[ccp4bb] dryshipper, MVE vs. CX100?

[Guenter Fritz]

Dear all,

we  have to "update" our dryshippers.

There are two models we are consifering:  the CX100 from Tayler Wharton 
or the MVE SC4/2. Has anybody experience with the dryshipper from MVE?


Thanks a lot in adavance for any insights!

Best wishes, Guenter



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Re: [ccp4bb] Structure prediction - waiting to happen

[Guenter Fritz]

Dear all,

taking AlphaFold models for " true" experimental structures seems to 
become a serious problem.
I am just returning from a meeting (not a structural biology meeting) 
and saw one model after other. And the non-structural biologist used 
terms like "we calculated a structure"  or "a AlphaFold crystal 
structure" or "the structure was accurate, is was all blue".
Alphafold models were used to predict ion channels, electron transfer 
pathways, enzyme mechanisms, and yes, not tested by experiments. Wide 
ranging conclusions were drawn on these pure models, which I would not 
dare to draw on limited experimental data.

There is something going severely wrong.
And don't get me wrong, I think AlphaFold and other prediction software 
is great to create testable models (like MR models) or try to figure out 
how proteins might assemble and so on.
But I get the impression that too many of our colleagues got the 
impression that pressing a button replaces experiments.
Seeing that grants are rejected by such arguments is alarming and we 
should do something.


Best wishes,
Guenter

Very sorry to hear about your grant. I've been there. It is crushing 
to be rejected, and frustrating when the reason given is ... wrong.


My journalist friends wonder why scientists don't like talking to 
journalists. This is why.  I remember when the first results from 
XFELs were published, and it was immediately declared that there was 
no longer a need for NASA, whose sole purpose (apparently) was to grow 
bigger and better crystals in space. (?!) I find the idea that 
AlphaFold has eliminated the need to solve any more structures equally 
ludicrous.


I think the best analogy for what has happened in structural biology 
is the same impact a Star Trek style "transporter device" would have 
on your daily commute. Except this "transporter" is only accurate 
enough to get you within a mile or two of your house.  Most of the 
time. Don't worry, its not going to beam you inside a rock or into the 
sky, as it was trained on data with good Clashscores (we think). But, 
you are on your own getting the rest of the way home. This "Last Mile" 
of transportation networks is actually the most challenging, and 
expensive, but also the most critical. In structural biology, the 
"Last Angstrom" between prediction and actuality is equally important, 
but also fraught with difficulty. It may seem like a short distance, 
until you have to walk it. So, despite amazing progress, it is still 
premature to dismantle infrastructure, and definitely a bad idea to 
nail your front door shut.


Personally, I see this structure prediction revolution as nothing more 
nor less than the fruition of Structural Genomics. It started in the 
final days of the 20th century. I was there! The stated goal of that 
worldwide initiative was to create the data set that would be needed 
by some future (at the time) homology modelling technology to do 
exactly what AlphaFold does: get us "close enough".  And then Greg 
Petsko asked: what is "close enough"?  He called it "The Grail 
problem". By what metric do you declare victory?  He made an excellent 
suggestion:


"But there is an obvious method of evaluation that will allow any 
structure prediction method to be assessed. It is simply to demand 
that the method produce a model that can be used to solve the 
corresponding protein crystal structure by the method of molecular 
replacement."

-Greg Petsko - June 9, 2000
https://doi.org/10.1186/gb-2000-1-1-comment002

This is the thing that just changed.  Structure prediction has finally 
crossed the "G-P threshold". Not 100% of the time, but impressively 
often now, the predictions can be used for MR. This is a massively 
useful tool!  Not the end of the field, but rather the beginning of an 
exciting new era where success rates skyrocket.


Scores like the GDT used in CASP were developed with this Grail 
Problem criterion in mind, and I think that is what John Moult and 
others meant when they said things that got quoted like this:
"Scores above 90 on the 100-point scale are considered on par with 
experimental methods, Moult says."

https://www.science.org/doi/full/10.1126/science.370.6521.1144

Meaning that the predicted models work as search models for MR about 
as often as search models derived from homologous (and yes, 
"experimentally determined") structures.  A GDT of 100 does NOT mean 
the model is better than the data. That is not even how it works.


But, unfortunately, this seems to have gotten paraphrased and 
sensationalized:


"generally considered to be competitive with the same results obtained 
via experimental methods"

https://www.sciencealert.com/ai-solves-50-year-old-biology-grand-challenge-decades-before-experts-predicted

"software predictions finally match structures calculated from 
experimental data"

https://www.science.org/doi/full/10.1126/science.370.6521.1144

"comparable in quality to experimental structures"

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] freezing in ethane / propane - unipucks ?

[Guenter Fritz]

Hi Steve,


Hi ,
1) If you are collecting the data at the APS  the samples frozen in propane can 
be legally shipped there (check APS web site for information)
2) The samples CANNOT be mounted using either the ACTOR or ALS Style auto 
mounter. But the samples can be shipped using either the Rigaku pucks or the 
Uni-pucks with a vial cover adapter. In any case you would need to manually 
mount and collect data or have the samples retrieved after the propane melted 
with the robotic mounter and mounted later by the robot for data collection.

I am in Europe, so I would go for ESRF / SLS.


 From my extensive experience quenching samples using propane it has been 
possible to freeze ALL in LN2 after a) perfecting cryoprotectant, b) perfecting 
freezing technique, c) modifying crystal growth conditions.


:)) yes better crystals is always the best solution. But we are happy 
that we have crystals at all after some efforts (insect cells, low 
solubility, crystallization in the glove box, )


Cheers Guenter



Steve Ginell



On Jan 26, 2022, at 1:31 PM, Joseph Ferrara  wrote:

When using Xe derivatization for SAD phasing, we (mostly Jim Pflugrath) used 
carbon tetrafluoride. CF4 has a melting point of 89.5K, just four degrees 
warmer than propane and is not flammable.

Joe Ferrara

-Original Message-
From: CCP4 bulletin board  On Behalf Of James Holton
Sent: Wednesday, January 26, 2022 13:07
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] freezing in ethane / propane - unipucks ?

I think the issue with propane at at least some light sources is that it is 
flammable.  Makes shipping and safety more complicated.

How hard would it be to let the propane melt off in a cryo gas stream at your 
home lab?  Then use tongs to transfer the pin into liquid N2 for handling as 
usual?  If you don't have a working N2 gas stream and are on a budget they are 
not that hard to build:
https://doi.org/10.1107/S0021889894006357

Cheers,

-James Holton
MAD Scientist


On 1/26/2022 9:25 AM, Guenter Fritz wrote:

Dear Dom,

thanks a lot. Yes, this might work sending a combipuck alongside with
a good bottle for the local contact.
I was wondering whether the grippers can handle a block of propane?

Best wishes,
guenter

Dear Guenter,

Would the use of vials inside combi-pucks
(https://www.mitegen.com/product/combipuck-system/) and some
arrangements with your local contact at the other end, perhaps help
with using propane remotely?

BW,

D

On 26/01/2022 16:53, Guenter Fritz wrote:

Dear all,

we have some  delicate crystals which might benefit from freezing in
propane. In former times (when I was still travelling to the
beamlines) we waited until the propane was solid in the vial and
then let the propane thaw in the cryo stream at the beamline.

But how can we  do this in these days with unipucks and no manual
mounting?

I was thinking about freezing in ethane and then transfer the loops
to liquid nitrogen, similarly we handle grids for cryo EM. Does
somebody has tried that? Any experience, tips & tricks would be very
welcome! Thanks in advance and best regards,

Guenter





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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] freezing in ethane / propane - unipucks ?

[Guenter Fritz]

Hi Joe,

thanks. I have never used CF4. Good point and worth a try.
Best,
Guenter

When using Xe derivatization for SAD phasing, we (mostly Jim Pflugrath) used 
carbon tetrafluoride. CF4 has a melting point of 89.5K, just four degrees 
warmer than propane and is not flammable.

Joe Ferrara

-Original Message-
From: CCP4 bulletin board  On Behalf Of James Holton
Sent: Wednesday, January 26, 2022 13:07
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] freezing in ethane / propane - unipucks ?

I think the issue with propane at at least some light sources is that it is 
flammable.  Makes shipping and safety more complicated.

How hard would it be to let the propane melt off in a cryo gas stream at your 
home lab?  Then use tongs to transfer the pin into liquid N2 for handling as 
usual?  If you don't have a working N2 gas stream and are on a budget they are 
not that hard to build:
https://doi.org/10.1107/S0021889894006357

Cheers,

-James Holton
MAD Scientist


On 1/26/2022 9:25 AM, Guenter Fritz wrote:

Dear Dom,

thanks a lot. Yes, this might work sending a combipuck alongside with
a good bottle for the local contact.
I was wondering whether the grippers can handle a block of propane?

Best wishes,
guenter

Dear Guenter,

Would the use of vials inside combi-pucks
(https://www.mitegen.com/product/combipuck-system/) and some
arrangements with your local contact at the other end, perhaps help
with using propane remotely?

BW,

D

On 26/01/2022 16:53, Guenter Fritz wrote:

Dear all,

we have some  delicate crystals which might benefit from freezing in
propane. In former times (when I was still travelling to the
beamlines) we waited until the propane was solid in the vial and
then let the propane thaw in the cryo stream at the beamline.

But how can we  do this in these days with unipucks and no manual
mounting?

I was thinking about freezing in ethane and then transfer the loops
to liquid nitrogen, similarly we handle grids for cryo EM. Does
somebody has tried that? Any experience, tips & tricks would be very
welcome! Thanks in advance and best regards,

Guenter





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Re: [ccp4bb] freezing in ethane / propane - unipucks ?

[Guenter Fritz]

Hi James,

I think the issue with propane at at least some light sources is that 
it is flammable.  Makes shipping and safety more complicated.


How hard would it be to let the propane melt off in a cryo gas stream 
at your home lab?  Then use tongs to transfer the pin into liquid N2 
for handling as usual?  If you don't have a working N2 gas stream and 
are on a budget they are not that hard to build:

https://doi.org/10.1107/S0021889894006357

Yes, that would be straightforward,  but was looking for a kind of 
shortcut :).


Cheers Guenter


Cheers,

-James Holton
MAD Scientist


On 1/26/2022 9:25 AM, Guenter Fritz wrote:

Dear Dom,

thanks a lot. Yes, this might work sending a combipuck alongside with 
a good bottle for the local contact.

I was wondering whether the grippers can handle a block of propane?

Best wishes,
guenter

Dear Guenter,

Would the use of vials inside combi-pucks 
(https://www.mitegen.com/product/combipuck-system/) and some 
arrangements with your local contact at the other end, perhaps help 
with using propane remotely?


BW,

D

On 26/01/2022 16:53, Guenter Fritz wrote:

Dear all,

we have some  delicate crystals which might benefit from freezing 
in propane. In former times (when I was still travelling to the 
beamlines) we waited until the propane was solid in the vial and 
then let the propane thaw in the cryo stream at the beamline.


But how can we  do this in these days with unipucks and no manual 
mounting?


I was thinking about freezing in ethane and then transfer the loops 
to liquid nitrogen, similarly we handle grids for cryo EM. Does 
somebody has tried that? Any experience, tips & tricks would be 
very welcome! Thanks in advance and best regards,


Guenter

 



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Re: [ccp4bb] freezing in ethane / propane - unipucks ?

[Guenter Fritz]

Dear Dom,

thanks a lot. Yes, this might work sending a combipuck alongside with a 
good bottle for the local contact.

I was wondering whether the grippers can handle a block of propane?

Best wishes,
guenter

Dear Guenter,

Would the use of vials inside combi-pucks 
(https://www.mitegen.com/product/combipuck-system/) and some 
arrangements with your local contact at the other end, perhaps help 
with using propane remotely?


BW,

D

On 26/01/2022 16:53, Guenter Fritz wrote:

Dear all,

we have some  delicate crystals which might benefit from freezing in 
propane. In former times (when I was still travelling to the 
beamlines) we waited until the propane was solid in the vial and then 
let the propane thaw in the cryo stream at the beamline.


But how can we  do this in these days with unipucks and no manual 
mounting?


I was thinking about freezing in ethane and then transfer the loops 
to liquid nitrogen, similarly we handle grids for cryo EM. Does 
somebody has tried that? Any experience, tips &  tricks would be very 
welcome! Thanks in advance and best regards,


Guenter



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[ccp4bb] freezing in ethane / propane - unipucks ?

[Guenter Fritz]

Dear all,

we have some  delicate crystals which might benefit from freezing in 
propane. In former times (when I was still travelling to the beamlines) 
we waited until the propane was solid in the vial and then let the 
propane thaw in the cryo stream at the beamline.


But how can we  do this in these days with unipucks and no manual mounting?

I was thinking about freezing in ethane and then transfer the loops to 
liquid nitrogen, similarly we handle grids for cryo EM. Does somebody 
has tried that? Any experience, tips &  tricks would be very welcome! 
Thanks in advance and best regards,


Guenter



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Re: [ccp4bb] update 7.1.016, very poor pyrogen restraints

[Guenter Fritz]

Hi Paul,

thanks a lot.

Yes, if refmac does not recognize the restrains properly, that would 
explain the funny behaviour. I will check the cif with a validator and 
compare the cifs from pyrogen and acedrg.


Hope you will have a working computer soon!
Best,
Guenter


On 08/11/2021 08:55, Guenter Fritz wrote:

Dear all,

with ccp4 update 7.1.016 pyrogen was updated from pyrogen-0.0-pre 
revision 10365 to pyrogen-0.0-pre revision 10625. I saw that the 
--MMFF (usage of Merck forcefield) option is now missing.


Yes, it's the default now (i.e. without mogul).

I get restraints from the latest version of pyrogen, which do not 
work in refmac. I added a screenshot of an refmac output  opened in 
coot below. Restraints were generated from smiles. I had also some 
trouble with some aromates in the previous version but not crumpled 
molecules like now.


My guess from your figure is that Refmac somehow failed to read the 
cif output of pyrogen and tried its best to make some bond restraints. 
Coot however *can* read the ouptput of pyrogen, which is why you get a 
nice chemical diagram. It might be worth your while to peruse the 
output of refmac and/or try to parse the output

from pyrogen in a mmCIF validator.

I am a bit hamstrung with helping you at the moment because my main 
computer has died and the backup laptop doesn't have an up to date 
CCP4 distribution.


In the meantime you might like to try Acedrg:

$ acedrg -i lig.smi

It's a bit slower but will give you more accurate bond lengths and 
angles (and an mmCIF file that Refmac can read).


Paul.





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[ccp4bb] update 7.1.016, very poor pyrogen restraints

[Guenter Fritz]

Dear all,

with ccp4 update 7.1.016 pyrogen was updated from pyrogen-0.0-pre 
revision 10365 to pyrogen-0.0-pre revision 10625. I saw that the --MMFF 
(usage of Merck forcefield) option is now missing.


I get restraints from the latest version of pyrogen, which do not work 
in refmac. I added a screenshot of an refmac output  opened in coot 
below. Restraints were generated from smiles. I had also some trouble 
with some aromates in the previous version but not crumpled molecules 
like now.


Please note, I have no access here to Mogul, so I am running pyrogen 
without Mogul. Might be that everything is fine if pyrogen is running 
with Mogul.


Has anybody else observed something similar? Any insights?

Thanks and best regards,

Guenter




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Re: [ccp4bb] software DS, fconv still available ? contact?

[Guenter Fritz]

Dear Manfred,

thanks a lot. I had checked the homepage before. There is a note that 
the software is not any more available  for download from their site. 
But no further hint whether it is available somewhere else.


Best regards,
Guenter

https://agklebe.pharmazie.uni-marburg.de/

Cheers,
Manfred

Am 14.09.2021 um 14:36 schrieb Guenter Fritz:

Dear all,

does somebody know whom to contact for the software  DSX or fconv of
the former group Klebe in Marburg?

Many thanks and best regards,

Guenter



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--
Dr. Manfred S. Weiss
Macromolecular Crystallography
Helmholtz-Zentrum Berlin
Albert-Einstein-Str. 15
D-12489 Berlin
Germany




Helmholtz-Zentrum Berlin für Materialien und Energie GmbH

Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher 
Forschungszentren e.V.


Aufsichtsrat: Vorsitzender Dr. Volkmar Dietz, stv. Vorsitzende Dr. 
Jutta Koch-Unterseher
Geschäftsführung: Prof. Dr. Jan Lüning (Sprecher), Prof. Dr. Bernd 
Rech, Thomas Frederking


Sitz Berlin, AG Charlottenburg, 89 HRB 5583

Postadresse:
Hahn-Meitner-Platz 1
14109 Berlin
Deutschland




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[ccp4bb] software DS, fconv still available ? contact?

[Guenter Fritz]

Dear all,

does somebody know whom to contact for the software  DSX or fconv of the 
former group Klebe in Marburg?


Many thanks and best regards,

Guenter



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[ccp4bb] EDSTATS

[Guenter Fritz]

Dear all, dear Ian,

I am trying to run edstats from command line. I have read the previous 
threads here and I was using the edstats.pl perl script. However I fail.


Here is what I type and what I get (same on a centos machine and my mac).


$ edstats.pl -hklin paefs_018_dimple_refmac.mtz -pdbin 
paefs_018_dimple_refmac.pdb -flabel F -log test1.log  -pdbout test1.pdb


WARNING: Experiment type is not defined, assuming 'X-RAY DIFFRACTION'

 OPENED INPUT MTZ FILE
 Logical Name: HKLIN   Filename: paefs_018_dimple_refmac.mtz

LABELS H K L FreeR_flag F SIGF FC PHIC FC_ALL PHIC_ALL FWT PHWT DELFWT 
PHDELWT FOM FC_ALL_LS PHIC_ALL_LS

TYPES  H H H I F Q F P F P F P F P W F P
XDATA   49.162   60.306  102.333  90.000  90.000 90.000   19.942   
1.200    19  1.0

 mtzfix:  WARNING: Output file is corrupt so deleted.

ERROR in mtzfix: stopping.

That's all I get, no log file.

Any insights are appreciated :).

Best wishes,

Guenter



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Re: [ccp4bb] Domain Motion Analysis

[Guenter Fritz]

Dear Antony,

similar to the approach Eleanor has described, lsqman will give you 
rotational matrix and translation vector.


http://xray.bmc.uu.se/usf/lsqman_man.html

Best regards, Guenter

Hi,

You can try elNemo server.


Regards,
Nasrin
PhD scholar




On Wed, Jul 8, 2020, 10:16 PM Antony Oliver 
mailto:antony.oli...@sussex.ac.uk>> wrote:


Dear CCP4bb

Is anyone kindly able to point me at a server or recommend
software that might help me analyse relative domain movements – by
comparison of two structures?

Structure 1 has molecules A and B.

Structure 2 has molecules A, B and C.

I am interested in the induced movements, and a description
thereof, that the presence of molecule C produces in molecules A
and B.

I am aware of DynDom – but I can’t seem to make it produce a
readily interpretable output (undoubtedly my fault).

Many thanks for your help in advance.

Antony.

*Antony W Oliver*FHEA, PhD
Faculty Senior Research Fellow

Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ









(office): +44 (0)1273 678349
(lab): +44 (0)1273 677512

*antony.oli...@sussex.ac.uk 
*https://www.sussex.ac.uk/lifesci/oliverlab
https://tinyurl.com/aw-oliver
https://orcid.org/-0002-2912-8273

http://www.sussex.ac.uk/lifesci/internal/staff/support


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Re: [ccp4bb] somewhat off-topic :)

[Guenter Fritz]

Dear Artem,

I am a fan of the former "Superformance" column from Merck. The 
production / selling  of these columns is sourced out to a small company 
called Goetec. The columns are not really cheap, but good quality, last 
forever, suited also for higher pressures, only glas and Teflon in 
contact with your sample. Packing is easy.

I just had a look at their website, https://www.goetec-labortechnik.de
unfortunately only in German and scarce info.
Here is a link to a pdf with the now so-called Supercompact system.
https://cdn.website-editor.net/844eacb718df4813ad43c226fccdb915/files/uploaded/Artikelliste_SuperCompact_de.pdf

Best, Guenter

CCP4 friends,

Sorry for the somewhat off-topic post!

For many years I've been a fan of the Pharmacia XK columns and heavily 
relied on them for the bulk of my work. Lately, however, GE has 
increased the prices so much that I am reluctant to buy new columns 
from them. And I don't mean filled columns - packing columns is a 
relatively trivial process if you know how to do it right.


So - before I spend a day searching and making calls - do you have an 
alternative suggestion that's more reasonable? Clearly the fittings 
etc. may need to be adapted, but that's not a big deal. I was already 
looking at Bio-Rad and found them almost as expensive, so I am 
actually looking for some sort of 'knockoff' manufacturer :)


Thank you

Artem

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[ccp4bb] dimple, skip pointless?

[Guenter Fritz]

Dear all,

I was going through the options in dimple. Is it possible to skip the 
checking of the space group by pointless?


Thanks in advance and best regards,

Guenter



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[ccp4bb] 2 Postdoc positions available

[Guenter Fritz]

On behalf of Huib Ovaa (h.o...@lumc.nl):

We are looking for postdoc candidates with a background in protein 
chemistry/ structural biology.


There are two projects: One project on MHC-T-cell interactions, and 
diagnostics/ immunotherapy using novel sets of protein reagents that we 
are developing.


The other project is on making Ub system probe molecules for various 
targets, requiring assay development, structures in a collaborative 
project with the screening and medicinal chemistry teams in the Ovaa lab 
(http://www.ovaalab.nl )


The candidates should have a strong background in expression and 
purification of proteins and structural biology / X-ray crystallography.


Please direct applications to Huib Ovaa:

Prof. Dr. Huib Ovaa │group leader chemical biology and small molecular 
screening │Oncode Institute & Leiden University Medical Center (LUMC) 
│Department of Cell and Chemical Biology │location T02.03 │


Einthovenweg 20 │2333 ZC Leiden, the Netherlands │email: h.o...@lumc.nl 
 │phone +31 71 5268721 │website lab 
http://www.ovaalab.nl  │website department 
https://ccb.lumc.nl/








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Re: [ccp4bb] Unusual monomer-monomer interface in crystal

[Guenter Fritz]

Dear Chris, are there any metal ions in your buffer or in your protein. 
We had a similar looking case. A Zn2+ ion bridged two monomers. Our 
protein is a Zn2+ binding protein. The Zn2+ originated from some 
denatured protein in the drop. No extra Zn2+ was in the crystallization 
buffer.


http://www.rcsb.org/structure/5CHT
https://www.nature.com/articles/nsmb.3371
HTH
Guenter

Dear CCP4BB Users,

I've recently solved the ~2.2 angstrom structure of a protein. In my 
electron density there are unusual monomer-monomer interfaces 
involving pairs of His and Cys residues (see https://ibb.co/wdWBcdk). 
Note the positive Fo-Fc density between the four side chains. As there 
is not adequate space for a water molecule or metal ion, perhaps the 
Cys residues are partially tied up disulfide bonds? However, the 
protein looks to be fully monomeric based on LC-MS measurements. Has 
anyone else observed crystal-driven formation of disulfide bridges?


Aside from this region, there is no extensive interface between 
momoners, and PDBePISA suggests a monomeric state.


Thanks in advance for any advice!

Best wishes,
Chris



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[ccp4bb] Summary: [ccp4bb] off topic: microscope in glove box

[Guenter Fritz]

Dear Artem, Marta, Arwen, Robin,

thank you all for the detailed info. We had a try today and it worked 
smoothly.

Thanks again and best regards,
Guenter
Short version: It should be OK, especially since the vacuum is 
transient and not particularly 'strong*' :)


Long version: if this is an older microscope there may be further 
delamination of optically bonded components if air is already admitted 
between glass planes (i.e. the optical cement is worn and old). Also 
some of the fancier models may have pneumatic balance elements for 
gross motion of the optical column - those may experience pressure 
differentials above their maximum tolerances. Finally, and very 
unlikely you may have a situation where multiple optical elements are 
sealed together in a single tube with air trapped between them - if 
the 'vent' is blocked (by e.g. old grease or something) then these may 
pop.


https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/ 



But the short version is right 99% of the time.

Artem

*"Professor Hubert Farnsworth 
<https://www.imdb.com/name/nm0921942/?ref_=tt_ch>: Well, it's a space 
ship, so I'd say anywhere between zero and one."

https://www.imdb.com/title/tt0584455/characters/nm0921942

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On Mon, Oct 21, 2019 at 10:00 AM Guenter Fritz 
<mailto:guenter.fritz.phenix.c...@gmail.com>> wrote:


Dear all,

I want to put one of our microscopes into the glove box. Does anybody
know whether some parts of the microscope optics do not like
vacuum in
the air lock ?

Thanks and best regards, Guenter



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[ccp4bb] off topic: microscope in glove box

[Guenter Fritz]

Dear all,

I want to put one of our microscopes into the glove box. Does anybody 
know whether some parts of the microscope optics do not like vacuum in 
the air lock ?


Thanks and best regards, Guenter



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Re: [ccp4bb] different residues as alternate occupancy?

[Guenter Fritz]

Dear all,
thank you all for feedback help.
Best wishes!
Guenter


Dear Guenter, yes look at crambin (pdbcode 1EJG).


This crashes Coot until you add*

  allow_duplicate_sequence_numbers()

to $HOME/.coot.py in OSX or the appropriate place on Windows. For
Windows, as there is no $HOME, Coot uses .coot.py or .coot-preferences/
directory for configuration - these can be found (added to) the
directory in which Coot was installed (e.g. C:\WinCoot).

To resolve your problem look at

https://www.phenix-online.org/newsletter/CCN_2015_07.pdf#page=12

example 5

Br, Georg.


Am 2019-06-24 um 4:22 PM schrieb Savvas Savvides:
The structure of milk proteins obtained from in vivo-grown crystals 
from a viviparous cockroach could also serve as an interesting case:


http://dx.doi.org/10.1107/S2052252516008903

best wishes
Savvas


On 24 Jun 2019, at 16:03, Holton, James M 
<270165b9f4cf-dmarc-requ...@jiscmail.ac.uk 
<mailto:270165b9f4cf-dmarc-requ...@jiscmail.ac.uk>> wrote:


A classic case of this is crambin. Residue 25 of 3nir.

-James Holton
MAD Scientist

On 6/24/2019 1:00 AM, Guenter Fritz wrote:

Dear all,

I am refining a multimer and mass spec data of the sample indicate
that there is a mixture of two variants which differ in one amino acid
residue. The density that we see is therefore most likely an average
of both variants. I have created a pdb file with "alternate residues"
each with 0.5 occupancy at this position to use it in refinement.
However, the programs detect this as an error in the pdb file.

Has anyone  faced such a problem previously? Any suggestions are very
much appreciated.

Thanks a lot and best regards, Guenter



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[ccp4bb] different residues as alternate occupancy?

[Guenter Fritz]

Dear all,

I am refining a multimer and mass spec data of the sample indicate that 
there is a mixture of two variants which differ in one amino acid 
residue. The density that we see is therefore most likely an average of 
both variants. I have created a pdb file with "alternate residues" each 
with 0.5 occupancy at this position to use it in refinement.  However, 
the programs detect this as an error in the pdb file.


Has anyone  faced such a problem previously? Any suggestions are very 
much appreciated.


Thanks a lot and best regards, Guenter



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Re: [ccp4bb] OT: Nvidia 3D vision 2 glasses with Ubuntu workstation

[Guenter Fritz]

Dear Adarsh,

just an add-on to the instructions of the others. Since GNOME is not 
anymore compatible with stereo, we use successfully Xubuntu (18.04) with 
NVIDIA drivers.

Best , Guenter

Hello everyone

We have just purchased a Dell workstation for crystallography data analysis. We 
were trying to use Nvidia 3D vision 2 glasses with it, but failed to do so. 
Please help me out with this one. Some relevant information is as follows:
OS: Ubuntu 16.04 LTS
Graphics : Quadro P4000 (unfortunately doesn't have a 3-pin DIN socket)
Monitor: Asus VG248QE

Thanks and regards
Adarsh Kumar
Suo Lab
Florida State University College of Medicine



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[ccp4bb] Refmac jelly body parameters

[Guenter Fritz]

Dear Refmac developers,

I am trying to reproduce some successful refmac runs but cannot get the 
previous results despite playing around with the parameters.


I did those runs in Feb 2014 (Refmac 5.8.0049) but unfortunately have 
overwritten the log files in a backup. I still have the pdbs, mtz and my 
notes.


I was using jelly body and Prosmart at a resolution of 3.1 A,  which 
worked superb and I could track some unexpected conformational changes. 
In several successive runs each with 100 cycles Rwork and Rfree were 
decreasing slowly and continuously and finally showed two rigid body 
movements.


When I now repeat those runs (same pdb, same mtz input files and using 
the prosmart restraint file from the former runs) I get very similar 
Rwork/Refree but not the rigid body movements of these domains again in 
the resulting pdb.


If I loosen the restraints in jelly body  Rwork decreases but Rfree 
stays, indicating some overfitting. And also in the resulting pdbs the 
previously observbed conformational change is not observed. I tried also 
to increase the number of cycles to some insane numbers (>600), but this 
did not improve the situation.


Any ideas what might go wrong? I am really fond of jelly body and would 
like now to check a series of new data sets at low resolution for the 
extent of the observed (and verified) conformational change.


Best regards,

Guenter







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Re: [ccp4bb] Acceptable range of CC1/2

[Guenter Fritz]

Dear Deepali,

the best way to define the resolution cut-off is to check whether the 
data still contribute to model quality. You can easily check that by the 
procedure called "paired refinement" as described by Diederichs and 
Karplus (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457925/  check 
Fig.1 and description).
You can prepare such a Figure for your refinement. This should convince 
every reviewer.

HTH
Guenter



Dear all,

I am trying to process a crystal data at *2.8Å* and having CC1/2 is 
0.771 (outer shell). I just want to know the acceptable range of 
CC1/2. These are the statistics of the process data.


Overall InnerShell OuterShell
Low resolution limit 74.67 74.67 2.95
High resolution limit 2.80 8.85 2.80
Rmerge 0.067 0.042 0.964
Rmerge in top intensity bin 0.043 - -
Rmeas (within I+/I-) 0.072 0.046 1.032
Rmeas (all I+ & I-) 0.072 0.046 1.032
Rpim (within I+/I-) 0.026 0.018 0.368
Rpim (all I+ & I-) 0.026 0.018 0.368
Fractional partial bias -0.025 -0.019 -0.071
Total number of observations 289022 8892 42020
Total number unique 37156 1218 5376
Mean((I)/sd(I)) 16.5 39.0 2.1
Mn(I) half-set correlation CC(1/2) 0.999 0.998 0.771
Completeness 99.3 98.7 99.0
Multiplicity 7.8 7.3 7.8
Anomalous completeness 99.3 98.5 98.9
Anomalous multiplicity 4.0 3.9 4.0
DelAnom correlation between half-sets -0.042 -0.036 0.015
Mid-Slope of Anom Normal Probability 0.937 - -

--
*Regards,*
*Deepali Verma*
*Junior Research Fellow*
*Jaypee Institute of Information Technology*




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[ccp4bb] Postdoctoral position available

[Guenter Fritz]

Dear all,

a postdoctoral position is available in a joint project between my group 
and the group of Julia Fritz-Steuber. The position is at the University 
of Stuttgart Hohenheim, where I have moved recently. The position is 
available immediately with preferred starting date 1.1.2018.


The project is on a membrane protein complex and its maturing factors 
(https://www.nature.com/articles/nature14003). A successful candidate is 
experienced in (membrane) protein biochemistry, biochemistry of oxygen 
sensitive proteins (work with a glove box) and X-ray crystallography.


Applications should be send to guenter.fr...@uni-hohenheim.de.

Please contact me for any further information.

Best regards,

Guenter Fritz

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] Interesting DNA contamination

[Guenter Fritz]

Dear Stefan,
just saw this after reading post:
http://www.nature.com/nature/journal/v522/n7557/full/nature14559.html
Best, Guenter

Pramod,

You already got good suggestions on how to handle DNA contamination in protein 
preparations.

Let me point out briefly that you haven't demonstrated yet that your 
contamination is DNA.

I had the same observation when purifying UvsX. A very persistent and strong 
contamination in all my preps at ~500kb. To test weather it was DNA or RNA I 
boiled the protein 30 minutes and incubated it with DNAase and RNAse and result 
was the same. I concluded it was neither RNA nor DNA and continued as if 
nothing had happened.

This publication is reporting the same observation:

Formosa and Alberts (1986) Purification and characterization of the T4 
bacteriophage uvsX protein. J Biol Chem. 1986 May 5;261(13):6107-18.

If you ever find out what it is that runs like 500kb DNA on Agarose, please let 
me know.
S.

[ccp4bb] generating and applying transformation matrix

[Guenter Fritz]

Dear all,

I want to have a closer look at a conformational change in a multidomain 
protein. We see clear changes from different structures but there is 
evidence that the conformational changes might be larger than observed.
My idea is to get the transformation matrix of each residue (or of each 
atom) to go from conformation 1 to conformation 2. These matrices could 
be scaled to see where the domains might move.
So its a kind of morphing but extrapolating from a certain structural 
change instead of interpolating between the two structures.


Has anybody came across this before and found a quick solution? Any hint 
is appreciated.


Best regards, Guenter

Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

[Guenter Fritz]

Hi Stephen,

we crystallized several proteins in a glove box placed in a room with 
air condition. We checked the temperature all the times and found that 
is had been quite stable.


For one protein that crystallized only at 4 deg C, we setup the 
crystallization plate within the glove box and put it into an gas-tight 
plexiglass box (built by our workshop). The box with the crystallization 
plate was then incubated at 4degC.


Best, Guenter


Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

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[ccp4bb] Configuring updates through a firewall

[Guenter Fritz]

Hi,
I am using CCP4 on Sci Linux machines  and running now into some trouble 
with update manager and our firewall.

I connect through a proxy and defined the proxy in the ccp4-setup.sh.
Running update-manager gives me an error message; a new window pops up 
telling me Downloaded data is corrupt, try again.


The proxy is as well as defined in the  ./config/CCP4/ccp4pm.conf file 
produced by the package manager. However, I had a forced passwd change 
here  and  the usrid  Ppxu= and the passwd pxw= for the proxy are 
not given in clear text, so I cannot edit those.


 I also tried simply to run the new package manager, but I cannot get 
through the firewall.


Can somebody point me how to get updates once more  running through the 
proxy?
Or, can somebody give me a hint how to edit the ccp4pm.conf file? That 
could resolve the issue as well.


Thanks, Guenter

Re: [ccp4bb] Presence of Fe3+ or Fe2+

[Guenter Fritz]

Hi Monica,

what is the reducing agent the protein gets the electron from?
Or is it simply unspecific oxidation of protein side chains?

Do you know what are the coordinating residues of Fe3+ / Fe2+ ?

Dependent on the coordination Fe3+ has some (often very weak) d-d 
transitions (400-500 nm) which are absent in Fe2+.


Best method of choice to check whether you have Fe3+ or Fe2+ is EPR.

Godd luck!
Guenter




I am working on a protein which binds to its ligand in a particular 
state i.e. Fe3+ state (active) and reduces it to Fe2+ state 
(inactive). I am trying to set up crystallisation trials with the 
ligand but seems like difficult to get them. However i just needed an 
advice here, is there any way to know whether the protein is in Fe2+ 
state or Fe3+ state or how can i make the protein to exist in more of 
Fe3+ state so that i increase the probability of ligand binding to the 
protein.


Your advice is highly appreciated.
Thanks in advance !!

Cheers
Monica

[ccp4bb] Aquarius2 ?

[Guenter Fritz]

Dear colleagues,

can somebody give me a hint, whether the program AQUARIUS2 or a similar 
/ successor program is available somewhere?

Thanks a lot in advance!
Best regards, Guenter

Re: [ccp4bb] Aquarius2 ?

[Guenter Fritz]

Hi Tim,
thanks for pointing me to that.
 Guenter

Dear Guenter,

my ixquick search for 'aquarius2 program protein' had a hit at
www.kuhnlab.bmb.msu.edu/publication_papers/pdf/raymerjmb1997.pdf that
mentions aquarius2 and sounds as though their program  consolv
(www.kuhnlab.bmb.msu.edu) was a replacement.

Regards,
Tim

On 11/07/2014 06:47 PM, Guenter Fritz wrote:

Dear colleagues,

can somebody give me a hint, whether the program AQUARIUS2 or a similar
/ successor program is available somewhere?
Thanks a lot in advance!
Best regards, Guenter

Re: [ccp4bb] Glutathione detection

[Guenter Fritz]

Dear Avinash,
you can use Ellman's reagent (DTNB) for GSH.
Have a look here:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides
For glutathione you don't have to use a buffer with GuHCl.
HTH, Guenter


Dear All,

Greetings and apology for an off topic query. may request you all to 
kindly share with me a conventional protocol, by which i can calculate 
a total Glutathione content (oxidized + reduced) in an 
/in-vitro/ reaction set up.


all i find over internet are kit-based methods, which turns out to be 
quite expensive for couple of reactions i would like to do.


thanks in advance

kind regards
Avinash Kale

Re: [ccp4bb] PyMol and Schrodinger

[Guenter Fritz]

Hi,
if one is hesitant  to compile it it, pymol is easily installed on a 
Linux box  via yum ;


enable EPEL repo ( in Redhat derivatives e.g. fedora, Centos, SciLinux)
yum install pymol will install version 1.6 (latest version by 
Schrödinger is 1.7)

Best, Guenter


Hi,
I have inquired at Schrodinger about the licensing for PyMol. I was 
surprised by their answer. The access to PyMol is only through a 
yearly licence. They do not offer the option of purchasing the 
software and using the obtained version without time limitation. This 
policy is very different from many other software packages, which one 
can use without continuing licensing fees and additional fees are only 
when an upgrade is needed. At least I believe that Office, EndNote, 
Photoshop and others are distributed this way.
I also remember very vividly the Warren’s reason for developing PyMol, 
and that was the free access to the source code. He later implemented 
fees for downloading binary code specific for one’s operating system 
but there were no time restrictions on its use.
As far as I recollect, Schrodinger took over PyMol distribution and 
development promising to continue in the same spirit. Please correct 
me if I am wrong.
I find the constant yearly licensing policy disturbing and will be 
looking for alternatives. I would like to hear if you have had the 
same experience and what you think about the Schrodinger policy.

Best wishes,

Mirek

Re: [ccp4bb] How to find the unfound part of a big protein

[Guenter Fritz]

Dear Sun,

I had a similar problem. If you have a good TaBr dataset this should 
give you good phases.

You can combine the TaBr phases and Molrepl phases in SHARP.
What worked in my hands very well and is easy to do: SAD using Phaser 
and the partial Mol Repl Model.


You find here a input file for such a scenario:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Phenix#phenix.phaser_-_SAD_phasing_with_Phaser

Or simply use the CCP4 gui Phaser and  'SAD with molecular replacement 
partial structure'

After DM or parrot you might get some interpretable density even at 6.5 A.

If you don't have a good Mol Repl Model for the missing part you might 
try http://toolkit.tuebingen.mpg.de/hhpred

to look for structures that are not that closely related.

SeMet can help a lot in this case too. You will get the SeMet positions 
at low resolution with Phaser as described above.
The SeMet positions will guide you to place a model into a very crude 
density.


HTH,
Guenter



Dear Crystallographers,

I'm working on a ~90KDa membrane protein, with big extracellular part, 
probably function as dimer.
Now we have dataset to ~4.2 Angstrom and using extracellular homolog 
structure we can find a solution for this part(~45% of the whole 
molecule MW) through molecular replacement, and the molecules are 
packed as layers, and the other part are presumably between these 
layers. However, we are having trouble to fit the rest of the protein 
even though there're some density between the solved part. Rfree is at 
40% now.


We're trying to do heavy atom soaking, such as TaBr. We collected data 
for MIR but it's not helping so far. (Can I combine these MIR data 
with the native dataset because the MIR set is only at ~6.5 Angstrom)?


Other information: this protein is expressed in Sf9 cells (so very 
hard to do Se-Met derivatives). The crystals is nice and big and cubic.


Any suggestions or examples? Thanks a lot.

Bingfa

[ccp4bb] Conserved water positions in low resolution refinement?

[Guenter Fritz]

Dear all,
and especially all of you involved in the development of refinement 
programs,
in low resolution refinement we always include structural information we 
actually do not see in the electron density (side chains, riding 
hydrogens) since we know that these atoms are there. But what about 
water molecules?
The inner shell water molecules are often tightly bound and reside on 
conserved positions.
I made a simple test and repeated a couple of refinement runs (refmac 
using the high res structure as ref model) with  or without water 
molecules. Indeed most water molecules stay almost at the same position 
and R factors using an input file with water molecules are significantly 
lower.


Shall we use such information in the future in low resolution refinement?

And if we decide to do so, are there already good tools to evaluate and 
find conserved water positions maybe even by comparison of closely 
related structures?


Best regards,
Guenter

Re: [ccp4bb] RE : [ccp4bb] Conserved water positions in low resolution refinement?

[Guenter Fritz]

Hi Sacha,
thanks a lot. I hadn't expected that the effect of missing  water 
molecules would be so severe.


Best, Guenter

Dear Guenter,

please look at at Fig. 7. It numerically confirms some of your worries.

Best regards,

Sacha

De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Guenter Fritz 
[guenter.fr...@uni-konstanz.de]
Date d'envoi : samedi 2 novembre 2013 16:04
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] Conserved water positions in low resolution refinement?

Dear all,
and especially all of you involved in the development of refinement
programs,
in low resolution refinement we always include structural information we
actually do not see in the electron density (side chains, riding
hydrogens) since we know that these atoms are there. But what about
water molecules?

[ccp4bb] TLS refinement refmac

[Guenter Fritz]

Dear all,

one gets different R values, if you re-read in the mtz written out by 
refmac after TLS refinement. I think this issue had been a while ago in  
ccp4bb, but I can't find the right track.


Here are the details.

1st run:
If we do TLS + restr. refinement in refmac
we get:
InitialFinal
R factor0.3010   0.2170
R free0.3175   0.2695

2nd run:
Now, if we use the same input pdb and the same input tls paramter file, 
but use the mtz written out by the first run, we get:

InitialFinal
R factor0.2274   0.1903
R free0.2482   0.2540


Apparently in the mtz file written out by the 1st refmac must contain 
some information that is re-read in the 2nd run. But one just defines 
FP, SIGFP and Rfree flags. Do FPs change in the output mtz after TLS 
refinement??


Any help to clarify this is appreciated.

Thanks, Guenter

Re: [ccp4bb] TLS refinement refmac

[Guenter Fritz]

Am 17.07.2013 11:59, schrieb Guenter Fritz:

Hi Stefan and Gottfried,
thanks a lot for the answers. This is the point. Wouldn't it make more 
sense to add an extra column that contains the changed Fs?

Best, Guenter


Hi,

it is strongly advised to use the original mtz e.g. scala.mtz as the 
refmac input mtz in all refmac runs,
as this contains the original Fs - Refmac applies some aniso 
corrections  to the Fs and puts them into the output.mtz.


so the output Fs are not the same as in the input F - therefore one 
should use the scala.mtz


cheers
Stefan


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag 
von Guenter Fritz

Gesendet: Mittwoch, 17. Juli 2013 10:39
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] TLS refinement refmac

Dear all,

one gets different R values, if you re-read in the mtz written out by 
refmac after TLS refinement. I think this issue had been a while ago 
in ccp4bb, but I can't find the right track.


Here are the details.

1st run:
If we do TLS + restr. refinement in refmac we get:
InitialFinal
R factor0.3010   0.2170
R free0.3175   0.2695

2nd run:
Now, if we use the same input pdb and the same input tls paramter 
file, but use the mtz written out by the first run, we get:

InitialFinal
R factor0.2274   0.1903
R free0.2482   0.2540


Apparently in the mtz file written out by the 1st refmac must contain
some information that is re-read in the 2nd run. But one just defines
FP, SIGFP and Rfree flags. Do FPs change in the output mtz after TLS
refinement??

Any help to clarify this is appreciated.

Thanks, Guenter

Re: [ccp4bb] Disulphide bonds and closed conformation

[Guenter Fritz]

Hi Jan,

you will find some methods for detection of thiols and/or disulfides there:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides

HTH,
Guenter

Dear all,

I am working on a protein where I have to stabilize the closed 
conformation of the protein using disulphide bond. The strategy to 
design the cysteine mutants is based on the molecular dynamic 
simulations, and accordingly the residues were chosen. The ultimate 
goal is to trap the ligand in closed conformation of protein and 
crystallize it. I am facing few issues: Is there some reliable assay 
that can check the formation of disulphide bonds in protein.  
Additionally, does anybody knows another method(s) that can be used to 
trap a closed conformation. I look forward for your suggestions and 
discussions on this issue.


Thanks very much!

Jan

Re: [ccp4bb] solvent mask from one crystal form applied to the other second ...

[Guenter Fritz]

Hi,

have a look into the supplement of the paper by Poul Nissen  pointed out.

There are some shell scripts exactly for that purpose.
I had to adapt the scripts a bit, but the instructions are very well to 
follow.


HTH
Guenter


Hi Pete

Thanks for the reply.

As for the transformation, I was going to use the refined mask operator (either 
from the end of a DM run that produces a better map  for features I know have 
to be there, or from a correlation of the averaging mask from  MAVE).

The interpolation is going to be a little more difficult. The 'good' solvent 
mask output after a dmmulti run
has different gridding, extent, and cell than the mask output by the other 
crystal forms.


However, the documentation for dmmulti says that SOLin can be on any grid or 
axis ordering..  not quite sure about the extent.

Furthermore, it seems that Poul Nissen was able to put in a generic mask for 
all his crystal forms 
(http://journals.iucr.org/d/issues/2010/03/00/kw5018/index.html) without regard 
for  interpolation (unit cell, extent, or gridding) nor transformation. So I'm 
not exactly sure how I'm getting an 'inconsistent cell info'.

Maybe it's because I'm getting my masks from dm instead of generating it from a 
my averaging mask (which came from a pdb).. hmmm...

Anyone know of a wiki page that talks about maps/masks and a description of 
their properties (gridding, extent, basically everything discussed in the 
output of mapinfo?) A wiki with examples (images) of different settings for 
these items would be especially useful.



F


On Oct 24, 2011, at 9:40 AM, Pete Meyer wrote:


Hi,

It's definitely possible, but the devil is in the details.  It's a two-step 
process: transform the coordinates of the mask so it's in the right spot in the 
new cell, and interpolate the mask into the appropriate grid for the new 
crystal form.

The last time I did this I was using O/PHASES masks (so I'm probably off on the details 
for CCP4 stuff): maprot should be able to handle the transformation and interpolation, 
but you may have to use a mask -  map -  maprot -  mask pathway.  This isn't 
ideal due to differences in mask interpolation vs map interpolation but could be a good 
starting point.

Determining the transformation may be tricker - I'd try placing pseudo-atoms at 
similar features in both maps and using lsqkab to get the transformation 
matrix; but this approach would probably need tuning to get a good alignment.

Good luck,

Pete

Francis E Reyes wrote:

Hi all
I'm using dmmulti and one of my crystal forms seems to have a better solvent 
mask (after outputting it with solout) than the other. (Probably due to better 
phased reflections at low res for the first crystal form). Is it possible to 
take the better solvent mask and use it as input for the solvent mask for the 
other? How's this done? (a direct input of the better xtal1 mask to xtal 2 
gives 'inconsistent cell info'). Thanks!
F
-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] IUCr committees, depositing images

[Guenter Fritz]

On the technical feasibility of storage of original data:

Sure, running Pilatus for an olympic record, we will go home with several T of 
data after 24 h (will we?).
  

Yes, we do already. I just checked the number of images from PILATUS 6M
we have collected so far this year : ca. 1.7 millions. End of the year
it will be more than 2 mio.
I compress everything and store it on  RAID systems. Disk space is
meanwhile so cheap and storage on disk is so easy compared to tapes. So
why bothering with the large number of images?

In the end for the determination of the structure only a few datasets
will be necessary. This means maybe 0.5 Tb  of compressed data to
deposit. I don't think this is too much.
But this is an abuse of the system. The final goal is the structure determination, and there are much less 
good crystals everywhere in one year that  one Pilatus could collect in one week.

But to decide fast if the crystal diffraction data from Pilatus is good for 
storage or even for measurement whatever the speed of data collection is, good 
data processing
software is needed. 

The special properties of PILATUS forces you to think more about your
data collection AND (!) it gives you the time to think at the beamline
about data collection. Fine phi slicing and high redundancy gives much
better data (important if your crystals are not that good).  The people
from Dectris will maybe add a note here.

Best,
Guenter

I personnaly think that there is only one, the one.
Anyhow, I think if the author wish to publish his structure, and it is 
important, and I am a reviewer, and it is going
to prestigious journal,  I will reprocess his data and will check his way to 
the final crystal structure solution from the beginning.
It is as in mathematics. If someone claim that he solved a long-staing problem from the past, he will not go away from his envious colleagues, who 
will drop everything and will sit and check, until they will find a mistake. What a pleasure!!!

And if there are no mistakes - chapeau !!!

FF
 
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology

Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Oct 16, 2011, at 20:38 , Frank von Delft wrote:

  

On the deposition of raw data:

I recommend to the committee that before it convenes again, every member should 
go collect some data on a beamline with a Pilatus detector [feel free to join 
us at Diamond].  Because by the probable time any recommendations actually 
emerge, most beamlines will have one of those (or similar), we'll be generating 
more data than the LHC, and users will be happy just to have it integrated, 
never mind worry about its fate.

That's not an endorsement, btw, just an observation/prediction.

phx.




On 14/10/2011 23:56, Thomas C. Terwilliger wrote:


For those who have strong opinions on what data should be deposited...

The IUCR is just starting a serious discussion of this subject. Two
committees, the Data Deposition Working Group, led by John Helliwell,
and the Commission on Biological Macromolecules (chaired by Xiao-Dong Su)
are working on this.

Two key issues are (1) feasibility and importance of deposition of raw
images and (2) deposition of sufficient information to fully reproduce the
crystallographic analysis.

I am on both committees and would be happy to hear your ideas (off-list).
I am sure the other members of the committees would welcome your thoughts
as well.

-Tom T

Tom Terwilliger
terwilli...@lanl.gov


  

This is a follow up (or a digression) to James comparing test set to
missing reflections.  I also heard this issue mentioned before but was
always too lazy to actually pursue it.

So.

The role of the test set is to prevent overfitting.  Let's say I have
the final model and I monitored the Rfree every step of the way and can
conclude that there is no overfitting.  Should I do the final refinement
against complete dataset?

IMCO, I absolutely should.  The test set reflections contain
information, and the final model is actually biased towards the
working set.  Refining using all the data can only improve the accuracy
of the model, if only slightly.

The second question is practical.  Let's say I want to deposit the
results of the refinement against the full dataset as my final model.
Should I not report the Rfree and instead insert a remark explaining the
situation?  If I report the Rfree prior to the test set removal, it is
certain that every validation tool will report a mismatch.  It does not
seem that the PDB has a mechanism to deal with this.

Cheers,

Ed.



--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

  

Re: [ccp4bb] MOLREP fails when using input fixed model

[Guenter Fritz]

Dear Zhong Chen and CCP4 users,
I get the very same error on a Centos 5 box. Is there a solution yet?
Best regards,
Guenter

Dear all,
   Recently, I used MOLREP to molecular replacement.
 My OS is fedora 14 and CCP4 version is the newest one ccp4-6.2.0 .
 When I run a pdb file and mtz by MOLREP without input fixed model, everything is right. 
  However, when I run it by MOLREP when using input fixed model. 
 The error is attached below.

 At line 1260 of file /usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f
Fortran runtime error: End of record
 Actually, my ccp4 is installed at /home/software/ccp4, not at  
/usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f, which does not exist in my 
linux.
In addition, I tried several published structures and tried to test MOLREP  by 
input fixed model method .The error are same.
 Moreover, I tried to run it in windows XP   using input fixed model,  Molrep 
run well.
 
 I thought that it is a bug in this version of MOLREP.

 Any comments are welcome.
  with best wishes
zhong chen
--
zhongzhou chen 
Room 2071, research center in life sciences,

 No. 2 yuanmingyuan west road, Haidian District, Beijing, 100193  P.R. China
Tel: (86)-10-62734078



--
PD Dr. Günter Fritz
Fachbereich Biologie
Universität Konstanz

[ccp4bb] Solved: [ccp4bb] MOLREP fails when using input fixed model

[Guenter Fritz]

Sorry,
I should have had a look also at the CCP4 site:
updated version of molrep from Aug 8.
http://www.ccp4.ac.uk/updates/linux/ccp4-6.2.0/bin/
Cheers
Guenter

Dear Zhong Chen and CCP4 users,
I get the very same error on a Centos 5 box. Is there a solution yet?
Best regards,
Guenter

Dear all,
   Recently, I used MOLREP to molecular replacement.
 My OS is fedora 14 and CCP4 version is the newest one ccp4-6.2.0 .
 When I run a pdb file and mtz by MOLREP without input fixed model, 
everything is right.   However, when I run it by MOLREP when using 
input fixed model.  The error is attached below.

 At line 1260 of file /usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f
Fortran runtime error: End of record
 Actually, my ccp4 is installed at /home/software/ccp4, not at  
/usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f, which does not exist 
in my linux.
In addition, I tried several published structures and tried to test 
MOLREP  by input fixed model method .The error are same.
 Moreover, I tried to run it in windows XP   using input fixed 
model,  Molrep run well.
 
 I thought that it is a bug in this version of MOLREP.

 Any comments are welcome.
  with best wishes
zhong chen
--
zhongzhou chen Room 2071, research center in life sciences,
 No. 2 yuanmingyuan west road, Haidian District, Beijing, 100193  
P.R. China

Tel: (86)-10-62734078

Re: [ccp4bb] off-topic: LCD monitors

[Guenter Fritz]

Dear Padmaja,
I have use  Samsung 2233RZ for stereo running on  Linux and  Windows 
systems. Perfomance is great.

HTH
Guenter
Pardon the off-topic query, but I would like to get some feedback 
about any personal preference for 3D LCD monitors. I am trying to 
decide between the following 3 monitors:



  *Samsung 2233RZ* 1680 x 1050 2D and 3d Widescreen LCD Monitor


  Asus VG236H 23 2ms 1920x1080 Full HD 120Hz 3D multimedia Height 
  Swivel Adjustable WideScreen LCD


Alienware OptX AW2310 23 3D Full HD Widescreen Monitor

The monitor will display off of a Mac Pro Two Quad-Core Intel Xeon 
computer running both Apple and Linux OS.


Any input will be much appreciated.

Padmaja

Re: [ccp4bb] Full-screen stereo with Coot/PyMol/etc on 3D LCD displays?

[Guenter Fritz]

Hi Amir,
I have VMware  WindowsXP (guest) running on Centos5 (host) running.
Open GL - 3D does not work. VMware emulates the graphics board. VMware 
6.5 supports only Direct X.

I don't know whether a newer version will do; I doubt.
Guenter

Hi,

Does newer 3-D hardware work using VMWare/Windows on a Mac?

Thanks!
Amir


___ _
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim Fairman 
[fairman@gmail.com]
Sent: Monday, January 24, 2011 6:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Full-screen stereo with Coot/PyMol/etc on 3D LCD displays?

Forgot to mention that this is only for the newer 120 Hz LCDs using the Nvidia 
3D Vision system.  You'll have to stick with your CRT for now or switch to 
Linux/Win for the newer hardware.

On Mon, Jan 24, 2011 at 1:10 AM, Jim Fairman 
fairman@gmail.commailto:fairman@gmail.com wrote:
Stereoscopic 3D driven by OpenGL via an Nvidia Quadro FX card is only available 
on Linux and Windows.  You'll have to beg the Nvidia gods for drivers for Mac.


On Mon, Jan 24, 2011 at 12:56 AM, Luecke, Hartmut 
hu...@uci.edumailto:hu...@uci.edu wrote:
My apologies if this has been discussed before.

Has anyone got this to work?  Preferably with a Mac?  We would love to hear
the configuration.


Cheers, Hudel

Hartmut Luecke
Director, Center for Biomembrane Systems
Depts. of Biochemistry, Biophysics  Computer Science
3205 McGaugh Hall
University of California
Irvine, CA 92697-3900
hu...@uci.edumailto:hu...@uci.edu http://bass.bio.uci.edu/~hudel/



This message contains confidential information and is intended only for the 
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does not accept liability for any errors or omissions in the contents of this 
message, which arise as a result of e-mail transmission.



--
Jim Fairman, Ph D.
Post-Doctoral Fellow
National Institutes of Health - NIDDK
The Buchanan Labhttp://www-mslmb.niddk.nih.gov/buchanan/index.html
Lab: 1-301-594-9229
E-mail: fairman@gmail.commailto:fairman@gmail.com 
james.fair...@nih.govmailto:james.fair...@nih.gov




--
Jim Fairman, Ph D.
Post-Doctoral Fellow
National Institutes of Health - NIDDK
The Buchanan Labhttp://www-mslmb.niddk.nih.gov/buchanan/index.html
Lab: 1-301-594-9229
E-mail: fairman@gmail.commailto:fairman@gmail.com 
james.fair...@nih.govmailto:james.fair...@nih.gov
  



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

e-mail: guenter.fr...@uni-konstanz.de

e-mail: guenter.fr...@uniklinik-freiburg.de
http://www.uniklinik-freiburg.de/neuropathologie/live/forschung/ag-g-fritz.html
Tel.: +49 761 270 5078
Fax.: +49 761 270 5050

Re: [ccp4bb] Mg2+ or water

[Guenter Fritz]

Hi,
you might have a look into the papers of MM Harding. There is a series 
of papers about metal ions in X-ray structures in Acta D.
Look at the number of coordination, geometry and distances. Then you can 
easily decide whether there is a metal ion and which metal ion it might be.
And of course the peak height of anomalous signal (e.g Ca2+, K+)  is a 
hint, but one has to be careful in the case there is not full occupancy.

HTH
Guenter




*Subject:* [ccp4bb] Mg2+ or water

 


Hi All,

I am refining a structure and encountered a problem of modeling a 
difference density as water or Mg2+, and would like to hear opinions 
from the community. It has the following coordinations (attached): the 
water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate 
group from the ligand, it also forms salt bridge/H-bonding interaction 
with a Glu residue from the protein, it is also within hydrogen 
bonding distance to the main chain N of another protein residue. In 
provious publication, it was modelled as a Mg2+ and the author 
reasoned the dual salt-bridge stabilizes the liganding binding, also 
the Mg2+ is present in the protein solution for crystallization. For 
my case, I have no Mg2+ present in the protein buffer, also modelling 
it with water refines perfectly with no indication of positive 
difference density even at 2.0 sigma cut off. Should I modelled this 
density as water or as Mg2+. Your opinions are appreciated.


JL
 




--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

e-mail: guenter.fr...@uni-konstanz.de

e-mail: guenter.fr...@uniklinik-freiburg.de
http://www.uniklinik-freiburg.de/neuropathologie/live/forschung/ag-g-fritz.html
Tel.: +49 761 270 5078
Fax.: +49 761 270 5050

Re: [ccp4bb] expression of Cys-rich small protein

[Guenter Fritz]

Hi Laurie,
nuclear protein, cysteines, my thumbs prickle it might be a zinc-binding 
protein.
Although 12 Cys per 257 aa is not yet that much. If you have always DTT 
present oxidation should be no problem.
Reconstitution of  zinc sites can be very tricky and is not 
straightforward. Also E.coli chaperones might not help in this case.
As you already  mentioned: I think expression insect cells is a good 
idea to test.

Best,
Guenter

All -

We are trying to express for structural studies a 257 AA eukaryotic 
intracellular (also possibly nuclear) protein (predicted to be single 
domain all-helical) that has 12 Cysteines.  No known metal-binding 
function not that it couldn't happen.  So far (E. coli) it expressed 
solubly as MBP fusion (with an N-terminal region deleted predicted 
disordered) until cleavage of MBP, then it's not soluble, including 
detergents added.  THe MBP fusion is usually soluble aggregate so we 
assume that our part is not folded right.  We have so far assumed it 
needs a lot of reducing agent (5 mM DTT or TCEP).Thinking of 
trying chaperones and insect cells next.


Any experience out there that might help?  Mostly I wonder about all 
the cysteines.  Don't really know if that is the problem.


Laurie Betts



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

e-mail: guenter.fr...@uni-konstanz.de

e-mail: guenter.fr...@uniklinik-freiburg.de
http://www.uniklinik-freiburg.de/neuropathologie/live/forschung/ag-g-fritz.html
Tel.: +49 761 270 5078
Fax.: +49 761 270 5050

Re: [ccp4bb] High Rmerge with thin frames

[Guenter Fritz]

Dear Ronnie,
we are working with weak diffracting crystals. Many tests (and taking 
into account  Marcus Muellers results) showed that 0.3-0.5 of XDS 
mosaicity, (very) low dose, and high redundancy give the best results. 
Our crystals diffract between 7 and 4 A. At low dose I do not check 
diffraction really by eye. I collect 200 images (fine slicing), process 
and check XDS statistics. (200 images is not much when the dataset 
comprises 9000-12000 images). Especially for anomalous data this 
strategy was pivotal.
We did also comparisons between CCD and PILATUS. We used one long 
crystal and collected on end end a MAD dataset using a  CCD and 
afterwards  at the other end a MAD dataset on PILATUS, same dose per 
degree.  Results were absolutely clear that the fine slicing give much 
better results.

Best,
Guenter

Dear Tassos,

I'm interested in your third point. Do you have any explanation for 
why 0.5-1 degrees oscillation gave better data? Purely due to the fact 
that the crystals survived longer and thus yielded higher redundancy 
data, or also other parameters?
Also do anyone know where the threshold lies for when /not/ to use 
fine phi slicing on the PILATUS? ie, at what level of diffraction 
would one need to increase the exposure (and oscillation in order to 
still get redundant data)?


We'll be in a similar position in the coming weeks with data 
collection using PILATUS detectors, and would like to maximize the 
potential data quality from our weak diffracting crystals. Any input 
on this would be greatly appreciated!


Cheers,
Ronnie Berntsson



On Nov 5, 2010, at 16:16, Anastassis Perrakis wrote:


three additional points:

1.


OTOH, if The diffraction is quite weak, one may be limited by counting
statistics.  This also cannot be overcome by processing.


As JIm suggests above then, maybe you should look if the 15% Rmerge 
is almost reasonable given the specific I/sigI at low resolution?



2. If there is one thing I do not like in XDS, is that there is no 
(or I have failed to find) statistics of I/sigI and Rmerge as 
function of image.

Have a look at the SCALA output. Maybe some images are bad?

3. making too fine slices of too weak diffraction images ends up with 
either too weak counting statistics or inability to 'lock' the 
refinement.
we did that for one crystal form, collecting 0.1, 0.2, 0.35, 0.5, 
0.7, 1.0 from various crystals (with the same dose per degree, at SLS 
using a PILATUS, mosaicity 0.4-0.6) in an attempt to get better Se 
signal. We miserably failed to get any useful signal at the end, but 
learned that for these very weak diffracting plates (submicron) 
collecting 0.5-1.0 degrees was actually giving at the end better data.


A.





--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

e-mail: guenter.fr...@uni-konstanz.de

e-mail: guenter.fr...@uniklinik-freiburg.de
http://www.uniklinik-freiburg.de/neuropathologie/live/forschung/ag-g-fritz.html
Tel.: +49 761 270 5078
Fax.: +49 761 270 5050

Re: [ccp4bb] protein turns brown

[Guenter Fritz]

Sandy,
like mentioned previously, sounds like FeS.
record an optical spectrum. Or even better, check whether there is 
somebody on the campus running an EPR machine (equipped for helium 
temperature measurements)
Maybe  a new FeS  protein? FeS is not necessarily required as redox 
cofactor. It can have any other function . See e.g. primase from 
eukaryotes (recent structures)
Or in your native (maybe eukarytic) protein is a Zn.  E.coli places 
sometimes a FeS instead of a Zn in the site.
Less likely but further possibility: Ni2+ bound to the protein. If DTT 
is added: Ni2+ -S coordinated is not redox stable and oxidizes to Ni3+ 
(brownish colour).

HTH
Guenter

Dear all,

I have purified protein from E.coli. expression system. the protein 
has been purified with three independant columns. Now during 
concentration step using amicon, the protein shows brown colour. what 
could be the reason.


best regards and Thanks,
sandy
http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle? 

Re: [ccp4bb] Is it possible for the Tris buffer to strip the Zn ions from the Zinc Finger motif of a protein?

[Guenter Fritz]

Dear Heng,
Tris forms only a very weak complex with Zn2+. It is unlikely that the
problem arises from Tris. It might be simply that the complex
dissociates in the presence of high imidazol when you elute the complex
from the nickel column. Imidazol is a pretty good Zn2+ coordinating
molecule. DTT in higher concentrations (1 mM should be now problem for a
zinc finger) can also strip of a Zn2+; it is a high affinity bidental
chelator.
HTH
Guenter


 
 Date: Sat, 22 May 2010 11:17:41 +0800
 From: rh_ibp2...@hotmail.com
 Subject: [ccp4bb]
 To: CCP4BB@JISCMAIL.AC.UK

 Dear all,

 Recently, I am working on a complex which includes two protein
 subunits. The interaction was based on the Zinc Finger motif of one
 protein. I co-purified the complex by nickel affinity column with one
 protein bearing a C terminal His tag and the other without any
 affinity tags. However, the complex was disassociated when applied to
 size exclusion chromatography. The buffer I use for SEC is 20mM
 Tris-HCl, 150mM NaCl, 1mM DTT, 5% Glycerol, pH 7.5, whearas the buffer
 I use for nickel affinity column is 50mM Na2HPO4, 10mM KH2PO4, 137mM
 NaCl, 2.7mM KCl, 10% Glycerol, pH7.4. So I am wondering is it possible
 for the Tris buffer to strip the Zn ions from the Zinc Finger motif of
 one protein that leads to the destruction of the complex?

 I will be very appreciated if anyone has some experience in such case
 and would like to share with me!


 Sincerely,

 Heng


 Institute of Biophysics,
 Chinese Academy of Sciences,
 Beijing 100101, China

 
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-- 
***

 Priv.Doz.Dr. Guenter Fritz
 Fachbereich Biologie
 Sektion Naturwissenschaften
 Universitaet Konstanz
 http://www.biologie.uni-konstanz.de/fritz

 Universitaetsstrasse 10
 Postfach M665
 D-78457 Konstanz

 e-mail: guenter.fr...@uni-konstanz.de

 Phone Office: +49-(0)7531 88 3205 
 Phone Lab   : +49-(0)7531 88 3733
 Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] Protein-antibody complex

[Guenter Fritz]

Hi Jan,

you might mutate the Cys residues which are oxidation sensitive,
you could block the Cys thiols  in your oxidation sensitive protein,
or you try crystallization at slightly acidic conditions where the Cys 
thiol should be more stable (might be bad for the ab-protein complex),

or try crystallization in a glove box.

But before doing all this you might check whether the thiols of your 
protein react with the disulfides in the ab. I don't think this happens 
frequenetly, but we had one case where a solvent exposed Cys thiol 
attacked the disulfide in an Ig domain. This happened only at high 
protein concentrations  (10 mg/ml). The not desired result was a 
covalently Cys bridged protein-Ig complex. For a test you can 
concentrate ab and protein separately, mix them, take aliquots at 
several time points, and run  non-reducing SDS-PAGE.  If you get a new 
high MW band you can do tryptic digest-ms analysis to identify the 
responsible Cys residue.
Another control experiment:  just incubate your protein without DTT,   
take aliquots at several time points, and run  non-reducing SDS-PAGE. 
Again mass-spec should give you an idea which Cys is reactive.

Mutating only this Cys might be already sufficient.

HTH
Guenter



Hi All,

 

 I have a simple question about the complex formation between 
macromolecular complex and antibody. My protein is stable in the 
presence of the 5mM DTT and under these conditions the reducing 
environment is too strong for the antibody to survive. I am also now 
trying to check the stability of the protein  in lower molar 
concentration of DTT, but as DTT being a strong reducing agent it 
might still pose a threat to the disintegrate  the antibody.


 

Does anybody have experience in handling protein-antibody complexes 
using other reducing agents? Your answers and help in this regard will 
be highly appreciated.


 


Thanks,

 


Jan




--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205 
Phone Lab   : +49-(0)7531 88 3733

Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] DSSP server

[Guenter Fritz]

Hi,
you can use the nice tool dssp2pdb by James Stroud 
(http://structure.usc.edu/dssp2pdb) to assign the ss of the DSSP output 
to your pdb file. See instructions there.

HTH
Guenter



I am sorry for this off-topic question. I see it has been discussed 
many times, but somehow I got stuck in the very beginning.


I want to assign ss to my pdb file in DSSP, but I could not find a 
server to run it. Does anybody know a webserver where I could run this 
kind of tasks? I use iMAC.


Many thanks!

Jane



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205 
Phone Lab   : +49-(0)7531 88 3733

Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] Anomalous map creating

[Guenter Fritz]

Hi,
I was looking recently for weak anomalous scatterers, when refined model 
is known.

I used phaser as described here:
http://www.phenix-online.org/pipermail/phenixbb/2008-July/001136.html

or running phaser from the ccp4 gui SAD with molecular replacement 
partial structure


Works very well, I could identify several ions which had been placed as 
water.


However, when I wanted to look at the anomalous LLG maps, I got a bit 
confused with the description on
http://www.phenix-online.org/pipermail/phenixbb/2008-July/001136.html. 
Using columns FLLG/PHLLG gave a map looking more like noise.


I got the anom. diff. map  using fft or directly in coot  (you first 
have to generate DANO from F+ and F- with sftools) using columns DANO, 
PHWT ,  and not PHLLG !?, can somebody comment on this?


This map looked clearly better than the anom.  diff. map generated using 
the phases of the refined model (CAD, FFT). 


Best,
Guenter

--
phenix.phaser  eof  SAD_LLG_initial.log
TITLE initial SAD LLG map
MODE EP_AUTO
HKLIN my_peak.mtz
LLGCOMPLETE CRYSTAL no77 COMPLETE OFF
LLGCOMPLETE CRYSTAL no77 SCATTERING ANOMALOUS
PARTIAL PDB ref.pdb IDENT 1.0
CRYSTAL no77 DATASET peak LABIN F+=F(+) SIGF+=SIGF(+) F-=F(-) SIGF-=SIGF(-)
COMPOSITION PROTEIN MW 68000 NUMBER 1
ROOT SAD_LLG_initial
eof


fftHKLIN my_peak_sftools1.mtz  MAPOUT my_peak_llg.map  EOF
TITLE  llg anom difference map
LABIN  DANO=DANO SIG1=SIGDANO PHI=PHWT W=FOM
resolution 50. 2.0
EOF

Hello everybody!

I am faced with a problem of calculating an anomalous map from a Se-Met
dataset, and
I cannot interpret the error message.

So, detailed problem description:

I was given a Se-Met dataset of my protein. I scaled it in Scala and made .mtz
file, but I do not phases.
And I cannot do a MR, but I have a coordinate file. This is my situation

So, what I did.
I made a copy of .mtz and did a refinement in refmac  - to generate phases.
During that I lost all anomalous data.
After I did CAD procedure - I took from original .mtz anomalous data (F(+),
F(-), DANO, IMEAN, I(+), I(-), with sigmas) and from refined mtz - H K L
FreeR_flag, F, SIGF, FC, PHIC, FC_ALL, PHIC_ALL, FWT, PHWT, DELFWT, PHDELWT,
FOM.
And then I did anomalous FFT
in the fields I put:
PHI - PHIC
Weight - FOM
DANO - DANO
Sigma - SIGDANO

I tried with and without excluding of R-free, but result was the same -
FAILED... And error message was
FFTBIG:  No reflexions pass acceptance criteria!  Check RESOLUTION,
EXCLUDE, missing data.
And I cannot find how to fix this.

It have also one more warning message -  * Missing value set to NaN in input
mtz file
but as I read it is not a problem - mtz is still readable.

I would be glad for any help or advice.
Thanks.

Sergii

P.S.   Please, find attached mtz and logs.



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205 
Phone Lab   : +49-(0)7531 88 3733

Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] Anomalous map creating

[Guenter Fritz]

Hi Randy,
thanks a lot! That explains everything.
best regards,
guenter

Hi,

We should probably clarify the documentation on this point.  When you 
complete the anomalous substructure in Phaser, it's an iterative 
process where LLG maps are computed looking for places where anomalous 
scattering should be added (or subtracted).  New sites are introduced, 
and then the next LLG map shows where further changes would be desired 
in the anomalous scatterer model.  Because the addition of earlier 
sites improves the model and the phases, second and subsequent LLG 
maps often lead to the addition of further sites.  After 3 or 4 
cycles, however, the process usually converges because there is no 
clear indication of further sites.  At this point, if all has gone 
well, the LLG map indeed should be relatively flat and should show 
only some noise, because all the information has been extracted to 
improve the anomalous scatterer model.  And this is the LLG map you 
get at the end of log-likelihood-gradient completion.


If you want to see the initial LLG map, you have to turn completion 
off, and then the map coefficients will show you what Phaser is 
interpreting in the first round of completion.  In our experience, 
this map alone will be clearer than a conventional anomalous 
difference Fourier, if you're starting from a protein model.  But if 
we stopped here, then we would lose the benefit of the iterative 
completion process.


All the best,

Randy Read

On 2 Nov 2009, at 09:24, Guenter Fritz wrote:


Hi,
I was looking recently for weak anomalous scatterers, when refined 
model is known.

I used phaser as described here:
http://www.phenix-online.org/pipermail/phenixbb/2008-July/001136.html

or running phaser from the ccp4 gui SAD with molecular replacement 
partial structure


Works very well, I could identify several ions which had been placed 
as water.


However, when I wanted to look at the anomalous LLG maps, I got a bit 
confused with the description on
http://www.phenix-online.org/pipermail/phenixbb/2008-July/001136.html. 
Using columns FLLG/PHLLG gave a map looking more like noise.


I got the anom. diff. map  using fft or directly in coot  (you first 
have to generate DANO from F+ and F- with sftools) using columns 
DANO, PHWT ,  and not PHLLG !?, can somebody comment on this?


This map looked clearly better than the anom.  diff. map generated 
using the phases of the refined model (CAD, FFT).

Best,
Guenter

--
phenix.phaser  eof  SAD_LLG_initial.log
TITLE initial SAD LLG map
MODE EP_AUTO
HKLIN my_peak.mtz
LLGCOMPLETE CRYSTAL no77 COMPLETE OFF
LLGCOMPLETE CRYSTAL no77 SCATTERING ANOMALOUS
PARTIAL PDB ref.pdb IDENT 1.0
CRYSTAL no77 DATASET peak LABIN F+=F(+) SIGF+=SIGF(+) F-=F(-) 
SIGF-=SIGF(-)

COMPOSITION PROTEIN MW 68000 NUMBER 1
ROOT SAD_LLG_initial
eof


fftHKLIN my_peak_sftools1.mtz  MAPOUT my_peak_llg.map  EOF
TITLE  llg anom difference map
LABIN  DANO=DANO SIG1=SIGDANO PHI=PHWT W=FOM
resolution 50. 2.0
EOF

Hello everybody!

I am faced with a problem of calculating an anomalous map from a Se-Met
dataset, and
I cannot interpret the error message.

So, detailed problem description:

I was given a Se-Met dataset of my protein. I scaled it in Scala and 
made .mtz

file, but I do not phases.
And I cannot do a MR, but I have a coordinate file. This is my 
situation


So, what I did.
I made a copy of .mtz and did a refinement in refmac  - to generate 
phases.

During that I lost all anomalous data.
After I did CAD procedure - I took from original .mtz anomalous data 
(F(+),
F(-), DANO, IMEAN, I(+), I(-), with sigmas) and from refined mtz - H 
K L
FreeR_flag, F, SIGF, FC, PHIC, FC_ALL, PHIC_ALL, FWT, PHWT, DELFWT, 
PHDELWT,

FOM.
And then I did anomalous FFT
in the fields I put:
PHI - PHIC
Weight - FOM
DANO - DANO
Sigma - SIGDANO

I tried with and without excluding of R-free, but result was the same -
FAILED... And error message was
FFTBIG:  No reflexions pass acceptance criteria!  Check RESOLUTION,
EXCLUDE, missing data.
And I cannot find how to fix this.

It have also one more warning message -  * Missing value set to NaN 
in input

mtz file
but as I read it is not a problem - mtz is still readable.

I would be glad for any help or advice.
Thanks.

Sergii

P.S.   Please, find attached mtz and logs.



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205 Phone Lab   : +49-(0)7531 88 3733
Fax:  +49-(0)7531 88 2966


--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj

[ccp4bb]

[Guenter Fritz]

Hi Sajid,
the NADP /NAD binding sites are often composed of two sites: one site 
that is specific for the adenine/adenosine moiety and another site for 
the nicotinamide moiety. It can happen that you see the adenine in the 
density  but not the nictoninamid tangling around.

HTH Guenter



I have used NADP in my crystallisation condition; and the crystal diffracted 
well; I built the model completely and the r-factor and r-free are 0.26 and 
0.29 respectively. The resolution is 2.3A. I am looking for NADP, in the 
binding region; I could see some density for NADP; But it is not continuous; 
there is no density for the middle phosphate group in fo-fc as well as 2fo-fc. 
Is it possible that NADP could hydrolyse? if so what could be the hydrolysed 
product


thank you

sajid



  Looking for local information? Find it on Yahoo! Local 
http://in.local.yahoo.com/
  



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205 
Phone Lab   : +49-(0)7531 88 3733

Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] Zinc or Iron binding protein, that is a question!

[Guenter Fritz]

Hi Xuan,
I guess your protein is not an E.coli protein. There are several 
examples that eukaryotic Zn-proteins expressed in E.coli contain Fe 
instead of Zn. I am sceptic whether IMAC with different metal ions will 
give the solution of the problem. If you really want to get information 
on the metal ion binding properties you will have to do some matallo 
biochemistry: preparing apo protein, reconstitution with metal ions, 
UV-Vis spectroscopy, EPR would be great, ...



Dear Sir or Madam,
 
The ICP-ES results indicated that 1 molar my protein purified from 
E.coli Origami(DE3) contained about a half molar Zinc and nearly a 
quarter molar Iron (whether II or III was not available). The protein 
carried a MBP tag on the N-terminal and the situation was similar with 
or without His tag at the C terminal. I want to determine whether my 
protein really bind Zinc or Iron. Does anyone have any experience 
about such problems?
 
Specifically, now I want to compare the binding efficiency on various 
IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or 
CuSO4(control). However,  considering the instability of Fe(II) in 
solution, the design still seemed problematic.
 
Sincerely,
 
Xuan Yang
 
National Laboratory of Biomacromolecules and

Center for Infection and Immunity,
Institute of Biophysics,
Chinese Academy of Sciences,
Room 1617, 15 DaTun Road,Chaoyang District,
Beijing, China, 100101
Tel: 86-10-64884329
Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn
We will either find a way or make one.
 

Re: [ccp4bb] off topic

[Guenter Fritz]

Jürgen Bosch schrieb:

Try TCEP as it does not interfere with the NiNTA resin whereas DTT does.
But why do you care if your column is brown or not - can you elute 
your protein ?

Are there other metal ions e.g. iron which bind to your resin perhaps ?

Jürgen

On 19 Jun 2009, at 02:25, Kn Ly wrote:


Hello everyone,

I am trying to purify a 13 KDa membrane protein using Ni NTA. The 
protein is
solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and 
binds

very well to the column. However, it also turns the column brownish.
The protein contains 4 cysteine residues so I suspect that this causes
cross-linking with other proteins and thus brownish precipitation on the
column. So I included 5 mM beta-ME in my buffer to prevent disulfide 
bond
formation but this doesn't help. I tried 1 mM DTT and this ruined the 
column.

Help!! Is there anyway to prevent this brownish problem?

Thanks a lot in advance
Kien


-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.me.com/bosch_lab/



--
***

Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Tel.: +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] CD spectroscopy sensitivity

[Guenter Fritz]

Dear Francisco,

Dear All

I got recently a comment on a paper review suggesting me to perform CD 
spectroscopy to determine the alpha-helical content of a mutant 
protein involved in a human disease. The diferences between the wild 
type and the mutant protein are just 17 aminoacids. My question is if 
CD spectroscopy could detect secondary structure differences between 
the wild-type and the mutant proteins based on this aminoacid 
difference. Let's say, is it possible to detect by CD the presence or 
absence of a 17 aa helix in a protein of 400 aa.
The answer is definitely yes. 17 out of 400 is ca. 4-5%. But your helix 
content in the 400 residues will not be 100%. If it is mainly 
alpha-helical, it will be ca. 50%. Then we are talking about 17 out of 
200 residues, what is 8%.


I think the more important  reason to analyze  your deletion mutant  by 
CD is whether the residual fold stays intact after removal of one helix.

HTH
Guenter


I would appreciate your opinion

All the best

Francisco J. Enguita
ITQB
Oeiras
Portugal



--
***
PD Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Tel. Office: +49-(0)7531 88 3205
Tel. Lab   : +49-(0)7531 88 3733
Fax:  +49-(0)7531 88 2966

[ccp4bb] Source Ta6Br12 for phasing

[Guenter Fritz]

Hi,
a follow up question: Which supplier offer  Ta6Br12?
Thanks,
Guenter

My post-doc recently produced a splendid (for its resolution)
~5A map of a medium sized protein-DNA complex using Ta6Br12
clusters.  And he's got a good toehold on a ~340kDa complex
using the same clusters.  So I'm recently converted to these
little nuggets.
   Phoebe

--

Re: [ccp4bb] Angle between two different helices from two different subunits

[Guenter Fritz]

Hi Peter,

you can do that with interhlx:
http://nmr.uhnres.utoronto.ca/ikura/resources/data+sw/interhlx/

There is also a way to determine the interhelical angle with molmol,
http://hugin.ethz.ch/wuthrich/software/molmol/
defining cylinders for the helices.
HTH
Guenter

Hi all

I would be interested to know about the programmes which can calculate 
the angle between the two helices.  I want to calculate the angle 
between the two different helices from two different subunits of the 
structure. I know a non-supported programme at ccp4 called helixang. 
Is this programme can be helpful? Furthermore, i am not able to 
understand , how this program calculates the angle between the two 
helices of different subunits, after reading its logfile. I will 
appreciate suggesstions.



Thanks in advance

Peter

[ccp4bb] coot add-residue without map

[Guenter Fritz]

Dear all,
I wanted to use COOT to add a few residues to a the N-term, just simple 
fantasy building. It should be straight forward with the 
add-terminal-residue command, however COOT insists on a e-density map.

Any idea to overcome this?
Thanks in advance,
Guenter

[ccp4bb] restraints for 4Fe4S in shelxl

[Guenter Fritz]

Dear All,
I am looking for a set of suitable restraints for  4Fe4S clusters in 
shelxl  refinement (the PRODRG server does not like Fe). So far I have 
put there distance restraints. Has somebody experience with angle 
restraints for the 4Fe4S clusters? 
Thanks,

Guenter

***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] cryoloops for X-ray data collection from protein crystals at room temperature

[Guenter Fritz]

Hi Cedric,
I used these, but just for testing crystals.  I was afraid that the 
crystal might move in the loop. For testing it worked pretty good.

http://www.jenabioscience.com/cms/en/1/catalog/733_microrttrade_room_temperature_mounting_system.html
There might be more suppliers. Please send a summary to ccp4bb.
Best
Guenter


cedric bauvois wrote:

Dear CCP4ers,

in their paper entitled  Using cryoloops for X-ray data collection 
from protein crystals at room temperature: A simple applicable method 
( *Journal of Crystal Growth* 
http://www.sciencedirect.com/science/journal/00220248
Volume 281, Issues 2-4 
http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%235302%232005%23997189997%23601824%23FLA%23_cdi=5302_pubType=Jview=c_auth=y_acct=C26678_version=1_urlVersion=0_userid=532047md5=9a4e7b2fc158c6d2396925c79d995e3d, 
1 August 2005, Pages 592-595.), the authors present a way to mount 
crystals using a cryoloop accompanied by a glass capillary cap (see 
abstract below).

Do you know if any commercial version of such system are now available ?

Abstract: Although cryoloops are now routinely used for X-ray data 
collection from protein crystals in cryocooling condition, it is still 
necessary to collect X-ray diffraction data from protein crystals at 
room temperature under such circumstances as to find resolution limit 
and/or to avoid damage of protein crystals at cryogenic temperature 
(e.g. 100 K). Here, we show that a cryoloop, which is accompanied by a 
glass capillary cap to maintain humid environment of crystal in the 
cryoloop, can be used not only to examine protein or non-protein 
crystals but also to collect X-ray diffraction data for structural 
analysis from protein crystals at room temperature. The size of 
cryoloop should be carefully chosen so that the crystal does not move 
in the cryoloop. This crystal mounting method can be time-saving 
compared to the traditional method to mount a crystal in a glass 
capillary tube.



Many thanks

--
Dr. Cedric Bauvois
Cristallographie des protéines
Institut de Recherches Microbiologiques JM Wiame -IRMW
Av E. Gryzon 1, 1070 Brussels (Belgium)  
tél: +32 (0)2 5273634

fax: +32 (0)2 5267273


--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

[ccp4bb] error in startup of ccp4i 6.1

[Guenter Fritz]

Dear all,
I have installed precompiled ccp4 61 (Redhat from download manager) on Centos5 
box, X86_64.

Running ccp4i gives:

[guen...@hypatia ~]$ ccp4i
Top level CCP4 directory is /usr/local/software/CCP4/61/ccp4-6.1.0
Using CCP4 programs from /usr/local/software/CCP4/61/ccp4-6.1.0/bin
Error in startup script: wrong # args: should be dbccp4i_open_project project 
args
   while executing
dbccp4i_open_project
   (eval body line 1)
   invoked from within
eval dbccp4i_open_project $project $args
   (procedure DbLoadFile line 12)
   invoked from within
DbLoadFile $project
   (procedure DbOpenDatabase line 13)
   invoked from within
DbOpenDatabase $project
   (procedure DbOpen line 30)
   invoked from within
DbOpen -init
   (procedure DbInitialise line 19)
   invoked from within
DbInitialise
   (procedure taskbrowser line 38)
   invoked from within
$system(RUN_MODE)
   (default arm line 9)
   invoked from within
switch  $system(RUN_MODE) \
 script {
# Run a script ($CCP4I/scripts/project.script) with parameters from def file

   source [file join $env(CCP4I_...
   (file /usr/local/software/CCP4/61/ccp4-6.1.0/ccp4i/bin/ccp4i.tcl line 163)
   invoked from within
source [file join $env(CCP4I_TOP) bin ccp4i.tcl]
   (file /usr/local/software/CCP4/61/ccp4-6.1.0/ccp4i/bin/ccp4i line 5)

-

If I run ccp4i as root everyting is fine. 


Moreover on another Centos5 box with similar architecture there are no problems 
usin the same installation files.




Albert Guskov has reported the same error on Mac OS X.
He edited the   configure.def file changing the value fro 
USE_DBCCP4I_ON_STARTUP from 1  to 0. 


Did not work in my case.

Any ideas?


Thanks a lot in advance,
Guenter





*
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fr...@uni-konstanz.de

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme

[Guenter Fritz]

Hi Michael,

Fraser et al writes that in case of  Synechococcus phytoene desaturase 
'NAD+ and NADP+ were observed to be involved, whilst FAD was an 
ineffective electron acceptor '

Biochem J. 1993 May 1;291 ( Pt 3):687-92
HTH
Guenter

PS The enzyme itself has no flavin bound?

michael nelson wrote:

Dear all,

Thank you for all your kind replies.

Here is a little bit more about the enzyme and how I carry out the 
assay at the first place.


My enzyme is a lipid desaturase, originally from plant but 
overexpressed in bacteria. FAD serves as a co-factor for this enzyme, 
in which FAD is reduced to FADH2.


My goal is set up an assay that would allows me to continuously 
monitor the progress of the reaction. And I didn't want to use HPLC to 
analyze the final product since that would take a lot time and we 
don't have an instrument readily available to us. I wish FAD could be 
an alternative way since FAD will have different Abs in reduced or 
oxidized forms.


I set up assay is a regular lab setting (not anaerobic), add FAD, 
substrate, ions and incubate. I finally add enzyme to initialize the 
reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't.


I have several concerns, one is the autooxidisability of FAD, how fast 
FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in 
solution. The second cocern is how fast the FAD reaction will go.


Please advise.

Thank you!

Mike




--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Semet in non-auxotrophic strains

[Guenter Fritz]

Hi Jacob,
you can express SeMet labeled protein in almost any E.coli expression 
strain if you use a defined minimal medium.

Here is a receipe that worked quite well in our hands:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
HTH
Guenter

Dear Crystallographers,

I have been using an E coli strain called Tuner (DE3) pLacI, which is 
not auxotrophic for methionine, nor is there an auxotrophic analogue 
available. It may be that my protein can be expressed in other 
strains, but this one works well right now, and it is usually better 
to stay with what works well. Has anybody tried growing semet protein 
in non-auxotrophic strains, or are there tricks to using 
non-auxotrophs for semet protein?


Thanks,

Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] losing zinc during crystallization

[Guenter Fritz]

Hi Andy,

 

I hope your offer for advice on crystallizing without O2 is still 
available.  I am working on crystallizing some proteins that are 
oxygen-sensitive.  We have a glove-box that I'm using and can make 
grid-screens using degassed solutions. 



What kind of glovebox is it?
There are several types,
there are glove boxes which use N2 and keep a pressurein the glove box
higher than the surrounding to keep oxygen out.
and there are glove boxes http://www.coylab.com/vinylgb.htm), which use
a mixture of N2/H2 at the same pressure as the surrounding and a
catalyst that  reduces O2 that diffusing into the glove box.
I prefer the latter type; see below

However, I would like to use some of the available commercial 
sparse-matrix screens if possible.  Is it possible to degas a large 
number of solutions (in a time-efficient way) to make this possible? 
 Do you primarily degas solutions and then try to crystallize your 
samples in a glove-box?



We usually fill aliquots of 0.5-1 ml of crystallization screen solutions
into 1.5 ml plastic screw cap vials (there are several types which have
a rubber sealing). O2 can diffuse through plastic. Using screw cap vials
prevents H2O to evaporate from your solutions. We keep those vials in
the glove box for several weeks until usage. In the Coy type of glove
box there is a steep gradient for O2 from the vial to the atmosphere of
the glovebox, since O2 is consumed by reacton with H2 at the catalyst.
We once measured the time how long it takes until the  oxygen level in
the solutions has dropped sufficiently but I forgot the exact numbers.
2-3 weeks should be fine. In the case of very sensitive proteins one can
still add reducing agents. You should use sterile plastic vials or add
azide to prevent microbial growth.


 Is there a way to limit the oxygen availability inside sealed crystal 
trays that would allow putting them in a cold-room for example (we 
can't fit an incubator in our glovebox so crystallizing at different 
temperatures is currently not possible).




This is a bit tricky. We use plexiglass boxes which were made by the
workshop here at the University. We prepare the  crystallization plates
in the glovebox and put them subsequently  into the boxes, seal them and
take them out for incubation at e.g. 4 deg C.  Moreover, you can use
plates with a sealing like the Qiagen/Nextal type and place them into
the box. Then you have two barriers. You can put  a vial into the box,
that contains contains a solution reacting with oxygen like e.g
dithionite or a solution of  glucose oxidase/glucose consuming O2 that
diffuses into the plexiglass box. With these boxes we can  check the
crystallization experiments under the microscope.
However, any measure you take to keep oxygen out, after 2 weeks there
will be already some oxygen. After 4 weeks even more. We followed the
diffusion of oxygen into the plexiglass boxes using reduced
methylviologen that is deep blue in the reduced state and reacts readily
with oxygen. It works well for short time periods but is not suited for
longer periods (1-3 months).

For longer incubation periods and very sensitive proteins you have to
crystallize in the glovebox.

HTH

Guenter


 

Thank you for your time, 

Best Regards, 

-Andy Torelli 

 

 

-Original Message- 

From: CCP4 bulletin board on behalf of Guenter Fritz 

Sent: Fri 9/26/2008 3:21 PM 

To: CCP4BB@JISCMAIL.AC.UK 

Subject: Re: [ccp4bb] losing zinc during crystallization 

 

Hi Sue, 

I fully agree with Thierry, you might have to cyrstallize the protein  

under exclusion of dioxygen. There are many metallo proteins which have  

to be crystallized that way. But a first attempt might be the TCEP as  

already suggested by many others. If you need advice regarding  

crystallization without O2, send me a note. 

Good luck! 

Guenter 




--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966

Re: [ccp4bb] losing zinc during crystallization

[Guenter Fritz]

Hi Sue,
I fully agree with Thierry, you might have to cyrstallize the protein 
under exclusion of dioxygen. There are many metallo proteins which have 
to be crystallized that way. But a first attempt might be the TCEP as 
already suggested by many others. If you need advice regarding 
crystallization without O2, send me a note.

Good luck!
Guenter

Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what 
seems to me to be an eternity. The protein contains stoichiometric 
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required 
for activity.  Each crystal we've obtained has lost the zinc and 
contains a disulfide bond between two cysteine residues that should be 
zinc ligands (based on structures of similar proteins).


We've tried crystalizing in the presence of reducing agents, 
crystallizing with substrate analogs, and supplementing the 
crystallization drops with zinc with no success (and combinations of 
these approaches).  We've obtained a variety of crystals and 
determined structures, but none contain any zinc.


Attempts to insert zinc into the crystal (zinc + reducting agent or 
zinc alone) have not been successful.


Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry  Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

Re: [ccp4bb] Imidazole's ability to chelate metal ions

[Guenter Fritz]

Dear Jacob,
this depends on type of metal ion, solvent accessibility of metal site 
and pH.
E.g. the log of  dissociation constants for histidine- Cu2+ / Ni2+/Zn2+ 
complexes are ca. 10.6, 8.7 and 6.6. Imidazol  might pull out easily 
these metals. Mg2+ and Ca2+ prefer coordination by oxygen. These should 
be not affected, even at such high concentrations of imidazol. Also many 
FeS proteins are successfully purified via IMAC without loss of Fe.

HTH
Guenter

Jacob Keller wrote:

Dear Crystallographers,
 
Does anybody happen to know whether imidazole is able to chelate metal 
ions in solution? It seems reasonable that since it can compete for 
binding to IMAC resins, it should have some affinity for at least Ni++ 
and Co++, but what about metal ions like Ca++ and Mg++? I assume that 
the affinity is weak, but at the concentrations at which we are wont 
to use it in our elutions (~100-500 mM), does it not seem likely that 
other metal ions are being competed away from our proteins as well?
 
Jacob Keller
 
 
***

Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED] mailto:[EMAIL PROTECTED]
***


--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Imidazole's ability to chelate metal ions

[Guenter Fritz]

Tabled stability constants are e.g. in  'NIST Critically Selected 
Stability Constants of Metal Complexes' an electronic database. I am 
sure you find also tables in the library.
Another source is 
http://old.iupac.org/publications/pac/1997/pdf/6907x1549.pdf; here 
enthalpies are given for several at different ionic strength etc. but 
not for imidazol with  Mg2+ or Ca2+.




Jacob Keller wrote:
Could those who responded with numbers for affinities of imidazole for 
metal ions please divulge their sources? It is not that I doubt their 
veracities, but it would be a nice reference to have on hand.


For those wondering about why I was asking about imidazole's affinity 
for metal ions, I was wondering whether the presence of imidazole 
would affect a metal-ion-dependent reaction. With, for example, 200 mM 
imidazole and 10 mM Ca++ or Mg++, what would be the amount of free 
metal? This can of course be calculated from imidazole's binding 
constant for these ions, which is another reason I ask for the sources 
of the numbers quoted in a couple of the responses.


Thanks for all of the helpful responses so far,

Jacob Keller


***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

- Original Message - From: Nadir T. Mrabet 
[EMAIL PROTECTED]

To: Jacob Keller [EMAIL PROTECTED]
Cc: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, July 18, 2008 8:55 AM
Subject: Re: [ccp4bb] Imidazole's ability to chelate metal ions


Imidazole can indeed complex (monodentate) metal ions but not chelate 
them (bidendate, at least).
However, the stability constant, K, of such complexes is rather low, 
eg log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu.
In comparison, metal chelates are formed with EDTA, for which log K = 
10.6 for Mg, 14.2 for Fe and 18.8 for Cu.

So the difference amounts to several orders of magnitude.

It should also be pointed out that the competitive effect of 
imidazole in IMAC does not involve binding to free metal ions,
but instead coordination to immobilized metal chelates, eg 
Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator).


In any situation where one assays a protein whose activity and/or 
stability and/or else is/are metal dependent, one should
rather use buffers (see below) that do not interfere (eg Good's 
buffers).


I suspect the imidazole in your case is either a buffer (pKa 7.0) or 
else results from competitive elution from an IMAC column.

What should be done depends on your exact conditions.

hth,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular  Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]



Jacob Keller wrote:

Dear Crystallographers,
 Does anybody happen to know whether imidazole is able to chelate 
metal ions in solution? It seems reasonable that since it can 
compete for binding to IMAC resins, it should have some affinity for 
at least Ni++ and Co++, but what about metal ions like Ca++ and 
Mg++? I assume that the affinity is weak, but at the concentrations 
at which we are wont to use it in our elutions (~100-500 mM), does 
it not seem likely that other metal ions are being competed away 
from our proteins as well?

 Jacob Keller
 ***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED] mailto:[EMAIL PROTECTED]
***




--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] how to promote folding for an unfolded protein

[Guenter Fritz]

Hi Jenny,
check on non-reducing SDS PAGE whether your disulfide bond (I assume 
there is one) is correct, i.e. you have monomeric protein on SDS-PAGE.
If there are disulfide linked oligomers, try expression in Origami 
strain at lower temperature, e.g. 25 deg C.Another possibility is to 
direct expression to the periplasm 
(http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides#Expression_of_proteins_containing_disulfides)

Good luck!
Guenter

Jenny wrote:

Dear CCP4 community,

Sorry for this off-topic protein purification problem.I'm trying to 
purify an immunoglobulin-like beta sheet protein  with a  c-terminus 
HIS construct. The protein expressed both in the supernat and pellet ( 
majority ). I purified the supernat and after run gel filtration, it's 
in the void volume. I also tried to purify from the pellet,and do 
dialysis refolding ( with and without L-arg ), after overnight 
dialysis, I run the gel filtration column, the apparent molecular 
weight looks like dimer/trimer. But when I did a CD scan of the 
protein, it's showing an unfolded protein profile.I was wondering if 
there is anyway to promote folding,or if anyway that can make some 
mutations to make it foldable.Any input would be useful.


Thanks a lot.

Jenny


--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Concentrating protein

[Guenter Fritz]

A mild and quick method is to use dry Sephadex G-25. The material will 
swell and take up all the liquid except molecules larger than ca. 5 kDa.



Dear All,

we have GCSF protein produced in inclusion bodies. we solubilise it refold 
it and then concentrate it using proflux system. still the concentration 
of the protein we get is less and volume is more for us to load in Ion 
exchange chromatography. is there any simple technique that can be 
performed in lab without using any hi-fi instrument to concentrate the 
protein in small volume of buffer. the protein we obtain is about 0.7 
mg/ml and we get 450 ml solution. our column is 110ml lab scale and we 
have to work in that only. i have heard of NH4SO4 precipitation. but it 
requires protein conc more than 1 mg/ml.


kindly help me to progress in my experiment.
  

Re: [ccp4bb] coordinates of TCEP

[Guenter Fritz]

Hi,
if a compound is not in the hic-up data base of the USF 
(http://xray.bmc.uu.se/hicup/)
I usually go to NCBI Pubchem database (TCEP e.g. at 
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=119411loc=ec_rcs) 
, download the sdf file, feed it into a programm that can read it and 
put out a pdb.

Regards,
Guenter

Re: [ccp4bb] pH gradient in Mono Q

[Guenter Fritz]

Matt, there should be also a material called PBE94 for pH range 9 to 4 
(and another one called Pharmalyte for high pH) to pack your own column. 
It worked pretty well for our protein and is not in the high price 
region of the MonoP column.

Best,
Guenter

Matthew Chu wrote:

Thanks Guenter and Andreas,

Yea, I have taken a look for the Mono P before, I thought the material 
they used in Mono P is basically the same as in Mono Q and I found the 
book Protein Purification Protocols: Second Edition  by Paul Cutler 
mentioned that phosphate buffer can also generate a continuous 
gradientthat's why I reckon we can also perform pH gradient in 
Mono Q. But I am worrying if it is a good idea to go for it, as both 
mono Q or mono P are quite expensive.


Matt

2008/6/24 Andreas Förster [EMAIL PROTECTED] 
mailto:[EMAIL PROTECTED]:


Hey Matt,

it seems to me that what you're asking for is chromatofocusing.
 See the official GE documentation:

http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin(260949098-R350)

http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin%28260949098-R350%29

The proprietary buffers are a bit expensive, but as you found out,
they're a bit complicated to make.  I don't know if you'd ruin
your Mono Q with a pH gradient.  If in doubt, buy one of the
dedicated Mono P column.

Hope that helps.


Andreas


Matthew Chu wrote:

Dear All,

Sorry for off-topic question. Does anyone have any experience
in purifying protein using pH gradient in Mono Q column?

I have been googling for a whole day, only one paper was found
to mention performing pH gradient in Mono Q, but in a mixture
of amine buffering species, which is a bit too complicated (J.
Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or
phosphate buffer give a linear pH gradient from pH 8.0 to 4.0?
Is it usual to perform pH gradient in Mono Q as I don't want
to ruin my Mono Q column...

Any suggestions are welcome. Thanks in advance!

Kind regards,
Matt

 

Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester






--

Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester



--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Protein binding to Zn and Ca

[Guenter Fritz]

Hi Neeraj,
a couple of S100 proteins bind Zn2+ and Ca2+. Ca2+ binds to EF-hands;  
Zn2+ binds to specific sites distinct of the EF-hands. We are 
investigating Zn2+-binding by spectroscopy and crystallography. We think 
that Zn2+ is like Ca2+ a kind of second messenger. If you have 
questions, I am happy to discuss these things.

Best,
Guenter


Hi all,
   I recently came across a question about an interesting idea. 
Does anyone know of an example of a protein binding to both Zn2+ and 
Ca2+ at the same time? Are there any known well studied precedents at 
all if any. Any help of insights would be very valuable.


thanks,
Neeraj



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***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Bacterial induction at 18C

[Guenter Fritz]

Raji,



I am working with E. coli cells co-transformed with two plasmids and I find 
that my cells lyse
following overnight inductions at 18C. 
Sounds more like a phage contamination. The phage becomes active as soon 
as the cells energy level decreases, e.g upon induction. We had once 
the same trouble. If it is a phage, autoclave everything and clean the 
lab thoroughly.

I suspect (among many things) that Ampicillin+
Chloramphenicol+ Kanamycin in the medium may be the source of my woes.

My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has 
anyone had
reasonable protein expression levels by inducing cultures at 18C for 6h? From 
what I understand, the
E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask.
  
Rule of the thumb is the Q10 rule, or in the case of e.coli it is a Q7 
rule. Doubling decreases twofold when temperature eis decreased by 7 deg C.


Godd luck,
Guenter

I am already playing with lowering and/or doing away with the antibiotics.

Any suggestions wrt 18C? The protein is insoluble at 30C.

Thanks.
Raji
  


--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966 

Re: [ccp4bb] Setting drops with proteins that are hard to concentrate

[Guenter Fritz]

Dear Matt,

make sure that you don't have slow or even fast formation of unspecific 
intermolecular disulfides by simple checks at several time points on 
SDS-PAGE using sample buffer without DTT. Check the content of free 
thiols and disulfides of your protein over a period of time. If you need 
easy and solid protocols, just send me a mail.

Guenter

Bottomley, Matthew wrote:


Dear All,

I have a 50kDa protein that is soluble and monodisperse at up to 
approx 1mg/ml (after Ni-affinity and size-exclusion chromatography).


However, it aggregates (probably both via disulphides and via 
'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, 
even in the presence of several detergents. I don't want to add DTT 
since my protein should have several intramolecular 
disulphidesalthough I do have 2 free Cysteines, partially exposed. 
I have already tried mutating the Cysteines, with little improvement.


Any suggestions for obtaining 5-10mg/ml?

Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 
50mM)  to aid concentrating (as in the Golovanov AP paper, JACS, 2004, 
pages 8933...)


Thanks for any input!

Yours,

Matt


Matthew J. Bottomley, Ph.D.
Senior Research Biochemist
IRBM / MRL-Rome


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--
***

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: [EMAIL PROTECTED]

Tel. Office: +49-(0)7531 88 3205 
Tel. Lab   : +49-(0)7531 88 3687
Fax:  +49-(0)7531 88 2966