[ccp4bb] AutoSol invalid MTZ column_types error

[Mintu Chandra]

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

[John Newitt]

Probably nucleic acids. Increase the number or volume of washes and
improve the washing of your inclusion bodies. Instead of sonication,
we use a Polytron homogenizer to resuspend the IBs pellet during
washing. This is faster and easier. Incorporate an additional
chromatography step such as Heparin-Sepharose or Cation-Exchange with
SP-Sepharose if the pI of your protein supports this. Alternatively,
try to flow your protein through DEAE or Q-Sepharose in the presence
of a moderate concentration of NaCl (e.g. 200-250 mM), where much of
the nucleic acids should be captured.

- John

On Wed, Jun 7, 2017 at 9:36 AM, 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.
>
>

Re: [ccp4bb] Problems with an exonuclease

[Edward A. Berry]

On 06/07/2017 10:46 AM, Bonsor, Daniel wrote:

It will either be two things. DNA or residual Triton-X-100.


Actually Triton X-100 absorbs more at 280 than 260 - in fact the hydroxyphenyl 
in TX-100 looks remarkably like tyrosine in RNase A. I long ago gave up hope of 
resolving triton from protein and DNA by spectral curve-fitting!
eab

Re: [ccp4bb] Conserved water

[Andres Libreros]

Thanks everyone for the tips. Actually, I need this information to improve my 
analysis on drug/protein interaction.
Best regards:)

Gerardo A. Libreros
Universidade de São Paulo
Laboratório de Biologia Estrutural Aplicada

Sent from my iPhone

> On 7 Jun 2017, at 18:34, Jared Sampson  wrote:
> 
> Hi Gerardo - 
> 
> Something based on distance alone (and not e.g. H-bonding network) could be 
> done in a straightforward fashion in PyMOL:
> 
> select close_wats, (object1 and solvent) within 1 of (object2 and solvent)
> hide everything, solvent
> as nb_spheres, close_wats
> 
> This would give you only the waters in object 1, but you could expand this 
> logic and include the object2 waters as well.
> 
> select close_wats, close_wats or ((object2 and solvent) within 1 of 
> (object1 and solvent))
> 
> Hope that helps.
> 
> Cheers,
> Jared
> 
> 
>> On Jun 6, 2017, at 7:30 PM, gerardo andres 
>> <130afa955101-dmarc-requ...@jiscmail.ac.uk> wrote:
>> 
>> Hi everyone, does anyone know any strategy or program (besides pywater) to 
>> identify conserved waters in a protein?
>> 
>> Thanks, 
>> 
>> Gerardo
> 

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

Several further notes after contemplation, lunch and a slow day. If the protein 
is His-tagged, you could stick the unfolded protein to Ni-Resin and extensively 
wash with 8M Urea to remove DNA/Triton-X-100, elute, and then refold. This may 
be easier than multiple sonications/centrifugations. Or you could stick the 
folded protein that you have purified to the Nickel resin and wash with high 
concentrations of salt to remove DNA/Triton-X-100.

To check if is really DNA-protein complex you have prepped, you can run two 
agarose gels. Stain one with ethidium bromide and the other one coomassie 
stain. The two bands should coincide with each other (protocol for native 
agarose gel electrophoresis can be found here 
http://www.sciencedirect.com/science/article/pii/S0003269700945986).

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

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.

Re: [ccp4bb] Problems with an exonuclease

[S. Mohanty]

Yes, washing IB with 10 % BPER  twice with sonication makes our IB (from 
various proteins) very clean before denaturation with 6M guanidine 
hydrochloride and further processing. Smita  

On Wednesday, June 7, 2017 12:03 PM, Nicole Thomas  
wrote:
 

 I've found that washing my IB's with B-PER helps dramatically to get rid of 
any impurities.
https://www.thermofisher.com/order/catalog/product/78248

Nicole ThomasUniversity of Wisconsin, MadisonGellman Group
On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers  
wrote:

I missed the Triton - that will be it!
On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers; 1. 20mM Tris, 500mM NaCl, 1% 
Triton-X-100, pH 7.52. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.53. 10mM 
Tris, 1M NaCl4. 20mM Tris, 500mM NaCl, pH 7.5 By resuspension and then 
sonication. This I find removes DNA and Triton-X-100. Also, if the pellet is 
very large, you may need to increase the number of washes, volume and length of 
sonication or split the pellet up. Other things to try…1.  Change the wash 
salt to KCl and use more, (3M). I was informed that KCl is a better disrupter 
of DNA than NaCl (I stand to be corrected if this is wrong).2.  At each 
wash stage, dissolve a small amount of IBs and measure the 260/280. The ratio 
should decrease in the latter washes, if they are working.3.  Does your 
exonuclease typically contain a divalent metal? You could try adding EDTA to 
the wash steps which may help in preventing DNA stick to your protein. All the 
best! Dan  Daniel A Bonsor PhD.Sundberg LabInstitute of Human 
VirologyUniversity of Maryland, Baltimore725 W Lombard Street 
N370BaltimoreMarylandMD 21201Tel: (410) 706-7457  From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK]On Behalf Of Mohammad Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease 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.  



-- 
Best wishes
Prof. Jon R Sayers, FRSBTel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.ukhttp://www.sheffield.ac.uk/ iicd/profiles/sayers





   

Re: [ccp4bb] Problems with an exonuclease

[Nicole Thomas]

I've found that washing my IB's with B-PER helps dramatically to get rid of
any impurities.

https://www.thermofisher.com/order/catalog/product/78248

Nicole Thomas
University of Wisconsin, Madison
Gellman Group

On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers 
wrote:

> I missed the Triton - that will be it!
>
> On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:
>
>> It will either be two things. DNA or residual Triton-X-100. When you say,
>> cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the
>> pellet and then centrifuged again? If the latter, try sonication. I wash my
>> IBs at least 4 times with the following buffers;
>>
>>
>>
>> 1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>>
>> 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>>
>> 3. 10mM Tris, 1M NaCl
>>
>> 4. 20mM Tris, 500mM NaCl, pH 7.5
>>
>>
>>
>> By resuspension and then sonication. This I find removes DNA and
>> Triton-X-100.
>>
>>
>>
>> Also, if the pellet is very large, you may need to increase the number of
>> washes, volume and length of sonication or split the pellet up.
>>
>>
>>
>> Other things to try…
>>
>> 1.   Change the wash salt to KCl and use more, (3M). I was informed
>> that KCl is a better disrupter of DNA than NaCl (I stand to be corrected if
>> this is wrong).
>>
>> 2.   At each wash stage, dissolve a small amount of IBs and measure
>> the 260/280. The ratio should decrease in the latter washes, if they are
>> working.
>>
>> 3.   Does your exonuclease typically contain a divalent metal? You
>> could try adding EDTA to the wash steps which may help in preventing DNA
>> stick to your protein.
>>
>>
>>
>> All the best!
>>
>>
>>
>> Dan
>>
>>
>>
>>
>>
>> Daniel A Bonsor PhD.
>>
>> Sundberg Lab
>>
>> Institute of Human Virology
>>
>> University of Maryland, Baltimore
>>
>> 725 W Lombard Street N370
>>
>> Baltimore
>>
>> Maryland
>>
>> MD 21201
>>
>> Tel: (410) 706-7457
>>
>>
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Mohammad Khan
>> *Sent:* Wednesday, June 07, 2017 9:37 AM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] Problems with an exonuclease
>>
>>
>>
>> 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.
>>
>>
>>
>>
>>
>
>
>
> --
> Best wishes
> Prof. Jon R Sayers, FRSB
> Tel: +44 (0) 114 2159552 <+44%20114%20215%209552>
> Email:  j.r.say...@shef.ac.uk
> http://www.sheffield.ac.uk/iicd/profiles/sayers
>
>

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Jon R Sayers]

 REFMAC and  COOT seem fine on my iMac running 10.12.5

On 7 June 2017 at 17:28, Diana Tomchick 
wrote:

> Hahahaha, that was a typo, I meant to write
>
> 10.12.5
>
> 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 7, 2017, at 11:25 AM, Scott Classen  wrote:
>
> 10.2 ?
> That OS is 15 years old.
>
> > On Jun 7, 2017, at 8:27 AM, Diana Tomchick  utsouthwestern.edu> wrote:
> >
> > I’ve not had any problems; the current version is 10.2.5, so it’s pretty
> stable now.
> >
> > 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 7, 2017, at 10:14 AM, Patrick Loll  wrote:
> >
> > I’m still running Yosemite on my Macs, both because I’m change-averse
> and because folks reported problems with some crystallographic software
> upon upgrading the OS.
> >
> > These reports have now faded into the haze of the past, and so I ask,
> have the issues been resolved? Is it safe to move to Sierra?
> >
> > Thanks as always,
> >
> > Pat
> >
> > 
> ---
> > Patrick J. Loll, Ph. D.
> > Professor of Biochemistry & Molecular Biology
> > Drexel University College of Medicine
> > Room 10-102 New College Building
> > 245 N. 15th St., Mailstop 497
> > Philadelphia, PA  19102-1192  USA
> >
> > (215) 762-7706
> > pat.l...@drexelmed.edu
> >
> >
> > ___
> > phenixbb mailing list
> > pheni...@phenix-online.org
> > http://phenix-online.org/mailman/listinfo/phenixbb
> > Unsubscribe: phenixbb-le...@phenix-online.org
> >
> >
> > 
> >
> > UT Southwestern
> >
> >
> > Medical Center
> >
> >
> >
> > The future of medicine, today.
> >
>
>
>


-- 
Best wishes
Prof. Jon R Sayers, FRSB
Tel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.uk
http://www.sheffield.ac.uk/iicd/profiles/sayers

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Diana Tomchick]

Hahahaha, that was a typo, I meant to write

10.12.5

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 7, 2017, at 11:25 AM, Scott Classen  wrote:

10.2 ?
That OS is 15 years old.

> On Jun 7, 2017, at 8:27 AM, Diana Tomchick 
>  wrote:
> 
> I’ve not had any problems; the current version is 10.2.5, so it’s pretty 
> stable now.
> 
> 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 7, 2017, at 10:14 AM, Patrick Loll  wrote:
> 
> I’m still running Yosemite on my Macs, both because I’m change-averse and 
> because folks reported problems with some crystallographic software upon 
> upgrading the OS.
> 
> These reports have now faded into the haze of the past, and so I ask, have 
> the issues been resolved? Is it safe to move to Sierra?
> 
> Thanks as always,
> 
> Pat
> 
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
> 
> (215) 762-7706
> pat.l...@drexelmed.edu
> 
> 
> ___
> phenixbb mailing list
> pheni...@phenix-online.org
> http://phenix-online.org/mailman/listinfo/phenixbb
> Unsubscribe: phenixbb-le...@phenix-online.org
> 
> 
> 
> 
> UT Southwestern
> 
> 
> Medical Center
> 
> 
> 
> The future of medicine, today.
> 

Re: [ccp4bb] Problems with an exonuclease

[Jon R Sayers]

I missed the Triton - that will be it!

On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:

> It will either be two things. DNA or residual Triton-X-100. When you say,
> cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the
> pellet and then centrifuged again? If the latter, try sonication. I wash my
> IBs at least 4 times with the following buffers;
>
>
>
> 1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>
> 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>
> 3. 10mM Tris, 1M NaCl
>
> 4. 20mM Tris, 500mM NaCl, pH 7.5
>
>
>
> By resuspension and then sonication. This I find removes DNA and
> Triton-X-100.
>
>
>
> Also, if the pellet is very large, you may need to increase the number of
> washes, volume and length of sonication or split the pellet up.
>
>
>
> Other things to try…
>
> 1.   Change the wash salt to KCl and use more, (3M). I was informed
> that KCl is a better disrupter of DNA than NaCl (I stand to be corrected if
> this is wrong).
>
> 2.   At each wash stage, dissolve a small amount of IBs and measure
> the 260/280. The ratio should decrease in the latter washes, if they are
> working.
>
> 3.   Does your exonuclease typically contain a divalent metal? You
> could try adding EDTA to the wash steps which may help in preventing DNA
> stick to your protein.
>
>
>
> All the best!
>
>
>
> Dan
>
>
>
>
>
> Daniel A Bonsor PhD.
>
> Sundberg Lab
>
> Institute of Human Virology
>
> University of Maryland, Baltimore
>
> 725 W Lombard Street N370
>
> Baltimore
>
> Maryland
>
> MD 21201
>
> Tel: (410) 706-7457
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Mohammad
> Khan
> *Sent:* Wednesday, June 07, 2017 9:37 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Problems with an exonuclease
>
>
>
> 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.
>
>
>
>
>



-- 
Best wishes
Prof. Jon R Sayers, FRSB
Tel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.uk
http://www.sheffield.ac.uk/iicd/profiles/sayers

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Scott Classen]

10.2 ?
That OS is 15 years old.

> On Jun 7, 2017, at 8:27 AM, Diana Tomchick 
>  wrote:
> 
> I’ve not had any problems; the current version is 10.2.5, so it’s pretty 
> stable now.
> 
> 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 7, 2017, at 10:14 AM, Patrick Loll  wrote:
> 
> I’m still running Yosemite on my Macs, both because I’m change-averse and 
> because folks reported problems with some crystallographic software upon 
> upgrading the OS.
> 
> These reports have now faded into the haze of the past, and so I ask, have 
> the issues been resolved? Is it safe to move to Sierra?
> 
> Thanks as always,
> 
> Pat
> 
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
> 
> (215) 762-7706
> pat.l...@drexelmed.edu
> 
> 
> ___
> phenixbb mailing list
> pheni...@phenix-online.org
> http://phenix-online.org/mailman/listinfo/phenixbb
> Unsubscribe: phenixbb-le...@phenix-online.org
> 
> 
> 
> 
> UT Southwestern
> 
> 
> Medical Center
> 
> 
> 
> The future of medicine, today.
> 

Re: [ccp4bb] Conserved water

[Edward A. Berry]

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.


If the number of waters is too large to inspect manually,
save the superposed structures to disk, grep out the waters from each into a 
separate files,
and use a program like http://www.cytbc1.com/berry/for/pdbdist3w.for
to compare files and write atoms closer than a specified threshold into a new 
pdb file

That program only compares two files, so you would have to do all possible 
pairs to get waters conserved
in at least 2 structures, or compare each file with the survivors from previous 
to get waters
conserved in all.
PS- It compiles with f77 (g77). May need touch-up for fortran90 (gfortran).
eab

On 06/07/2017 02:59 AM, Eleanor Dodson wrote:

Many years ago I wrote code to label waters with a code related to the 
residue/atom  they were Hbonded to , so then you could check whether all OH TYR 
227 in each chain  had an associated water.. But it used non-standard water 
naming ..

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.

Eleanor

On 7 June 2017 at 00:30, gerardo andres <130afa955101-dmarc-requ...@jiscmail.ac.uk 
> wrote:

Hi everyone, does anyone know any strategy or program (besides pywater) to 
identify conserved waters in a protein?

Thanks,

Gerardo

Re: [ccp4bb] [phenixbb] comfortable OS X level

[Diana Tomchick]

I’ve not had any problems; the current version is 10.2.5, so it’s pretty stable 
now.

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 7, 2017, at 10:14 AM, Patrick Loll  wrote:

I’m still running Yosemite on my Macs, both because I’m change-averse and 
because folks reported problems with some crystallographic software upon 
upgrading the OS.

These reports have now faded into the haze of the past, and so I ask, have the 
issues been resolved? Is it safe to move to Sierra?

Thanks as always,

Pat

---
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu


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Medical Center



The future of medicine, today.

[ccp4bb] comfortable OS X level

[Patrick Loll]

I’m still running Yosemite on my Macs, both because I’m change-averse and 
because folks reported problems with some crystallographic software upon 
upgrading the OS.

These reports have now faded into the haze of the past, and so I ask, have the 
issues been resolved? Is it safe to move to Sierra? 

Thanks as always,

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers;

1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
3. 10mM Tris, 1M NaCl
4. 20mM Tris, 500mM NaCl, pH 7.5

By resuspension and then sonication. This I find removes DNA and Triton-X-100.

Also, if the pellet is very large, you may need to increase the number of 
washes, volume and length of sonication or split the pellet up.

Other things to try…

1.   Change the wash salt to KCl and use more, (3M). I was informed that 
KCl is a better disrupter of DNA than NaCl (I stand to be corrected if this is 
wrong).

2.   At each wash stage, dissolve a small amount of IBs and measure the 
260/280. The ratio should decrease in the latter washes, if they are working.

3.   Does your exonuclease typically contain a divalent metal? You could 
try adding EDTA to the wash steps which may help in preventing DNA stick to 
your protein.

All the best!

Dan


Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

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.

Re: [ccp4bb] Problems with an exonuclease

[Jon R Sayers]

Dear Mohammad,
If your protein is purified from insoluble material there could be some DNA
in there though if it were stoichiometric your 26 would be >> than your
280, as the former has a much higher extinction co-efficient. A ratio  of 2
is could be RNA contamination. I'd also check the mass spec of your protein
to see if it has any unusual modification - urea is notorious for cause
carbamylation of N-terminal amino groups,  and of lysine and arginine
residues.  What that does to UV I'm not sire sure but irrespective of the
UV anomaly, I'd always get the beast mass-specced!

On 7 June 2017 at 14:36, 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.
>
>
>


-- 
Best wishes
Prof. Jon R Sayers, FRSB
Tel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.uk
http://www.sheffield.ac.uk/iicd/profiles/sayers

[ccp4bb] Problems with an exonuclease

[Mohammad Khan]

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.

Re: [ccp4bb] Conserved water

[benjamin bax]

How about 
WONKA and OOMMPPAA: analysis of protein–ligand interaction data to direct 
structure-based drug design?
Ben 



On 7 Jun 2017, at 07:59, Eleanor Dodson  wrote:

Many years ago I wrote code to label waters with a code related to the 
residue/atom  they were Hbonded to , so then you could check whether all OH TYR 
227 in each chain  had an associated water.. But it used non-standard water 
naming ..

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.

Eleanor

On 7 June 2017 at 00:30, gerardo andres 
<130afa955101-dmarc-requ...@jiscmail.ac.uk 
> wrote:
Hi everyone, does anyone know any strategy or program (besides pywater) to 
identify conserved waters in a protein?

Thanks, 

Gerardo



Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

[ccp4bb] CryoEM postdoctoral position at the Well one Centre for Cell Biology, Edinburgh, UK

[ARULANANDAM Jeyaprakash]

A postdoctoral position is available in Jeyapraksh (JP) Arulanandam’s lab at 
the Wellcome Centre for Cell Biology, University of Edinburgh. The JP lab 
(http://jeyaprakash.bio.ed.ac.uk) aims to 
understand the structural level mechanistic details of processes regulating 
error-free chromosome segregation. The successful candidate will combine 
protein biochemistry and structural biology with mammalian cell based assays to 
dissect the molecular details of how specific intermolecular protein 
interactions achieve stable centromere maintenance and accurate chromosome 
segregation during cell division.

Applicants must have (or will shortly be awarded) a PhD with training in 
biochemistry, structural biology and cell biology. The successful candidate 
will be a highly motivated and enthusiastic individual with an outstanding 
academic track record, good communication skills and the ability to work as a 
team. Applicants with experience in single particle cryoEM, biochemistry and 
mammalian cell based siRNA/CRISPR rescue assays are encouraged to apply.

The post is for 12 months in the first instance with the possibility of 
extension.

Closing Date: 16 June 2017 at 5pm GMT
For further particulars and to apply for this post, please follow  
https://www.vacancies.ed.ac.uk/pls/corehrrecruit/erq_jobspec_version_4.jobspec?p_id=039927


Dr. A. Jeyaprakash Arulanandam
Wellcome Senior Research Fellow
Wellcome Trust Centre for Cell Biology
University of Edinburgh
Michael Swann building (S5.18)
Max Born Crescent
Edinburgh EH9 3BF
UK
Tel: +44 131 6507113
Fax: +44 131 6080414
Web: http://jeyaprakash.bio.ed.ac.uk/
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[ccp4bb] CryoEM Postdoctoral position at the Wellcome Centre for Cell biology, Edinburgh, UK

[ARULANANDAM Jeyaprakash]

A postdoctoral position is available in Jeyapraksh (JP) Arulanandam’s lab at 
the Wellcome Centre for Cell Biology, University of Edinburgh. The JP lab 
(http://jeyaprakash.bio.ed.ac.uk) aims to 
understand the structural level mechanistic details of processes regulating 
error-free chromosome segregation. The successful candidate will combine 
protein biochemistry and structural biology with mammalian cell based assays to 
dissect the molecular details of how specific intermolecular protein 
interactions achieve stable centromere maintenance and accurate chromosome 
segregation during cell division.

Applicants must have (or will shortly be awarded) a PhD with training in 
biochemistry, structural biology and cell biology. The successful candidate 
will be a highly motivated and enthusiastic individual with an outstanding 
academic track record, good communication skills and the ability to work as a 
team. Applicants with experience in single particle cryoEM, biochemistry and 
mammalian cell based siRNA/CRISPR rescue assays are encouraged to apply.

The post is for 12 months in the first instance with the possibility of 
extension.

Closing Date: 16 June 2017 at 5pm GMT
For further particulars and to apply for this post, please follow  
https://www.vacancies.ed.ac.uk/pls/corehrrecruit/erq_jobspec_version_4.jobspec?p_id=039927


Dr. A. Jeyaprakash Arulanandam
Wellcome Senior Research Fellow
Wellcome Trust Centre for Cell Biology
University of Edinburgh
Michael Swann building (S5.18)
Max Born Crescent
Edinburgh EH9 3BF
UK
Tel: +44 131 6507113
Fax: +44 131 6080414
Web: http://jeyaprakash.bio.ed.ac.uk/
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

Re: [ccp4bb] post-doctoral position and PhD position structural biology of ion channels

[Chris Ulens]

A post-doctoral position and PhD position are available at the laboratory of 
Structural Neurobiology, KU Leuven Belgium. The lab website is available at 
http://www.xtal.be. The research in our laboratory is aimed at understanding 
the molecular mechanism of ligand-gated ion channels. Our method relies on 
X-ray crystallography and cryo-EM in combination with electrophysiological 
recordings and fluorescence spectroscopy. Our laboratory hosts an integrated 
macromolecular crystallization facility with a Mosquito crystallization robot 
and two RockImagers. Current structural targets include the family of 
pentameric ligand-gated ion channels (Cys-loop receptors), TRP channels and CNG 
channels.

We would like to recruit a post-doc and/or PhD student with prior experience in 
expression, purification and crystallization of membrane proteins. Prior 
experience with X-ray crystallography or cryo-EM is considered a major 
advantage.

The successful candidate:
- easily communicates in an open verbal manner and has a team player spirit
- has an ambitious work ethic
- critically contributes to writing of manuscripts and grant proposals
- makes efforts to obtain independent funding
- meets intermediate deadlines and brings projects to a successful end
- fuels internal discussions of recent publications in the field
- sets an example to younger PhD students in the lab
- identifies directions for future scientific research

The position is open immediately and applications will be accepted until the 
position is filled. Applicants are requested to submit a CV with publication 
list and two references. E-mail to chris.ul...@kuleuven.be

Selected lab publications:

Molecular blueprint of allosteric binding sites in a homologue of the 
agonist-binding domain of the α7 nicotinic acetylcholine receptor. Spurny R, 
Debaveye S, Farinha A, Veys K, Vos AM, Gossas T, Atack J, Bertrand S, Bertrand 
D, Danielson UH, Tresadern G, Ulens C.
Proc Natl Acad Sci U S A. 2015 May 12;112(19):E2543-52.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by 
benzodiazepines. Spurny R, Ramerstorfer J, Price K, Brams M, Ernst M, Nury H, 
Verheij M, Legrand P, Bertrand D, Bertrand S, Dougherty DA, de Esch IJ, 
Corringer PJ, Sieghart W, Lummis SC, Ulens C. Proc Natl Acad Sci U S A. 2012 
Oct 30;109(44):E3028-34.

A structural and mutagenic blueprint for molecular recognition of strychnine 
and d-tubocurarine by different cys-loop receptors. Brams M, Pandya A, Kuzmin 
D, van Elk R, Krijnen L, Yakel JL, Tsetlin V, Smit AB, Ulens C.
PLoS Biol. 2011 Mar;9(3):e1001034.

[ccp4bb] MoRDa update

[Alexey Vagin]

Dear All

Morda at ccp4online has been updated. 
The structure solution program is improved and database is extended .

The update is also available for existing local Morda installations and can be 
installed from command line: change to installation directory (by default 
MoRDa_DB) and type ./update_morda.

Both update and installation instructions are available from Morda homepage.

Best regards
Alexey

Re: [ccp4bb] Conserved water

[Eleanor Dodson]

Many years ago I wrote code to label waters with a code related to the
residue/atom  they were Hbonded to , so then you could check whether all OH
TYR 227 in each chain  had an associated water.. But it used non-standard
water naming ..

Easiest way is to line up molecule pairs or chain pairs in COOT and see if
there are equivalent waters.

Eleanor

On 7 June 2017 at 00:30, gerardo andres <
130afa955101-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi everyone, does anyone know any strategy or program (besides pywater) to
> identify conserved waters in a protein?
>
> Thanks,
>
> Gerardo
>