehow only to each person individually.
I am summarizing my answers since yesterday here:

I have tried refolding it at pH 7, 7.5 and 8. I add 4mM TCEP to my urea
buffers and reduce it to 2 mM on refolding. I have tried in presence of DTT
and also in absence of any reductant.
My refolded wild type enzyme shows activity, whereas the mutant doesn't.

Yes, I have got my protein mass-speced. Didn't see any modification!

I have tried to remove the contamination by affinity and ion exchange but
with no avail. I have to try heparin-sepharose though.

I sonicate my samples in the washing steps.
I also suspected Triton X as the contaminant. But I then figured out that
TX-100 has a high absorbance at 280 nm, rather than at 260. I will surely
try the KCl and EDTA suggestions.

I have also tried purifying in absence of TX-100, but with same results!

As for running an agarose gel, I have done so but couldn't really see
anything on an EtBr gel.

I will try the suggestions of washing with other solutions as suggested.

Thank you all for the great suggestions!

On Wed, Jun 7, 2017 at 3:36 PM, Mohammad Khan 
wrote:

> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with
> urea, I refold the protein by rapid dilution and get an aggregate and
> monomer peak of the same on GFC. and have checked CD as well as activity,
> both of which are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>
>

[ccp4bb] Call for group leader et IPBS, Toulouse, France

[maveyrau]

Dear CCP4ers,

The Institute of Pharmacology and Structural Biology (IPBS) in Toulouse, 
France, is seeking new group leaders. Details of the call are available at 
http://www.ipbs.fr/call2017. Please note that the application deadline is July 
15, 2017. Address inquiries related to the call at: recr...@ipbs.fr (not to 
me!).

Founded in 1996, the Institute of Pharmacology and Structural Biology (IPBS) is 
a leading research institute of the French National Centre for Scientific 
Research (CNRS) and the University of Toulouse. Located on the main Campus of 
the Université Toulouse III-Paul Sabatier in Toulouse, southwest France, the 
IPBS offers multidisciplinary education in the fields of Science, Health, 
Engineering and Technology, developing one of the most important scientific 
research clusters in France.
Our Institute is a world leader in the discovery, characterization and 
validation of novel important pathways and pharmacological targets in the 
fields of cancer and infectious diseases, through the use of molecular and 
cellular biology approaches, together with in vivo experiments. It conducts 
state-of-the-art research in structural biology, proteomics, biophysics, 
cancerology, immunology and microbiology (http://www.ipbs.fr). The IPBS brings 
together more than 250 scientists and supporting staffs, including more than 60 
national and international postdoctoral fellows and PhD students. The IPBS 
offers outstanding scientific and stimulating research environment and several 
cutting-edge core facilities with highly qualified staff. These include mass 
spectrometry and proteomics, macromolecular crystallography, liquid- and 
solid-state NMR, biophysical characterization of proteins and complexes, 
virtual screening, whole body, tissue and cellular imaging, flow cytometry and 
cell sorting in standard or BSL3 environments, single particle tracking and 
tethered particle motion analysis, and animal facilities.

In order to reinforce its research endeavors in an inspiring, collaborative and 
cutting-edge environment, the IPBS is seeking new talented junior group leaders 
addressing fundamental questions within the spectrum of its research fields. 
Young scientists of any nationality, at junior or midcareer level, and with an 
excellent track record of publications in internationally recognized journals, 
are encouraged to apply. International experience as well as capacity to 
interact with other research groups within the institute are highly 
recommended. Successful candidate(s) will be provided laboratory and office 
space for 5-8 people, a technical personnel and free access to the institute’s 
core facilities for a period of 2 years, together with a starting package for 
basic equipment and consumables. In addition, strong support will be provided 
from the IPBS to obtain tenured positions at CNRS, INSERM or the University of 
Toulouse. Outstanding candidates are expected to establish independent and 
vigorous national and international extramurally-funded research programs (ANR, 
ERC, etc.) that fit at least with one of the priority topics listed below. 
Researchers already holding a permanent position are also welcome to apply.

IPBS Priority topics
See full details for each topic at: http://www.ipbs.fr/call2017
• Tumor microenvironment – Cancer immunology - Interactions of immune cells 
with stromal cells, the extracellular matrix and/or other immune cells; immune 
response to cancer.
• DNA repair/chromatin remodeling in cancer - DNA damage response (DDR) in 
connection with cancer through epigenetics, transcriptional regulation, DNA 
repair pathologies, chromosomal translocations, DDR- based drug discovery, or 
tumor resistance to clastogenic agents.
• Pulmonary infections - Biology of bacterial respiratory pathogens, with a 
strong focus on mechanisms underlying pathogen’s persistence, drug resistance 
or tolerance within the host.
• Drug discovery & structural biology - Development of biological and chemical 
drugs of the future, with the aim of strengthening the pharmacological aspects 
of the IPBS research framework; characterization of drug-target interactions 
and mechanism of action; “hit to lead” strategies and new approaches for drug 
target deconvolution or vectorization will be favored. A particular attention 
will be given to candidates willing to create a group in biological NMR.

Application (about 5 pages in English) should include a cover letter describing 
previous research experience, an outline of the future research project, 
motivation for joining the institute and names and e-mail address of 3 
referees, together with an updated CV (including a complete list of 
publications). Application should be sent to recr...@ipbs.fr in a single PDF 
file named LASTNAME_FIRSTNAME_IPBS2017.pdf. Other formats will not be 
considered.

kind regards,
Laurent
--
Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr
P I C