Re: [ccp4bb] low-resolution and zinc
Are you trying to have Coot display the Zn-ligand bonds? That's a different issue from having refmac recognize and use the metal-ligand restraints in refinement. (I don't bother to have Coot do this.) Looking at your environment distances, it looks like refmac has done a restrained refinement, as your Zn-ligand bond distances look right. You can tell in the refmac log file if you get a message describing that a Zn-ligand distance was found and it is not listed as "not be used." (If you get this latter message, you have the wrong link usage setting chosen in the CCP4i GUI.) I just had an undergraduate student do the procedure yesterday to do a restrained refinement for a ZnCys2HisAsp environment. Refmac picks up the Zn-Cys link in the library without any intervention, and the Zn-His and Zn-Asp bonds have to be taken care of in the refmac-generated .cif file. All the links show up in the LINKR records. There may be a more elegant way to do this but it works. ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/9/2012 1:09 PM, SD Y wrote: Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the images https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. Thanks SDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics. Watch CHI2 :-) 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 <mailto:mbfro...@post.tau.ac.il> Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com <mailto:herman.schreu...@sanofi.com> wrote: Then we agree. I got confused because you mentioned"space group" and not "point group" in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board. Herman
Re: [ccp4bb] low-resolution and zinc
I am using Refmac_5.7.0029 in CCP4ThanksSDY Date: Fri, 9 Nov 2012 20:35:48 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK What program do you use for refinement?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 Nov 9, 2012, at 20:09 , SD Y wrote:Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the imageshttps://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.pnghttps://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. ThanksSDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics.Watch CHI2 :-) 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 Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote:Then we agree. I got confused because you mentioned"space group" and not "point group" in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board.Herman <74_refmac5_1.log>
Re: [ccp4bb] low-resolution and zinc
What program do you use for refinement? 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 Nov 9, 2012, at 20:09 , SD Y wrote: > Dear All, > > Thanks for all the suggestions on low resolution, SG and Zn and I learned a > lot. I am not done yet. > > I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger > Rowlett. Also an excellent protocol is available at > http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. > Protocol seems to be very straight forward. I am not getting the result I > wanted. > > 1. When ever I generate cif file, its not writing any for ZN-SG links at all. > > 2. After refmac refinement it only draw in LINK for His-ZN. Please see the > images > https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png > https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png > > 3. I get this His-Zn link without using .cif file, so what stage do I use > this .cif file? > > 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing > LINK in PDB. I only see > LINKR ZNZN H 1 SG CYS A 83ZN-CYS > > I dont know what is missing, I also attached the log file which generated the > ZN-His coordination. > > Any help is highly appreciated. > > Thanks > SDY > > Date: Thu, 8 Nov 2012 14:36:59 +0200 > From: mbfro...@post.tau.ac.il > Subject: Re: [ccp4bb] low-resolution and zinc > To: CCP4BB@JISCMAIL.AC.UK > > The experiment should be very problematic if I can't determine point group on > the base of the symmetry merging statistics. > Watch CHI2 :-) > 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 Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote: > > Then we agree. I got confused because you mentioned"space group" and not > "point group" in your phrase about PHASER and MOLREP and was afraid others > might have gotten confused as well. Also, in case of twinning or almost > crystallographic non-crystallographic symmetry, determining the point group > on the basis of processing statistics alone can be inconclusive or even > misleading. If I recall correctly, there has recently been a thread about > this in the bulletin board. > Herman > > > > > <74_refmac5_1.log>
Re: [ccp4bb] low-resolution and zinc
The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics. Watch CHI2 :-) 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 Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote: > Then we agree. I got confused because you mentioned"space group" and not > "point group" in your phrase about PHASER and MOLREP and was afraid others > might have gotten confused as well. Also, in case of twinning or almost > crystallographic non-crystallographic symmetry, determining the point group > on the basis of processing statistics alone can be inconclusive or even > misleading. If I recall correctly, there has recently been a thread about > this in the bulletin board. > Herman > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix > Frolow > Sent: Thursday, November 08, 2012 1:14 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] low-resolution and zinc > > I do not see with what you do not agree in what was written (maybe not very > carefully). One determine space group looking on systematic absences, this I > know. > Space groups P41 - P43 ( I do not go to more general discussion for the sake > of argument) are not distinguishable and one have to try molecular > replacement in both. > Most probably the correct space group will give a sensible solution and wrong > one will not. I was talking about distinguishing between point groups P4 and > P422. > I hope you grew that they CAN be distinguished on the level prior to > molecular replacement? > And I hope that you agree that starting refinement of demanding project at > relatively low resolution such as 3.4 Angstrom it is advisable to > characterise space group, twinning status etc.? > If you agree also to that, with what you do not agree? > 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 Nov 8, 2012, at 12:20 , herman.schreu...@sanofi.com wrote: > >> Dear Dr. Frolow, >> >> I do not agree. In the absence of heavy atom/anomalous data, the only way to >> distinguish e.g. between P41 and P43 is with molecular replacement. On could >> do it automatically, like is implemented in modern programs, or run MR in >> different space groups manually, but one has to test the various >> possibilities. >> >> Herman >> >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix >> Frolow >> Sent: Thursday, November 08, 2012 10:36 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] low-resolution and zinc >> >> Yogi, >> I was not mentioning MY book, it is not written yet :-) >> Zn an Ca are different element. Zn is transition element 24'th most abundant >> on earth, and Ca is alkaline element 5 most abundant on earth. >> But in the case of affinity to very strong binding site they behave >> similarly - they are picked by the molecule from the surrounding even if >> they are present in very low concentrations. >> >> Beeng in your position I will stop refinement and will take time to define >> space group properly. Difference P43 and P43212 (forget about screw axes - >> the point groups are important - P4 or P422) >> MUST be visible during data processing. If you did not inherited your data >> from the source going back in time and collected them (data) by yourself, >> difference between merging your data in P4 >> or P422 will be VERY visible. If the difference between them is negligible ( >> Rmerge factors say 0.04 in one case and 0.05 in another) you have space >> group P422 (or merohedral twinning in P4, I can't think clearly in this >> time of the day if such twinning is possible). If your space group is P4 >> and you try to merge it in space group P422, your Rmerge will be 0.4 -0.5. >> Generally, elegant practice of crystallography does not require >> determination of the space group using PHASER or MOLREP :-\ These >> facilities were inserted into molecular replacement programs for younger >> generation who come to protein crystallography with 0 (zero) of mathematics >> and physics
Re: [ccp4bb] low-resolution and zinc
Then we agree. I got confused because you mentioned"space group" and not
"point group" in your phrase about PHASER and MOLREP and was afraid
others might have gotten confused as well. Also, in case of twinning or
almost crystallographic non-crystallographic symmetry, determining the
point group on the basis of processing statistics alone can be
inconclusive or even misleading. If I recall correctly, there has
recently been a thread about this in the bulletin board.
Herman
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Felix Frolow
Sent: Thursday, November 08, 2012 1:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] low-resolution and zinc
I do not see with what you do not agree in what was written
(maybe not very carefully). One determine space group looking on
systematic absences, this I know.
Space groups P41 - P43 ( I do not go to more general discussion
for the sake of argument) are not distinguishable and one have to try
molecular replacement in both.
Most probably the correct space group will give a sensible
solution and wrong one will not. I was talking about distinguishing
between point groups P4 and P422.
I hope you grew that they CAN be distinguished on the level
prior to molecular replacement?
And I hope that you agree that starting refinement of demanding
project at relatively low resolution such as 3.4 Angstrom it is
advisable to characterise space group, twinning status etc.?
If you agree also to that, with what you do not agree?
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 Nov 8, 2012, at 12:20 , herman.schreu...@sanofi.com wrote:
Dear Dr. Frolow,
I do not agree. In the absence of heavy atom/anomalous
data, the only way to distinguish e.g. between P41 and P43 is with
molecular replacement. On could do it automatically, like is implemented
in modern programs, or run MR in different space groups manually, but
one has to test the various possibilities.
Herman
From: CCP4 bulletin board
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix Frolow
Sent: Thursday, November 08, 2012 10:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] low-resolution and zinc
Yogi,
I was not mentioning MY book, it is not written
yet :-)
Zn an Ca are different element. Zn is transition
element 24'th most abundant on earth, and Ca is alkaline element 5 most
abundant on earth.
But in the case of affinity to very strong
binding site they behave similarly - they are picked by the molecule
from the surrounding even if they are present in very low
concentrations.
Beeng in your position I will stop refinement
and will take time to define space group properly. Difference P43 and
P43212 (forget about screw axes - the point groups are important - P4 or
P422)
MUST be visible during data processing. If you
did not inherited your data from the source going back in time and
collected them (data) by yourself, difference between merging your data
in P4
or P422 will be VERY visible. If the difference
between them is negligible ( Rmerge factors say 0.04 in one case and
0.05 in another) you have space group P422 (or merohedral twinning in
P4, I can't think clearly in this time of the day if such twinning is
possible). If your space group is P4 and you try to merge it in space
group P422, your Rmerge will be 0.4 -0.5.
Generally, elegant practice of crystallography
does not require determination of the space group using PHASER or MOLREP
:-\ These facilities were inserted into molecular replacement programs
for younger generation who come to protein crystallography with 0 (zero)
of mathematics and physics and are surrounded by similar flock.
In the moment you will know what your space
group is and you will know if the twinning is present, you can
concentrate only on refinement. In your case (3.4 Angstrom resolution)
you will find DEN extremely useful.
FF
Dr Felix Frolow
Professor of Structural Biology and
Biotechnology, Department
Re: [ccp4bb] low-resolution and zinc
I do not see with what you do not agree in what was written (maybe not very carefully). One determine space group looking on systematic absences, this I know. Space groups P41 - P43 ( I do not go to more general discussion for the sake of argument) are not distinguishable and one have to try molecular replacement in both. Most probably the correct space group will give a sensible solution and wrong one will not. I was talking about distinguishing between point groups P4 and P422. I hope you grew that they CAN be distinguished on the level prior to molecular replacement? And I hope that you agree that starting refinement of demanding project at relatively low resolution such as 3.4 Angstrom it is advisable to characterise space group, twinning status etc.? If you agree also to that, with what you do not agree? 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 Nov 8, 2012, at 12:20 , herman.schreu...@sanofi.com wrote: > Dear Dr. Frolow, > > I do not agree. In the absence of heavy atom/anomalous data, the only way to > distinguish e.g. between P41 and P43 is with molecular replacement. On could > do it automatically, like is implemented in modern programs, or run MR in > different space groups manually, but one has to test the various > possibilities. > > Herman > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix > Frolow > Sent: Thursday, November 08, 2012 10:36 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] low-resolution and zinc > > Yogi, > I was not mentioning MY book, it is not written yet :-) > Zn an Ca are different element. Zn is transition element 24'th most abundant > on earth, and Ca is alkaline element 5 most abundant on earth. > But in the case of affinity to very strong binding site they behave similarly > - they are picked by the molecule from the surrounding even if they are > present in very low concentrations. > > Beeng in your position I will stop refinement and will take time to define > space group properly. Difference P43 and P43212 (forget about screw axes - > the point groups are important - P4 or P422) > MUST be visible during data processing. If you did not inherited your data > from the source going back in time and collected them (data) by yourself, > difference between merging your data in P4 > or P422 will be VERY visible. If the difference between them is negligible ( > Rmerge factors say 0.04 in one case and 0.05 in another) you have space group > P422 (or merohedral twinning in P4, I can't think clearly in this time of > the day if such twinning is possible). If your space group is P4 and you try > to merge it in space group P422, your Rmerge will be 0.4 -0.5. > Generally, elegant practice of crystallography does not require determination > of the space group using PHASER or MOLREP :-\ These facilities were inserted > into molecular replacement programs for younger generation who come to > protein crystallography with 0 (zero) of mathematics and physics and are > surrounded by similar flock. > In the moment you will know what your space group is and you will know if the > twinning is present, you can concentrate only on refinement. In your case > (3.4 Angstrom resolution) you will find DEN extremely useful. > 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 Nov 8, 2012, at 05:27 , SD Y wrote: > >> Dear Prof. Frolow, >> >> Our library has that book and I will look read it. I will also look in to >> your book too. >> I haven't been able to differentiate between P43 and P43 21 2, all refining >> results in similar numbers. P43 is slightly better with R work/Rfree is >> 30.5/37%. But its stuck there. >> I have built everything except Zn co-ordination. I will read your chapters >> to learn about SG. >> I understand that Zn is same as Ca as you are suggesting. >> I will also follow the other suggestion you have made regarding Anomolous >> signal. >> >> I sincerely appreciate your time and help which I was very much in need of. >> >> Thanks >> Yogi >> >> From: mbfro...@post.tau.ac.il >> Subject: Re: [ccp4bb] low-resolution and zinc >> Date: Wed, 7 Nov 20
Re: [ccp4bb] low-resolution and zinc
Dear Dr. Frolow,
I do not agree. In the absence of heavy atom/anomalous data, the only
way to distinguish e.g. between P41 and P43 is with molecular
replacement. On could do it automatically, like is implemented in modern
programs, or run MR in different space groups manually, but one has to
test the various possibilities.
Herman
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Felix Frolow
Sent: Thursday, November 08, 2012 10:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] low-resolution and zinc
Yogi,
I was not mentioning MY book, it is not written yet :-)
Zn an Ca are different element. Zn is transition element 24'th
most abundant on earth, and Ca is alkaline element 5 most abundant on
earth.
But in the case of affinity to very strong binding site they
behave similarly - they are picked by the molecule from the surrounding
even if they are present in very low concentrations.
Beeng in your position I will stop refinement and will take time
to define space group properly. Difference P43 and P43212 (forget about
screw axes - the point groups are important - P4 or P422)
MUST be visible during data processing. If you did not inherited
your data from the source going back in time and collected them (data)
by yourself, difference between merging your data in P4
or P422 will be VERY visible. If the difference between them is
negligible ( Rmerge factors say 0.04 in one case and 0.05 in another)
you have space group P422 (or merohedral twinning in P4, I can't think
clearly in this time of the day if such twinning is possible). If your
space group is P4 and you try to merge it in space group P422, your
Rmerge will be 0.4 -0.5.
Generally, elegant practice of crystallography does not require
determination of the space group using PHASER or MOLREP :-\ These
facilities were inserted into molecular replacement programs for younger
generation who come to protein crystallography with 0 (zero) of
mathematics and physics and are surrounded by similar flock.
In the moment you will know what your space group is and you
will know if the twinning is present, you can concentrate only on
refinement. In your case (3.4 Angstrom resolution) you will find DEN
extremely useful.
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 Nov 8, 2012, at 05:27 , SD Y wrote:
Dear Prof. Frolow,
Our library has that book and I will look read it. I
will also look in to your book too.
I haven't been able to differentiate between P43 and P43
21 2, all refining results in similar numbers. P43 is slightly better
with R work/Rfree is 30.5/37%. But its stuck there.
I have built everything except Zn co-ordination. I will
read your chapters to learn about SG.
I understand that Zn is same as Ca as you are
suggesting.
I will also follow the other suggestion you have made
regarding Anomolous signal.
I sincerely appreciate your time and help which I was
very much in need of.
Thanks
Yogi
From: mbfro...@post.tau.ac.il
Subject: Re: [ccp4bb] low-resolution and zinc
Date: Wed, 7 Nov 2012 23:35:35 +0200
To: ccp4...@hotmail.com
It is THE BOOK published in 1976! There is a chapter
about determination of a space group (actually speaking enantiomeric
space groups such as P3121 or P3112). But it can be
expanded to anything, as in Blandell (and mine) times we have used to
take so called presses ion phortographs from which the space groups
where easily and swiftly determined and only so called enantiomorphic
space groups remained elusive
As far as Zn is concerned I am talking about traces. We
work with calcium binding proteins and never add calcium. Calcium is
taken from who knows where, but it is there, in the
binding site. We actually know where from - from traces of the elements.
As I believe it can be anything.
FF
FF
Dr Felix Frolow
Professor of Structural Biology and Biotechnology,
Department of Molecular Microbiology and Biotechnology
Tel Aviv University 69978, Israel
Acta Crystallographi
Re: [ccp4bb] low-resolution and zinc
Yogi, I was not mentioning MY book, it is not written yet :-) Zn an Ca are different element. Zn is transition element 24'th most abundant on earth, and Ca is alkaline element 5 most abundant on earth. But in the case of affinity to very strong binding site they behave similarly - they are picked by the molecule from the surrounding even if they are present in very low concentrations. Beeng in your position I will stop refinement and will take time to define space group properly. Difference P43 and P43212 (forget about screw axes - the point groups are important - P4 or P422) MUST be visible during data processing. If you did not inherited your data from the source going back in time and collected them (data) by yourself, difference between merging your data in P4 or P422 will be VERY visible. If the difference between them is negligible ( Rmerge factors say 0.04 in one case and 0.05 in another) you have space group P422 (or merohedral twinning in P4, I can't think clearly in this time of the day if such twinning is possible). If your space group is P4 and you try to merge it in space group P422, your Rmerge will be 0.4 -0.5. Generally, elegant practice of crystallography does not require determination of the space group using PHASER or MOLREP :-\ These facilities were inserted into molecular replacement programs for younger generation who come to protein crystallography with 0 (zero) of mathematics and physics and are surrounded by similar flock. In the moment you will know what your space group is and you will know if the twinning is present, you can concentrate only on refinement. In your case (3.4 Angstrom resolution) you will find DEN extremely useful. 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 Nov 8, 2012, at 05:27 , SD Y wrote: > Dear Prof. Frolow, > > Our library has that book and I will look read it. I will also look in to > your book too. > I haven't been able to differentiate between P43 and P43 21 2, all refining > results in similar numbers. P43 is slightly better with R work/Rfree is > 30.5/37%. But its stuck there. > I have built everything except Zn co-ordination. I will read your chapters to > learn about SG. > I understand that Zn is same as Ca as you are suggesting. > I will also follow the other suggestion you have made regarding Anomolous > signal. > > I sincerely appreciate your time and help which I was very much in need of. > > Thanks > Yogi > > From: mbfro...@post.tau.ac.il > Subject: Re: [ccp4bb] low-resolution and zinc > Date: Wed, 7 Nov 2012 23:35:35 +0200 > To: ccp4...@hotmail.com > > It is THE BOOK published in 1976! There is a chapter about determination of a > space group (actually speaking enantiomeric > space groups such as P3121 or P3112). But it can be expanded to anything, as > in Blandell (and mine) times we have used to take so called presses ion > phortographs from which the space groups where easily and swiftly determined > and only so called enantiomorphic space groups remained elusive…. > As far as Zn is concerned I am talking about traces. We work with calcium > binding proteins and never add calcium. Calcium is > taken from who knows where, but it is there, in the binding site. We actually > know where from - from traces of the elements. > As I believe it can be anything. > FF > 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 Nov 7, 2012, at 20:13 , SD Y wrote: > > Dear Prof. Frolow, > > During sample development I have not used anything related to Zn but could be > from traces of contamination from Tris, NaCl, DTT, EDTA, LiSO4, HEPES and > other salt which were part of auto induction media. > > I am trying to search the refence in google. Are you refering to the Book > published in 1976 titled "protein crystallography", if not could you please > kindly direct me to right reference. > > I sincerely appreciate your time and suggestion. > > Warm reagrds, > SDY > > > > > > > > > From: mbfro...@post.tau.ac.il > Subject: Re: [ccp4bb] low-resolution and zinc > Date: Wed, 7 Nov 2012 19:35:21 +0200 > To: ccp4...@hotmail.com > > Zn is always there as anything else. > If you have high affinity binding site, it will be
Re: [ccp4bb] low-resolution and zinc
Well, galvanised iron is an old story of Zn in insuline… :-\ 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 Nov 7, 2012, at 23:58 , Katherine Sippel wrote: > As a follow up to Roger's statement if you are doing any sort of analytical > metal analysis be careful with the controls (metal-free water/acid washed > glassware). Also most AC/heating systems include galvanized steel in the duct > work that spits out Zn like crazy and can screw up your measurements. If you > have access to a neurotically OCD analytical chemist to assist I'd suggest > plying them with coffee and complements. > > Cheers, > Katherine > > On Wed, Nov 7, 2012 at 3:41 PM, Felix Frolow wrote: > As far as sigma level is concerned, and if I remember that you are working at > 3.4 angstrom resolution - this 6 sigma is VERY STRONG. > I am sure it is a metal atom. But you can re-process your data preserving > anomalous signal and calcule anomalous map easily done in > PHENIX, less so in CCP4 and then displaying anomalous map as a difference map > in COOT you MUST see strong peak on this map in the heavy atom location. > 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 Nov 7, 2012, at 20:13 , SD Y wrote: > >> Dear Prof. Frolow, >> >> During sample development I have not used anything related to Zn but could >> be from traces of contamination from Tris, NaCl, DTT, EDTA, LiSO4, HEPES and >> other salt which were part of auto induction media. >> >> I am trying to search the refence in google. Are you refering to the Book >> published in 1976 titled "protein crystallography", if not could you please >> kindly direct me to right reference. >> >> I sincerely appreciate your time and suggestion. >> >> Warm reagrds, >> SDY >> >> >> >> >> >> >> >> >> From: mbfro...@post.tau.ac.il >> Subject: Re: [ccp4bb] low-resolution and zinc >> Date: Wed, 7 Nov 2012 19:35:21 +0200 >> To: ccp4...@hotmail.com >> >> Zn is always there as anything else. >> If you have high affinity binding site, it will be filled with Zn (or >> similar) on the various stages of your >> sample development. >> BTW use old Blandell&Johnson popular in my time (70-90's) approach - in the >> correct space group the peak hight of this heavy atom will be the highest >> comparing to other space groups >> 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 Nov 7, 2012, at 19:29 , SD Y wrote: >> >> Dear all, >> >> I have a related question to the one I have posted "low resolution and SG", >> on which I am still working based on the suggestions I have got. >> >> The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 >> cys and 1 His residues. They have add Zn in to their experiment. >> In my 3.4 A structure (I am still working on right SG), initial maps show >> very strong positive density (sigma=6.5) at the place of Zn ( >> https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not >> used Zn in my experiment. I could only suspect Tryptone and yeast extract >> which I used to make media. >> >> I would like to know how likely this positive density belongs to Zn? How to >> reason the presence of Zn when its not been used? >> Is there is any way to confirm if its Zn. If this is not Zn, what else could >> it be? Any thing I could try to rule out or in Zn or other ions. >> I appreciate your help and suggestions. >> >> Sincerely, >> SDY >> >> > >
Re: [ccp4bb] low-resolution and zinc
As a follow up to Roger's statement if you are doing any sort of analytical metal analysis be careful with the controls (metal-free water/acid washed glassware). Also most AC/heating systems include galvanized steel in the duct work that spits out Zn like crazy and can screw up your measurements. If you have access to a neurotically OCD analytical chemist to assist I'd suggest plying them with coffee and complements. Cheers, Katherine On Wed, Nov 7, 2012 at 3:41 PM, Felix Frolow wrote: > As far as sigma level is concerned, and if I remember that you are working > at 3.4 angstrom resolution - this 6 sigma is VERY STRONG. > I am sure it is a metal atom. But you can re-process your data preserving > anomalous signal and calcule anomalous map easily done in > PHENIX, less so in CCP4 and then displaying anomalous map as a difference > map in COOT you MUST see strong peak on this map in the heavy atom location. > 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 Nov 7, 2012, at 20:13 , SD Y wrote: > > Dear Prof. Frolow, > > During sample development I have not used anything related to Zn but could > be from traces of contamination from Tris, NaCl, DTT, EDTA, LiSO4, HEPES > and other salt which were part of auto induction media. > > I am trying to search the refence in google. Are you refering to the Book > published in 1976 titled "protein crystallography", if not could you please > kindly direct me to right reference. > > I sincerely appreciate your time and suggestion. > > Warm reagrds, > SDY > > > > > > > > -- > From: mbfro...@post.tau.ac.il > Subject: Re: [ccp4bb] low-resolution and zinc > Date: Wed, 7 Nov 2012 19:35:21 +0200 > To: ccp4...@hotmail.com > > Zn is always there as anything else. > If you have high affinity binding site, it will be filled with Zn (or > similar) on the various stages of your > sample development. > BTW use old Blandell&Johnson popular in my time (70-90's) approach - in > the correct space group the peak hight of this heavy atom will be the > highest comparing to other space groups > 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 Nov 7, 2012, at 19:29 , SD Y wrote: > > Dear all, > > I have a related question to the one I have posted "low resolution and > SG", on which I am still working based on the suggestions I have got. > > The model I have used, has Zn co-ordinated well in tetrahydral fashion by > 3 cys and 1 His residues. They have add Zn in to their experiment. > In my 3.4 A structure (I am still working on right SG), initial maps > show very strong positive density (sigma=6.5) at the place of Zn ( > https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have > not used Zn in my experiment. I could only suspect Tryptone and > yeast extract which I used to make media. > > I would like to know how likely this positive density belongs to Zn? How > to reason the presence of Zn when its not been used? > Is there is any way to confirm if its Zn. If this is not Zn, what else > could it be? Any thing I could try to rule out or in Zn or other ions. > I appreciate your help and suggestions. > > Sincerely, > SDY > > > > >
Re: [ccp4bb] low-resolution and zinc
As far as sigma level is concerned, and if I remember that you are working at 3.4 angstrom resolution - this 6 sigma is VERY STRONG. I am sure it is a metal atom. But you can re-process your data preserving anomalous signal and calcule anomalous map easily done in PHENIX, less so in CCP4 and then displaying anomalous map as a difference map in COOT you MUST see strong peak on this map in the heavy atom location. 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 Nov 7, 2012, at 20:13 , SD Y wrote: > Dear Prof. Frolow, > > During sample development I have not used anything related to Zn but could be > from traces of contamination from Tris, NaCl, DTT, EDTA, LiSO4, HEPES and > other salt which were part of auto induction media. > > I am trying to search the refence in google. Are you refering to the Book > published in 1976 titled "protein crystallography", if not could you please > kindly direct me to right reference. > > I sincerely appreciate your time and suggestion. > > Warm reagrds, > SDY > > > > > > > > > From: mbfro...@post.tau.ac.il > Subject: Re: [ccp4bb] low-resolution and zinc > Date: Wed, 7 Nov 2012 19:35:21 +0200 > To: ccp4...@hotmail.com > > Zn is always there as anything else. > If you have high affinity binding site, it will be filled with Zn (or > similar) on the various stages of your > sample development. > BTW use old Blandell&Johnson popular in my time (70-90's) approach - in the > correct space group the peak hight of this heavy atom will be the highest > comparing to other space groups > 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 Nov 7, 2012, at 19:29 , SD Y wrote: > > Dear all, > > I have a related question to the one I have posted "low resolution and SG", > on which I am still working based on the suggestions I have got. > > The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 > cys and 1 His residues. They have add Zn in to their experiment. > In my 3.4 A structure (I am still working on right SG), initial maps show > very strong positive density (sigma=6.5) at the place of Zn ( > https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not > used Zn in my experiment. I could only suspect Tryptone and yeast extract > which I used to make media. > > I would like to know how likely this positive density belongs to Zn? How to > reason the presence of Zn when its not been used? > Is there is any way to confirm if its Zn. If this is not Zn, what else could > it be? Any thing I could try to rule out or in Zn or other ions. > I appreciate your help and suggestions. > > Sincerely, > SDY > >
Re: [ccp4bb] low-resolution and zinc
Tryptone and/or yeast extract is loaded with metals, including zinc. It is almost impossible to keep zinc contamination at bay without using carefully defined media and taking heroic measures. (I know this because of the difficulty of making overexpressed metallo-substituted zinc-enzymes when this is the only way to do it). So, yes, it is very possible to observe zinc coordination in heterologously expressed proteins when exogenous zinc has not be explicitly included. I don't put that much stock in the sigma level. It is too variable and depends on the resolution, the current state of the maps (initial or nearly final phases), and occupancy. You should have very obvious positive difference density, however. Some clues to Zn-coordination include the following: (1) Zn(II) is often in a tetrahedral or pseudo-tetrahedral geometry, and typically accepts a mixture of hard to soft ligands; (2) Zn-S bonds are approximately 2.3 A; Zn-O or Zn-N bonds are approximately 2.0 A. If this is compatible with your unconstrained fit of the data to the density using a Zn(II) ion, then there is a strong possibility of Zn-coordination. To confirm Zn coordination, you can measure the Zn content of the protein sample using ICP-OES, ICP-MS, or TXRF. For ICP-OES, I usually dilute a protein sample at 10-50 mg/mL 1:50 to 1:150 and analyze a 1.5 mL sample. You can do multiple metals at the same time, including Zn. I usually also look for Fe, Ni, Cu, Mn and do Co as a control (Co should be zero or nearly so.) Once you measure metal content, you can estimate occupancy by measuring the Zn/protein molar ratio. If you do this, use a non-idiosyncratic (sequence-insensitive) protein assay. I would recommend a UV-based method using A210 in 0.01% detergent (e.g. Triton or Brij) For homogeneous proteins, the A210 method is usually accurate within 5%, which is sufficient to get usable metal:protein ratios. If you must use a colorimetric method, the only one I trust not to be idiosyncratic is the microbiuret method, which is as old as dirt. It usually agrees with the A210 method. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/7/2012 12:29 PM, SD Y wrote: Dear all, I have a related question to the one I have posted "low resolution and SG", on which I am still working based on the suggestions I have got. The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 cys and 1 His residues. They have add Zn in to their experiment. In my 3.4 A structure (I am still working on right SG), initial maps show very strong positive density (sigma=6.5) at the place of Zn ( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not used Zn in my experiment. I could only suspect Tryptone and yeast extract which I used to make media. I would like to know how likely this positive density belongs to Zn? How to reason the presence of Zn when its not been used? Is there is any way to confirm if its Zn. If this is not Zn, what else could it be? Any thing I could try to rule out or in Zn or other ions. I appreciate your help and suggestions. Sincerely, SDY
Re: [ccp4bb] low-resolution and zinc
On Wed, 2012-11-07 at 11:29 -0600, SD Y wrote: > https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png Your sigma level of 6.5 seems a bit low, so maybe it is a different metal. But on your main question - yes, metal binding proteins do pick up metals from the media. Once metal is coordinated, it is not very likely to leave, unless you remove it on purpose. Place Zn there and refine. Then see if you have a) residual density and b) B-factor mismatch with coordinating residues. If so, you may try different metals and see what fits better, although at this resolution it's tricky. It is also possible that you have partial metal occupancy. Depending on the wavelength you used you may be able partially confirm or completely reject zinc option by looking at anomalous difference maps. If you have more crystals and access to tunable beam, you should be able to run fluorescence scan to see what metal you got. If not, there is ICP-MS for metal identification, you would have to run it with your protein sample and you need access to an instrument. Cheers, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
[ccp4bb] low-resolution and zinc
Dear all, I have a related question to the one I have posted "low resolution and SG", on which I am still working based on the suggestions I have got. The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 cys and 1 His residues. They have add Zn in to their experiment. In my 3.4 A structure (I am still working on right SG), initial maps show very strong positive density (sigma=6.5) at the place of Zn ( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not used Zn in my experiment. I could only suspect Tryptone and yeast extract which I used to make media. I would like to know how likely this positive density belongs to Zn? How to reason the presence of Zn when its not been used?Is there is any way to confirm if its Zn. If this is not Zn, what else could it be? Any thing I could try to rule out or in Zn or other ions. I appreciate your help and suggestions. Sincerely, SDY
