Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Frank Von Delft]

Absolutely. But don't bother with SIRAS:  straight SAD will do the job 9/10 
times these days, thanks to magnificent beamlines and algorithms.

Sent from tiny silly touch screen

From: "Whitley, Matthew J" 
Sent: 22 Jun 2017 1:09 am
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling


I second (or third) the suggestion that others have given to try soaking 
mercury compounds into your crystal.  Mercury absolutely loves free cysteines, 
and if you have 5, you have a great chance of getting binding.  If isomorphism 
is maintained after soaking, the isomorphous differences will be huge, and if 
you can collect data at a synchrotron, then the anomalous signal also opens up 
the possibility of SIRAS phasing.  Everybody seems to have their own favorite 
mercury compound to recommend, and mine is potassium tetraiodomercurate 
(K2HgI4).  Just recently I was able to get mercury binding to three free 
cysteines in my protein with this compound with absolutely no effort 
whatsoever.  I simply dissolved a few grains of compound in an 
acetonitrile/water solution, added a drop to my crystal drop, waited 30 
minutes, and then collected the data.  SHELX was able to locate the sites and 
solve the phases by SIRAS in mere moments.  It couldn't have been easier, and I 
thank the experimental phasing gods for their generosity.  In any event, your 
protein has a large number of free cysteines, so I think you probably have an 
above average chance for some flavor of phasing based on mercury to be 
successful.


Good luck!


Matthew


---
Matthew J. Whitley, Ph.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine



From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Wednesday, June 21, 2017 7:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 20 Jun 2017 to 21 Jun 2017 (#2017-172)

--

Date:Wed, 21 Jun 2017 17:46:56 +0200
From:Vito Calderone 
Subject: Se-Met and Se-Cys double labelling

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity…I suppose MR would be very unlikely to
work…so I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Whitley, Matthew J]

I second (or third) the suggestion that others have given to try soaking 
mercury compounds into your crystal.  Mercury absolutely loves free cysteines, 
and if you have 5, you have a great chance of getting binding.  If isomorphism 
is maintained after soaking, the isomorphous differences will be huge, and if 
you can collect data at a synchrotron, then the anomalous signal also opens up 
the possibility of SIRAS phasing.  Everybody seems to have their own favorite 
mercury compound to recommend, and mine is potassium tetraiodomercurate 
(K2HgI4).  Just recently I was able to get mercury binding to three free 
cysteines in my protein with this compound with absolutely no effort 
whatsoever.  I simply dissolved a few grains of compound in an 
acetonitrile/water solution, added a drop to my crystal drop, waited 30 
minutes, and then collected the data.  SHELX was able to locate the sites and 
solve the phases by SIRAS in mere moments.  It couldn't have been easier, and I 
thank the experimental phasing gods for their generosity.  In any event, your 
protein has a large number of free cysteines, so I think you probably have an 
above average chance for some flavor of phasing based on mercury to be 
successful.


Good luck!


Matthew


---
Matthew J. Whitley, Ph.D.
Research Associate
Department of Structural Biology
University of Pittsburgh School of Medicine



From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Wednesday, June 21, 2017 7:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 20 Jun 2017 to 21 Jun 2017 (#2017-172)

--

Date:Wed, 21 Jun 2017 17:46:56 +0200
From:Vito Calderone 
Subject: Se-Met and Se-Cys double labelling

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity…I suppose MR would be very unlikely to
work…so I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] Rcrane error, update

[Ursula Schulze-Gahmen]

I figured it out. Rcrane works fine if I remove the hydrogens before going
into Coot.

Ursula

On Wed, Jun 21, 2017 at 12:04 PM, Ursula Schulze-Gahmen <
uschulze-gah...@lbl.gov> wrote:

> I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane
> window opens and I am able to calculate new conformations for my nucleic
> acid. But when I am trying to accept a conformation, I am getting an error
> message:
>
> Traceback (most recent call last):
>   File "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.
> 7/site-packages/coot/rcrane/reviewSuitesGui.py", line 1041, in __accept
> self.__pseudoMolecule.finalMoleculeCleanup(fixSegids =
> bool(self.__pseudoMolecule.hasSavedCoordinates()))
>   File "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.
> 7/site-packages/coot/rcrane/pseudoMolecule.py", line 1735, in
> finalMoleculeCleanup
> curRes[3].sort(key = lambda x: self.__reorderAtomsOrder[str(
> curRes[2])][x[0][0].strip()])
>   File "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.
> 7/site-packages/coot/rcrane/pseudoMolecule.py", line 1735, in 
> curRes[3].sort(key = lambda x: self.__reorderAtomsOrder[str(
> curRes[2])][x[0][0].strip()])
> KeyError: "H5'"
>
> Any suggestions what might be wrong, and how I can fix this?
>
> Thanks
>
> --
> Ursula Schulze-Gahmen, Ph.D.
> Project Scientist
> UC Berkeley, QB3
> 360 Stanley Hall #3220
> Berkeley, CA 94720-3220
> (510) 643 9491 <(510)%20643-9491>
>



-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Bonsor, Daniel]

To perform double labelling on your protein in an NON auxotrophic strain may be 
difficult. For the seleomet side, you can shut down the biosynthesis of Met by 
the addition of lysine, phenylalanine, threonine, isoleucine and valine; 
various protocols exist online. However to shutdown biosynthesis of cysteine, 
you need to add cysteine (acts as a negative feedback inhibitor on itself ), 
which defeats the point. I cannot find online if selenocys can inhibit 
biosynthesis of cysteine, like cysteine can. You could perform a small test 
expression by adding all the amino acids (lysine, phenylalanine, threonine, 
isoleucine, valine, selenomet, selenocys) 30 mins before induction, do your 
typical expression and purification and confirm by mass spec of labelling.

If not you could trying sulfur SAD, the various derivatives suggested today, 
soaks with halides, magic triangle. Mutate leucine residues to methionine. 

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



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Vito 
Calderone
Sent: Wednesday, June 21, 2017 11:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Se-Met and Se-Cys double labelling

I am working on a protein having 360 residues. In its sequence there are 3 Met 
and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the closest 
homologue has 20% identity...I suppose MR would be very unlikely to work...so I 
would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply the 
threshold to get a good anomalous signal. For this reason I would like to 
exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double mutant 
protein in NON auxotrophic strains of E. coli which you have experienced 
working efficiently?
Thanks

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Guenter Fritz]

Dear Vito,

for SeMet have a look here:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
 Worked like a charm for E.coli and also for other expression hosts 
with minor modifications 
(https://www.nature.com/nature/journal/v516/n7529/full/nature14003.html#methods). 


HTH
Guenter

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity…I suppose MR would be very unlikely to
work…so I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] off-topic:fluorescence polarization displacement assay

[Tristan Croll]

Looks like positive cooperativity to me. If binding at one site increases 
affinity at the second site, then intermediate concentrations of your 
unlabelled ligand can (seemingly paradoxically) *increase* binding of your 
tracer. At higher levels still, the unlabelled ligand outcompetes the tracer as 
expected. See figure 1 in http://molpharm.aspetjournals.org/content/70/5/1783.

Hope this helps,

Tristan

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 21 Jun 2017, at 20:20, megha abbey  wrote:
> 
> Hello All,
> 
> This is an off-topic question. I have some issues regarding Fluorescence 
> Polarization competitive displacement assay and would need some advice. 
> 
> I have developed an in vitro fluorescence polarization based assay using a 
> N-terminus labelled FITC peptide. The peptide is 21 amino acids long and the 
> binding protein is 50 Kda in size. The Kd for interaction is 1 uM. I am 
> further running a competitive displacement assay using the exactly same 
> unlabelled peptide. Here, with increasing concentration of the unlabeled 
> peptide, the polarization signal first increases and then shows a sharp 
> decline. Attached is the raw data file for the above. 
> 
> I believe that if the unlabelled peptide had been aggregating, the 
> polarization signal would increase but not drop. If the binding would have 
> been non-specific, then the unlabelled peptide should not displace at all, 
> but here I see an increase followed by a decrease in the signal. What does 
> this increase and sharp drop in polarization signify and how do I fix this? 
> Please help.
> 
> I have checked the polarization for titration of the unlabelled peptide mixed 
> with fixed conc. of FITC-peptide (no protein added). Here, the polarization 
> signals are the same for the entire range of unlabeled peptide. I have also 
> tried incubating the unlabelled peptide with the protein (for ~15min) first 
> followed by addition of FITC-peptide, but the results are the same.  
> 
> Thank you,
> Megha
> 

[ccp4bb] off-topic:fluorescence polarization displacement assay

[megha abbey]

Hello All,

This is an off-topic question. I have some issues regarding Fluorescence
Polarization competitive displacement assay and would need some advice.

I have developed an in vitro fluorescence polarization based assay using a
N-terminus labelled FITC peptide. The peptide is 21 amino acids long and
the binding protein is 50 Kda in size. The Kd for interaction is 1 uM. I am
further running a competitive displacement assay using the exactly same
unlabelled peptide. Here, with increasing concentration of the unlabeled
peptide, the polarization signal first increases and then shows a sharp
decline. Attached is the raw data file for the above.

I believe that if the unlabelled peptide had been aggregating, the
polarization signal would increase but not drop. If the binding would have
been non-specific, then the unlabelled peptide should not displace at all,
but here I see an increase followed by a decrease in the signal. What does
this increase and sharp drop in polarization signify and how do I fix this?
Please help.

I have checked the polarization for titration of the unlabelled peptide
mixed with fixed conc. of FITC-peptide (no protein added). Here, the
polarization signals are the same for the entire range of unlabeled
peptide. I have also tried incubating the unlabelled peptide with the
protein (for ~15min) first followed by addition of FITC-peptide, but the
results are the same.

Thank you,
Megha


unlabelled displ..xlsx
Description: MS-Excel 2007 spreadsheet

Re: [ccp4bb] Rcrane error, update

[Francis Reyes]

I'm thinking a nomenclature issue. 

H5 ? Should be H5' no? 

Have you run your structure through a pdb remediator? 

http://kinemage.biochem.duke.edu/software/remediator.php is my favorite. 

You will want to play around with the flags to get the right output. I think 
rCrane likes old style nomenclature (asterisks instead of apostrophes). 

F

On Jun 21, 2017, at 3:04 PM, Ursula Schulze-Gahmen  
wrote:

> I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane 
> window opens and I am able to calculate new conformations for my nucleic 
> acid. But when I am trying to accept a conformation, I am getting an error 
> message:
> 
> Traceback (most recent call last):
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/reviewSuitesGui.py",
>  line 1041, in __accept
> self.__pseudoMolecule.finalMoleculeCleanup(fixSegids = 
> bool(self.__pseudoMolecule.hasSavedCoordinates()))
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/pseudoMolecule.py",
>  line 1735, in finalMoleculeCleanup
> curRes[3].sort(key = lambda x: 
> self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/pseudoMolecule.py",
>  line 1735, in 
> curRes[3].sort(key = lambda x: 
> self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
> KeyError: "H5'"
> 
> Any suggestions what might be wrong, and how I can fix this?
> 
> Thanks
> 
> -- 
> Ursula Schulze-Gahmen, Ph.D.
> Project Scientist
> UC Berkeley, QB3
> 360 Stanley Hall #3220
> Berkeley, CA 94720-3220
> (510) 643 9491

[ccp4bb] Rcrane error, update

[Ursula Schulze-Gahmen]

I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane
window opens and I am able to calculate new conformations for my nucleic
acid. But when I am trying to accept a conformation, I am getting an error
message:

Traceback (most recent call last):
  File
"/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/reviewSuitesGui.py",
line 1041, in __accept
self.__pseudoMolecule.finalMoleculeCleanup(fixSegids =
bool(self.__pseudoMolecule.hasSavedCoordinates()))
  File
"/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/pseudoMolecule.py",
line 1735, in finalMoleculeCleanup
curRes[3].sort(key = lambda x:
self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
  File
"/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/pseudoMolecule.py",
line 1735, in 
curRes[3].sort(key = lambda x:
self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
KeyError: "H5'"

Any suggestions what might be wrong, and how I can fix this?

Thanks

-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

[ccp4bb] rcrane key error

[Ursula Schulze-Gahmen]

I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane
window opens and I am able to calculate new conformations for my nucleic
acid. But when I am trying to accept a conformation, I am getting an error
message: Key Error "H5". Any suggestions what might be wrong, and how I can
fix this?

Thanks

Ursula

-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

[ccp4bb] VS: [ccp4bb] Se-Met and Se-Cys double labelling

[Kajander, Tommi A]

Something like Balbes for MR is worth a shot? I wouldnt bother manually with MR 
anymore unless really clear. Or try Hg and Pt for Cys, His and Met. I would try 
the Pt chlorides. All depends on pH etc...

Tommi


 Alkuperäinen viesti 
Lähettäjä: "Keller, Jacob" 
Päivämäärä: 21.06.2017 19.51 (GMT+02:00)
Saaja: CCP4BB@JISCMAIL.AC.UK
Aihe: Re: [ccp4bb] Se-Met and Se-Cys double labelling

Halide soaks anyone? Cs or NaI?

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J 
van Raaij
Sent: Wednesday, June 21, 2017 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

On 21 Jun 2017, at 17:46, Vito Calderone 
> wrote:

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity匢 suppose MR would be very unlikely to
work卻o I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Keller, Jacob]

Halide soaks anyone? Cs or NaI?

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J 
van Raaij
Sent: Wednesday, June 21, 2017 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

On 21 Jun 2017, at 17:46, Vito Calderone 
> wrote:

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity匢 suppose MR would be very unlikely to
work卻o I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Mark J van Raaij]

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

> On 21 Jun 2017, at 17:46, Vito Calderone  wrote:
> 
> I am working on a protein having 360 residues. In its sequence there are 3
> Met and 5 free Cys.
> I will need MAD to solve the structure since based on the sequence the
> closest homologue has 20% identity匢 suppose MR would be very unlikely to
> work卻o I would like to express a selenium derivative to exploit MAD.
> Looking in the literature 1 Se-Met every 120 residues seems not to comply
> the threshold to get a good anomalous signal. For this reason I would like
> to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
> Could somenone suggest a reference to a protocol to express the double
> mutant protein in NON auxotrophic strains of E. coli which you have
> experienced working efficiently?
> Thanks

[ccp4bb] Se-Met and Se-Cys double labelling

[Vito Calderone]

I am working on a protein having 360 residues. In its sequence there are 3
Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity…I suppose MR would be very unlikely to
work…so I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to comply
the threshold to get a good anomalous signal. For this reason I would like
to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks

[ccp4bb] Blend error

[zheng zhou]

Hi, all

I am running  Blend on a new machine (Redhat 6.9 Santiago) and met the
following X11 module and R code error. Thanks for your help.

When I run
$  X -version

It returns:
X.Org X Server 1.17.4
Release Date: 2015-10-28
X Protocol Version 11, Revision 0

Build ID: xorg-x11-server 1.17.4-16.el6
Current version of pixman: 0.32.8

I checked that libpng12.so.0 is in /usr/lib64

***
* Information from CCP4Interface script
***
The program run with command: ccp4-7.0/bin/blend -a data
has failed with error message
Error in png(file = "./tree.png", height = 1000, width = 1000) :
  X11 module cannot be loaded
Execution halted

 EXECUTION ERROR!
An error occurred in the execution of R code associated with BLEND.
***


#CCP4I TERMINATION STATUS 0 "Error in png(file = "./tree.png", height
= 1000, width = 1000) :X11 module cannot be loaded Execution
halted   EXECUTION ERROR! An error occurred in the execution of R code
associated with BLEND."
#CCP4I TERMINATION TIME 03 Jun 2017  22:33:34

Re: [ccp4bb] Weak density of RNA in a complex structure

[Reza Khayat]

Dear Chen,

Is this an icosahedral virus crystal structure, or a highly symmetric structure 
where the RNA may have different binding modes to each subunit and thus 
averaged out in the crystal?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Nicolas FOOS 
[nicolas.f...@esrf.fr]
Sent: Wednesday, June 21, 2017 4:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Weak density of RNA in a complex structure

Dear Chen,

I will answer with some question :

- How is the refinement going ? Is the RNA properly taking in count ? Which 
soft do you use ?

- How do you solve the structure ? MR ? If it's MR did you use a model which 
contain both protein and RNA ? Did you try to solve with the protein only ? 
Maybe you are "forging" RNA.

- Depending of your data (wavelenght and redundancy) you can try to calculate 
an anomalous difference map to see the phosphorus of the RNA to be more 
confident.

- Last point, it could be the occupancy connected with the stability of the 
complex, maybe sometimes RNA is here sometimes not. I see that one time with 
two complex in the same ASU, one has one missing partner.

For my side, I really like to refine my DNA-protein complex with Buster from 
global Phasing. It gives you a very nice and informative density map. If you 
have this possibility let's try.

Hope to help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19


On 21/06/2017 09:34, Chen WeiFei wrote:

Dear All,


We have get a complex crystal and the resolution can be refined to nearly 1.84 
Å. But the electron density of the RNA is very weak.

In some datasets we can't find any density of the RNA and in other datasets we 
can see more or less some RNA density.

For now we can build 6-7 nucleotides but we can't distinguish the rigth 
sequence.

If anyone has the same problem and how to solve this problem.

Best Regards,


Dr Wei-Fei Chen

College of Life Sciences,

Northwest A University

Re: [ccp4bb] Protein or DNA crystals

[Patrick Shaw Stewart]

We have sometimes used Michael Garavito's excellent suggestion of diffusing
glutaraldehyde into the drops - mentioned on this board a few days ago.
Our protein crystals either turned brown or turned into brown goo.

Michael and others, what effect would you expect glutaraldehyde to have on
DNA crystals?

Patrick


On 19 June 2017 at 15:20, Joseph Ho  wrote:

> Dear all:
>
> I would like to seek your opinion on our crystal hits. We are working
> on protein/dsDNA complex. By changing different protein and DNA
> (14-22bp) constructs, we recently got some hits from commercial
> screens using sitting drop vapor diffusion (very small xtals). The
> precipitant is PEG and the picture of crystals are attached. In this
> particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
> 100mM NaCl. The crystal seems floating and sit in the bottom. We do
> some test shot from other conditions and it is not salt crystals. The
> crystals can suck in izit dye.  I do some google and it seems izit dye
> also turns dsDNA crystal into blue. We also do UV/Vis microscope but
> no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.
>
> This is our first time to work on protein/DNA complex crystals and we
> are not certain if this is just DNA or protein/DNA crystals. Can you
> provide your comments on our hits?
>
> Thank you for your help
>
> Joseph
>



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Re: [ccp4bb] Weak density of RNA in a complex structure

[Nicolas FOOS]

Dear Chen,

I will answer with some question :

- How is the refinement going ? Is the RNA properly taking in count ? 
Which soft do you use ?


- How do you solve the structure ? MR ? If it's MR did you use a model 
which contain both protein and RNA ? Did you try to solve with the 
protein only ? Maybe you are "forging" RNA.


- Depending of your data (wavelenght and redundancy) you can try to 
calculate an anomalous difference map to see the phosphorus of the RNA 
to be more confident.


- Last point, it could be the occupancy connected with the stability of 
the complex, maybe sometimes RNA is here sometimes not. I see that one 
time with two complex in the same ASU, one has one missing partner.


For my side, I really like to refine my DNA-protein complex with Buster 
from global Phasing. It gives you a very nice and informative density 
map. If you have this possibility let's try.


Hope to help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 21/06/2017 09:34, Chen WeiFei wrote:


Dear All,


We have get a complex crystal and the resolution can be refined to 
nearly 1.84 Å. But the electron density of the RNA is very weak.


In some datasets we can't find any density of the RNA and in other 
datasets we can see more or less some RNA density.


For now we can build 6-7 nucleotides but we can't distinguish the 
rigth sequence.


If anyone has the same problem and how to solve this problem.

Best Regards,


Dr Wei-Fei Chen

College of Life Sciences,

Northwest A University

[ccp4bb] Weak density of RNA in a complex structure

[Chen WeiFei]

Dear All,


We have get a complex crystal and the resolution can be refined to nearly 1.84 
Å. But the electron density of the RNA is very weak.

In some datasets we can't find any density of the RNA and in other datasets we 
can see more or less some RNA density.

For now we can build 6-7 nucleotides but we can't distinguish the rigth 
sequence.

If anyone has the same problem and how to solve this problem.

Best Regards,


Dr Wei-Fei Chen

College of Life Sciences,

Northwest A University