Re: [ccp4bb] Questions about PanDDA modelling and refining
**Question:**
After step 11 in my pandda-export folder I have the following files:
File name:
Remarks:
1
ensemble-model.log
2
ensemble-model.pdb
Contains both bound and ground-state
3
ensemble-model-restraints.log
4
ensemble-model-restraints.phenix.params
5
ensemble-model-restraints.refmac.params
6
pandda-input.mtz
Does not contain the "event density" of ligand
7
pandda-input.pdb
Does not contain ligand
8
pandda-model.pdb
Contains bound state of ligand
9
pandda-output.mtz
Does not contain the "event density" of ligand
10
pandda-output-event-001.mtz
Contains the "event density" of ligand
In the file ensemble-model.pdb I see water and ligand on the same spot. My
protein normally has water in that site but if the ligand is bound - there
is no water.
**Answer:**
Hello, I recently went through several challenges with ligand
identification using PanDDA, but I hope I can now help you.
Firstly, explaining your files after pandda.export:
1. The ensemble model is the file that contains a combination of the
unbound file (pandda-input) and the file you modeled during the
pandda.inspect step (pandda-model). In the final step of pandda.export, it
automatically runs giant.merge_conformations, which generates the ensemble
model, the restraints, and their respective logs.
2. The pandda-input files are those you submitted to pandda.analyse, so
they are the inputs for pandda.
3. The pandda model is the file you modeled and saved during the
pandda.inspect step.
The only thing I don't understand about your pandda export output files is
that your event map “pandda-output-event-001.mtz” is in mtz format when it
should be in .native.ccp4 format. Additionally, the z-map was not extracted
by pandda.export, and it is crucial for analyzing blobs in the event map. I
also can't understand what this output.mtz refers to.
These were my output files (very similar to yours except for the
differences I pointed out). Notice that I have the event map and the z-map
in .native.ccp4 format:
The first question then is: what version of PanDDA and CCP4 are you using?
This might help us understand why these differences were observed.
---
**Question:**
Further modeling is required for my structures, and thus I move on to step
13 where I get confused. I have highlighted in bold the instructions of the
tutorial.
Generation of restraints for refinement (giant.make_restraints) It says:
"...The output restraint files can then be fed to giant.quick_refine which
then either runs phenix or refmac (see below)." Should I skip this step as
I already have the phenix.params and refmac.params files?
**Answer:**
Yes, the output files from pandda.export already contain the restraints you
will need for refinement.
---
**Question:**
Quick-and-easy refinement (giant.quick_refine) To make refinement more
straightforward giant.quick_refine can be used to refine the models. A
normal refinement will look like
giant.quick_refine input.pdb input.mtz ligand.cif restraints.params
Which files should I use here? The pandda-input.pdb pandda-input.mtz
ligand.cif and ensemble-model-refmac.params? If the .params file is for the
ensemble, should I rather use the ensemble-model.pdb
pandda-output-event-001.mtz ligand.cif and ensemble-model-refmac-params?
**Answer:**
In the first refinement cycle using giant.refine, you should use the files:
ensemble-model.pdb, pandda-input.mtz, ligand.cif, and either
ensemble-model-refmac.params or ensemble-model-phenix.params.
---
**Question:**
If I want to include additional parameterization for the refmac, do I
manually add instructions in the .params file? For example, if I want to
increase the weight parameter?
**Answer:**
I don't know. If anyone else can clarify this question, it would be greatly
appreciated.
---
**Question:**
Splitting the ensemble model (giant.split_conformations) Should it be done
on the output of step 13.2?
**Answer:**
After the first refinement cycle, a folder “refine001” will be created with
the following files:
This output.pdb is the refined ensemble model. If adjustments to the ligand
are necessary, it is advisable to split the ensemble into bound and unbound
models because working with the ensemble model can be confusing due to the
multiple alternate conformations it contains. After the split, you can
separately model the unbound state using the output mtz normally, and the
bound state using the event map.
---
**Question:**
(Re-)modelling of the bound-/ground-states (coot) The ground-state
conformations should be modeled into a ground-state ("reference")
dataset/map (e.g., "*-ground-state-average-map.native.ccp4" from
pandda.analyse). I do not have any file in the format native.ccp4
Should I do it with the file pandda-input.pdb with pandda-input.mtz? The
bound-state conformations of the protein are modeled into the appropriate
event maps as in pandda.inspect. Should I use the files pandda-model.pdb
with pandda-ouput-event-001.mtz?
[ccp4bb] Questions about PanDDA modelling and refining
Hello,
I am learning how to use PanDDA and would appreciate some clarifications on the
modeling and refining steps.
I realize that maybe some questions will be too obvious, but I would appreciate
the confirmation of my guesses so I might learn to do it the correct way.
I managed to get to step 13 of the tutorial
(https://pandda.bitbucket.io/pandda/tutorials.html#) (but with my data).
After step 11 in my pandda-export folder, I have the following files:
File name:
Remarks:
1
ensemble-model.log
2
ensemble-model.pdb
Contains both bound and ground-state
3
ensemble-model-restraints.log
4
ensemble-model-restraints.phenix.params
5
ensemble-model-restraints.refmac.params
6
pandda-input.mtz
Does not contain the "event density" of ligand
7
pandda-input.pdb
Does not contain ligand
8
pandda-model.pdb
Contains bound state of ligand
9
pandda-output.mtz
Does not contain the "event density" of ligand
10
pandda-output-event-001.mtz
Contains the "event density" of ligand
In the file ensemble-model.pdb I see water and ligand on the same spot. My
protein normally has water in that site, but if the ligand is bound - there is
no water.
Further modeling is required for my structures and thus I move on to step 13
where I get confused. I have highlighted in bold the instructions of the
tutorial.
1.
Generation of restraints for refinement (giant.make_restraints)
It says: "...The output restraint files can then be fed to giant.quick_refine,
which then either runs phenix or refmac (see below)."
Should I skip this step as I already have the phenix.params and refmac.params
files?
1.
Quick-and-easy refinement (giant.quick_refine)
To make refinement more straightforward, giant.quick_refine can be used to
refine the models.
A normal refinement will look like
giant.quick_refine input.pdb input.mtz ligand.cif restraints.params
Which files should I use here? The pandda-input.pdb, pandda-input.mtz
ligand.cif and ensemble-model-refmac.params?
If the .params file is for the ensemble, should I rather use then
ensebmle-model.pdb, padda-output-event-001.mtz ligand.cif and
ensemble-model-refmac-params?
If I want to include additional parameterization for the refmac, do I manually
add instructions in the .params file? For example, if I want to increase the
weight parameter?
2.
Splitting the ensemble model (giant.split_conformations)
Should it be done on the output of the step 13.2?
3.
(Re-)modelling of the bound-/ground-states (coot)
The ground-state conformations should be modelled into a ground-state
("reference") dataset/map (e.g. "*-ground-state-average-map.native.ccp4" from
pandda.analyse).
I do not have any file in the format native.ccp4
Should I do it with the file pandda-input.pdb with pandda-input.mtz?
The bound-state conformations of the protein are modeled into the appropriate
event maps, as in pandda.inspect.
Should I use the files pandda-model.pdb with pandda-ouput-event-001.mtz?
However, if you want to edit both structures simultaneously (e.g. to add a
molecule that is the same in both states), then this should be done to the
combined ensemble model.
Should I use ensemble-model.pdb with pandda-output-event-001.mtz?
4.
Re-merging single-state models for refinement.
After you have re-modelled the bound- and ground-states of the protein, you
need re-assemble the ensemble model for refinement.
giant.merge_conformations ground-state.pdb bound-state.pdb
Maybe it will be more clear which exact files to use here once I go through
steps 1-4, but if there is something I should be aware of during this step -
please let me know.
Please let me know if you any of my questions should be clarified more.
I will greatly appreciate any help I can get with this!
Best regards,
Vladyslav
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Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Hi Joao Can you post the mtzdmp output? Cheers Ian On Tue, 16 Jun 2020, 09:30 Joao Ramos, wrote: > Dear Ian, > > I'm trying to use the edstats.pl script but I'm having problems with the > column labels. mtzdmp can read the mtz file, but edstats.pl gives me a > message saying that mtzfix cannot find the labels. I'm using a mtz file > from a refinement job in Phenix. > > Best regards, > Joao Ramos > > Ian Tickle escreveu no dia terça, 16/06/2020 à(s) > 09:39: > >> Hi João >> >> In cases like this it's always a good idea to check whether you can read >> the same MTZ file with mtzdmp. If that fails with the same error message >> it means that the pathname/filename was indeed incorrectly specified, or it >> could possibly be a permissions issue (the file needs to be given read >> permission). >> >> Cheers >> >> Ian >> >> >> On Mon, 15 Jun 2020, 19:09 Ian Tickle, wrote: >> >>> >>> Hi Joao >>> >>> The top of the page was cut off in your screenshot so I can't see the >>> command line you typed. It would be better to copy/paste the entire text, >>> including the input command line and the output, into the email rather than >>> taking a screenshot. 'Cannot open file' most likely means that the input >>> filename was not typed correctly or the path was not specified correctly if >>> it's in another directory. Anyway you really shouldn't be calling the >>> MTZFIX or EDSTATS programs directly. Using the Perl script 'edstats.pl >>> ' http://www.ccp4.ac.uk/dist/checkout/edstats/edstats.pl is much >>> easier. It should be in your path after running the CCP4 setup script. >>> >>> Cheers >>> >>> -- Ian >>> >>> >>> On Mon, 15 Jun 2020 at 18:41, Joao Ramos >>> wrote: >>> Dear Ian, Thanks for the reply! I've sourced the setup script, but now I get a 'cannot open file' error message (screenshot attached) when trying to run MTZFIX. Best regards, Joao Ramos Ian Tickle escreveu no dia segunda, 15/06/2020 à(s) 16:57: > > Hi Joao > > For all CCP4 programs you need to source your CCP4 setup script. > > Cheers > > -- Ian > > > On Mon, 15 Jun 2020 at 15:29, Joao Ramos > wrote: > >> Dear CCP4bb, >> >> I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a >> Phenix refinement job, in order to have a more reliable analysis of model >> quality. From what I read, one should go through the program MTZFIX and >> then create the FFT maps (density and difference maps). However, while >> attempting to run the Phenix output mtz file in MTZFIX, I receive an >> error >> message saying: "Cannot open environ.def". >> >> My questions are: >> 1) Is the way to go from the Phenix refinement output files to >> EDSTATS described earlier correct? Are there alternative programs >> included >> in Phenix? >> 2) How to fix the error and run MTZFIX? >> >> I'm currently using the new ccp4 v7.1, in macOS v10.14.5. >> >> Best regards, >> Joao Ramos >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 >> > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Dear Ian, I'm trying to use the edstats.pl script but I'm having problems with the column labels. mtzdmp can read the mtz file, but edstats.pl gives me a message saying that mtzfix cannot find the labels. I'm using a mtz file from a refinement job in Phenix. Best regards, Joao Ramos Ian Tickle escreveu no dia terça, 16/06/2020 à(s) 09:39: > Hi João > > In cases like this it's always a good idea to check whether you can read > the same MTZ file with mtzdmp. If that fails with the same error message > it means that the pathname/filename was indeed incorrectly specified, or it > could possibly be a permissions issue (the file needs to be given read > permission). > > Cheers > > Ian > > > On Mon, 15 Jun 2020, 19:09 Ian Tickle, wrote: > >> >> Hi Joao >> >> The top of the page was cut off in your screenshot so I can't see the >> command line you typed. It would be better to copy/paste the entire text, >> including the input command line and the output, into the email rather than >> taking a screenshot. 'Cannot open file' most likely means that the input >> filename was not typed correctly or the path was not specified correctly if >> it's in another directory. Anyway you really shouldn't be calling the >> MTZFIX or EDSTATS programs directly. Using the Perl script 'edstats.pl' >> http://www.ccp4.ac.uk/dist/checkout/edstats/edstats.pl is much easier. >> It should be in your path after running the CCP4 setup script. >> >> Cheers >> >> -- Ian >> >> >> On Mon, 15 Jun 2020 at 18:41, Joao Ramos >> wrote: >> >>> Dear Ian, >>> >>> Thanks for the reply! >>> >>> I've sourced the setup script, but now I get a 'cannot open file' error >>> message (screenshot attached) when trying to run MTZFIX. >>> >>> Best regards, >>> Joao Ramos >>> >>> Ian Tickle escreveu no dia segunda, 15/06/2020 à(s) >>> 16:57: >>> Hi Joao For all CCP4 programs you need to source your CCP4 setup script. Cheers -- Ian On Mon, 15 Jun 2020 at 15:29, Joao Ramos wrote: > Dear CCP4bb, > > I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a > Phenix refinement job, in order to have a more reliable analysis of model > quality. From what I read, one should go through the program MTZFIX and > then create the FFT maps (density and difference maps). However, while > attempting to run the Phenix output mtz file in MTZFIX, I receive an error > message saying: "Cannot open environ.def". > > My questions are: > 1) Is the way to go from the Phenix refinement output files to EDSTATS > described earlier correct? Are there alternative programs included in > Phenix? > 2) How to fix the error and run MTZFIX? > > I'm currently using the new ccp4 v7.1, in macOS v10.14.5. > > Best regards, > Joao Ramos > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Hi João In cases like this it's always a good idea to check whether you can read the same MTZ file with mtzdmp. If that fails with the same error message it means that the pathname/filename was indeed incorrectly specified, or it could possibly be a permissions issue (the file needs to be given read permission). Cheers Ian On Mon, 15 Jun 2020, 19:09 Ian Tickle, wrote: > > Hi Joao > > The top of the page was cut off in your screenshot so I can't see the > command line you typed. It would be better to copy/paste the entire text, > including the input command line and the output, into the email rather than > taking a screenshot. 'Cannot open file' most likely means that the input > filename was not typed correctly or the path was not specified correctly if > it's in another directory. Anyway you really shouldn't be calling the > MTZFIX or EDSTATS programs directly. Using the Perl script 'edstats.pl' > http://www.ccp4.ac.uk/dist/checkout/edstats/edstats.pl is much easier. > It should be in your path after running the CCP4 setup script. > > Cheers > > -- Ian > > > On Mon, 15 Jun 2020 at 18:41, Joao Ramos > wrote: > >> Dear Ian, >> >> Thanks for the reply! >> >> I've sourced the setup script, but now I get a 'cannot open file' error >> message (screenshot attached) when trying to run MTZFIX. >> >> Best regards, >> Joao Ramos >> >> Ian Tickle escreveu no dia segunda, 15/06/2020 à(s) >> 16:57: >> >>> >>> Hi Joao >>> >>> For all CCP4 programs you need to source your CCP4 setup script. >>> >>> Cheers >>> >>> -- Ian >>> >>> >>> On Mon, 15 Jun 2020 at 15:29, Joao Ramos >>> wrote: >>> Dear CCP4bb, I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a Phenix refinement job, in order to have a more reliable analysis of model quality. From what I read, one should go through the program MTZFIX and then create the FFT maps (density and difference maps). However, while attempting to run the Phenix output mtz file in MTZFIX, I receive an error message saying: "Cannot open environ.def". My questions are: 1) Is the way to go from the Phenix refinement output files to EDSTATS described earlier correct? Are there alternative programs included in Phenix? 2) How to fix the error and run MTZFIX? I'm currently using the new ccp4 v7.1, in macOS v10.14.5. Best regards, Joao Ramos -- To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 >>> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Hi Joao The top of the page was cut off in your screenshot so I can't see the command line you typed. It would be better to copy/paste the entire text, including the input command line and the output, into the email rather than taking a screenshot. 'Cannot open file' most likely means that the input filename was not typed correctly or the path was not specified correctly if it's in another directory. Anyway you really shouldn't be calling the MTZFIX or EDSTATS programs directly. Using the Perl script 'edstats.pl' http://www.ccp4.ac.uk/dist/checkout/edstats/edstats.pl is much easier. It should be in your path after running the CCP4 setup script. Cheers -- Ian On Mon, 15 Jun 2020 at 18:41, Joao Ramos wrote: > Dear Ian, > > Thanks for the reply! > > I've sourced the setup script, but now I get a 'cannot open file' error > message (screenshot attached) when trying to run MTZFIX. > > Best regards, > Joao Ramos > > Ian Tickle escreveu no dia segunda, 15/06/2020 à(s) > 16:57: > >> >> Hi Joao >> >> For all CCP4 programs you need to source your CCP4 setup script. >> >> Cheers >> >> -- Ian >> >> >> On Mon, 15 Jun 2020 at 15:29, Joao Ramos >> wrote: >> >>> Dear CCP4bb, >>> >>> I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a >>> Phenix refinement job, in order to have a more reliable analysis of model >>> quality. From what I read, one should go through the program MTZFIX and >>> then create the FFT maps (density and difference maps). However, while >>> attempting to run the Phenix output mtz file in MTZFIX, I receive an error >>> message saying: "Cannot open environ.def". >>> >>> My questions are: >>> 1) Is the way to go from the Phenix refinement output files to EDSTATS >>> described earlier correct? Are there alternative programs included in >>> Phenix? >>> 2) How to fix the error and run MTZFIX? >>> >>> I'm currently using the new ccp4 v7.1, in macOS v10.14.5. >>> >>> Best regards, >>> Joao Ramos >>> >>> -- >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 >>> >> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions regarding MTZFIX and EDSTATS
Hi Joao For all CCP4 programs you need to source your CCP4 setup script. Cheers -- Ian On Mon, 15 Jun 2020 at 15:29, Joao Ramos wrote: > Dear CCP4bb, > > I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a > Phenix refinement job, in order to have a more reliable analysis of model > quality. From what I read, one should go through the program MTZFIX and > then create the FFT maps (density and difference maps). However, while > attempting to run the Phenix output mtz file in MTZFIX, I receive an error > message saying: "Cannot open environ.def". > > My questions are: > 1) Is the way to go from the Phenix refinement output files to EDSTATS > described earlier correct? Are there alternative programs included in > Phenix? > 2) How to fix the error and run MTZFIX? > > I'm currently using the new ccp4 v7.1, in macOS v10.14.5. > > Best regards, > Joao Ramos > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Questions regarding MTZFIX and EDSTATS
Dear CCP4bb, I'm trying to obtain RSC metrics using the CCP4 program EDSTATS for a Phenix refinement job, in order to have a more reliable analysis of model quality. From what I read, one should go through the program MTZFIX and then create the FFT maps (density and difference maps). However, while attempting to run the Phenix output mtz file in MTZFIX, I receive an error message saying: "Cannot open environ.def". My questions are: 1) Is the way to go from the Phenix refinement output files to EDSTATS described earlier correct? Are there alternative programs included in Phenix? 2) How to fix the error and run MTZFIX? I'm currently using the new ccp4 v7.1, in macOS v10.14.5. Best regards, Joao Ramos To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/wa.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Questions about antibody in crystallization
Yes, the hinge region between Fab and Fc is highly flexible. If you look at IgG by EM you will see many different conformations. Best to use Fab or scFv. Jarrod Mousa On Thu, Jun 22, 2017 at 1:39 PM, Cheng Zhang wrote: > Hi all, > > I have a naive question about antibodies. Many people used Fab fragments > in crystallization. I am wondering if it is possible to use the whole IgG > molecule with Fc fragment as well. Or it would be too flexible and bad for > crystallization? > > Thanks, > > Cheng > > > -- > - > Cheng Zhang >
Re: [ccp4bb] Questions about antibody in crystallization
The fact that the PDB holds hundreds of FABs and a handful whole ABs suggests to me that the latter are hard to get crystals for. So, all the more reason for us bioinformaticians that you, experimentalists try it :-) Gert On 22-6-2017 20:39, Cheng Zhang wrote: Hi all, I have a naive question about antibodies. Many people used Fab fragments in crystallization. I am wondering if it is possible to use the whole IgG molecule with Fc fragment as well. Or it would be too flexible and bad for crystallization? Thanks, Cheng -- - Cheng Zhang
[ccp4bb] Questions about antibody in crystallization
Hi all, I have a naive question about antibodies. Many people used Fab fragments in crystallization. I am wondering if it is possible to use the whole IgG molecule with Fc fragment as well. Or it would be too flexible and bad for crystallization? Thanks, Cheng -- - Cheng Zhang
Re: [ccp4bb] questions
Thanks to all of those that sent in their comments. For those that have an interest in the consensus (after ~24 hours): For question one about the PDB deposition code- out of 10: 3 said in footnote, 2 said they had either seen or had put the deposition code in the materials and methods, and a majority indicated that it shouldn't matter, but from a pragmatic standpoint the "editor is always right" or "house rules". There is no indication in the notes to authors for this journal, which would have been the obvious way to find out where this information should go. It was also pointed out that the IUCr journals helpfully put this on the front page, so again it shouldn't matter where it is in the text. For question two on the 'rotamer-quality score' from MolProbity- 6 guessed that what was meant was the number associated with poor rotamers (i.e. in this case 12) and possibly the percentage (2.3%), but no one was actually sure that this was the case. So although I guessed incorrectly, at least it was a bit of a sanity check for me, as I hate to miss the obvious. I also got some helpful hints as to how to improve this number. I'll go with the consensus and see whether I get another berating or relief at getting it correct. Thanks again to everyone for their comments. Cheers, tom From: Peat, Tom (CMSE, Parkville) Sent: Thursday, 17 October 2013 1:59 PM To: 'ccp4bb@jiscmail.ac.uk' Subject: questions Dear CCP4 community, I would like to tap into the collective wisdom of you folks on two questions, both of which have put me into the bad graces of a particular editor. The first question seems trivial, but I will ask it anyway- where would you put the PDB deposition code in a manuscript? I may be old fashioned, but I have put it in the footnotes just prior to the references (which also ends up being the acknowledgments section in some journals), into Table 1 or in some more chemistry oriented journals in the footnotes on the first page (often near the author information). I've been told that it obviously goes into the Materials and Methods section (where I cannot ever remember seeing it, but my memory seems to be fading with old age). I find this a little strange as I consider the final model to be a result and not a material used to produce data nor a method. But maybe people are now putting their results into the methods section. So opinions on this question are welcome. The second question is hopefully straight-forward. I was also asked to put a number in Table 1 which I am happy to do, but I don't understand how to get this number. I was asked to put the 'rotamer-quality score' from MolProbity into the table. I don't run MolProbity often, but the output I got from the server doesn't have a 'rotamer-quality score' that I can find (see attachment). Is there some option that I am missing that gives this elusive factor? I also took a look at the Chen et al paper (Acta Cryst D, 2010) on MolProbity and it mentions a rotamer-quality score for specific residues but doesn't refer to an overall score (which is what I am assuming is needed for a table). I already have the well known Ramachandran percentages (favourable, allowed /poor and outliers) in the table, so the editor clearly wants something different. When I took a guess by putting in the 'MolProbity score' I was basically called an idiot that can't follow directions. Help on this front would be appreciated as although I have been called worse things, it would be nice to eventually get what is being referred to. Thanks, tom Tom Peat CSIRO, Melbourne, Australia
Re: [ccp4bb] Questions on unknown density?
Hi, Try modeling a zinc close to the histidine and then do one round of refinement the density will improve close to that area, generally a zinc ion coordinated to histidine also has a water molecule close to the zinc. HTH, Shya On Tue, Jun 19, 2012 at 5:47 AM, Lianying Jiao wrote: > Dear all, > > As in *Screenshot2.png*, some unknown density exists around a histidine. > The crystallization condition contains Zn(OAc)2, PEG3350, pH4.5. Do anyone > tell me what is it? Thanks in advance. > > Sincerely, > Lianying Jiao >
Re: [ccp4bb] Questions on unknown density?
Check anomalous map. Likely a Zn Jürgen .. 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.mac.com/bosch_lab/ On Jun 19, 2012, at 5:58, "Lianying Jiao" wrote: > Dear all, > > As in Screenshot2.png, some unknown density exists around a histidine. The > crystallization condition contains Zn(OAc)2, PEG3350, pH4.5. Do anyone tell > me what is it? Thanks in advance. > > Sincerely, > Lianying Jiao >
Re: [ccp4bb] Questions on unknown density?
Hi, Without looking at your model/maps it is difficult to make a really educated guess. By looking at the single screen shot you attach, I would say it could be a couple of different things, in order of likelihood based on a single screenshot (at whatever level of confidence this assures): 1-Zn coordinate to His, and some waters around it. 2-Phosphorylation of the His. Is this expected or known for this enzyme? Although I dont really see nice tetrahedral geometry, and I dont know that you really would at your resolution. 3-Alternate His side chain conformation. I cant really say if this fits your density with just that one screen shot. It doesnt really look like it to me. I would inspect distances and angles for each of the scenarios presented above and then build a model that makes good chemical and physical sense. Hope his helps. yuri
[ccp4bb] Questions about cryoprotectants
Hi, Thank you for all your suggestions! I will test them and let you know what happens! Yuan
Re: [ccp4bb] Questions about cryoprotectant
Different suggestion: I assume you don't have problems obtaining crystals, so I'd setup a 24 or 96 well plate with your preferred condition then add to each row or column a different know cryo at low concentration e.g. 1-10%. Then under the conditions in which you still get crystals in the presence of the cryo I would soak the crystals in a proper cryo condition for this particular additive and flash freeze the crystal in liquid nitrogen and not in the stream. You seem to be very close to cracking your problem, don't give up and try a lot of things. As someone mentioned already do flash annealing and you can do this also with the liquid nitrogen by lifting your crystal out of the bath before plunging it back in - sure you're missing a data point there what would the diffraction have been if I had not done this. In my hands I ALWAYS try flash annealing with every crystal - you can only learn, there's nothing to loose. The question is only what do you call a bad diffracting crystal 3Å ? 4 Å 6 Å? Here's a more recent example July 2009 http://web.mac.com/bosch_lab/jbosch/Labfun.html#4 The crystals are tiny ~20x20x60 (not really visible in the image on the web) and before flash annealing diffracted to maybe 5A. I brought this data set home with 2.2Å longest cell 220 Å (shot at SSRl 9-2) Good luck, Jürgen On Oct 7, 2009, at 4:54 PM, ycheng wrote: - 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.mac.com/bosch_lab/
Re: [ccp4bb] Questions about cryoprotectant
re : Paratone-N oil. 1) I find it much easier to work with if you cut it 50:50 with light mineral oil. 2) he stress of moving mechanically weak crystals (needles or thin plates) from a crystallisation liquor to the more viscous Paratone can cause the crystal to visibly bend - which might not be such a good thing. But I find that paratone works really well for most chunky, solid crystals. re: the high mosiacity issues - maybe you could try some gentle dehydration of the crystals? Maybe preincubate the crystals in mother liquor + 10>20% PEG20k prior to freezing? HTH Dave 2009/10/7 Janet Newman : > One thing to do here is to work out where you have your problem. > > Is the crystal mosaic after you have introduced your potential cryo > solutions, but still at room temperature? If it is, there is very little > chance (snowballs in hell type chance) that it will get less mosaic when you > flash cool it. > > Can you transfer your crystal from the high AmPO4 to high AmSO4 ? or LiSO4 or > malonate (all known cryo-salts) and flash cool from there? > > Janet > > From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of ycheng > [ych...@email.unc.edu] > Sent: 08 October 2009 07:54 > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Questions about cryoprotectant > > Hi, > I am trying to find an appropriate cryo-condition for my protein crystals. > The mother liquid is 2-2.5M Ammonium phosphate dibasic > 100mM TrisHCL pH8. The room-temperature diffraction looks not bad > (mosaicity 0.8, resolution 2.6) But the diffraction turned to be very > mosaic if I freeze the crytals in the absence of cryos or in the presence > of mother liquid plus different concentration of glycerol (5%,10%,15%.20%). > I don't think the ice formation is the problem since I didn't see any ice > by my eyes or ice diffraction in the presence or absence of cryos. Also, I > didn't see any cracks on my crystals when I transfered them to the cryo > conditions I have already tried. > My question here is: > 1)what's the role of cryo? I know it helps prevent ice formation. Based on > my case, it looks like cryo might also help to keep the crytal packing good > when frozed. > 2) What do I need to do to find a good cryo? What in my mind is to try > other cryos like sucrose, PEG400, ethylene glycol. > > Thanks a lot for your attention! > > Yuan > -- Science knows it doesn't know everything...otherwise it would stop. - Dara O'Briain David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB
Re: [ccp4bb] Questions about cryoprotectant
One thing to do here is to work out where you have your problem. Is the crystal mosaic after you have introduced your potential cryo solutions, but still at room temperature? If it is, there is very little chance (snowballs in hell type chance) that it will get less mosaic when you flash cool it. Can you transfer your crystal from the high AmPO4 to high AmSO4 ? or LiSO4 or malonate (all known cryo-salts) and flash cool from there? Janet From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of ycheng [ych...@email.unc.edu] Sent: 08 October 2009 07:54 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions about cryoprotectant Hi, I am trying to find an appropriate cryo-condition for my protein crystals. The mother liquid is 2-2.5M Ammonium phosphate dibasic 100mM TrisHCL pH8. The room-temperature diffraction looks not bad (mosaicity 0.8, resolution 2.6) But the diffraction turned to be very mosaic if I freeze the crytals in the absence of cryos or in the presence of mother liquid plus different concentration of glycerol (5%,10%,15%.20%). I don't think the ice formation is the problem since I didn't see any ice by my eyes or ice diffraction in the presence or absence of cryos. Also, I didn't see any cracks on my crystals when I transfered them to the cryo conditions I have already tried. My question here is: 1)what's the role of cryo? I know it helps prevent ice formation. Based on my case, it looks like cryo might also help to keep the crytal packing good when frozed. 2) What do I need to do to find a good cryo? What in my mind is to try other cryos like sucrose, PEG400, ethylene glycol. Thanks a lot for your attention! Yuan
Re: [ccp4bb] Questions about cryoprotectant
I have had a fair amount of success using 2.1-2.3 M Na Malonate as a cryo protectant for crystals from high salt conditions like yours. Fluorinated oils are also an option, they generally are a little easier to work with then Paratone. Good Luck Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 On Oct 7, 2009, at 3:54 PM, ycheng wrote: Hi, I am trying to find an appropriate cryo-condition for my protein crystals. The mother liquid is 2-2.5M Ammonium phosphate dibasic 100mM TrisHCL pH8. The room-temperature diffraction looks not bad (mosaicity 0.8, resolution 2.6) But the diffraction turned to be very mosaic if I freeze the crytals in the absence of cryos or in the presence of mother liquid plus different concentration of glycerol (5%,10%, 15%.20%). I don't think the ice formation is the problem since I didn't see any ice by my eyes or ice diffraction in the presence or absence of cryos. Also, I didn't see any cracks on my crystals when I transfered them to the cryo conditions I have already tried. My question here is: 1)what's the role of cryo? I know it helps prevent ice formation. Based on my case, it looks like cryo might also help to keep the crytal packing good when frozed. 2) What do I need to do to find a good cryo? What in my mind is to try other cryos like sucrose, PEG400, ethylene glycol. Thanks a lot for your attention! Yuan
Re: [ccp4bb] Questions about cryoprotectant
You have options: 1. Paratone-N (rarely works for me when other methods don't also work) 2. ML + 25-30% glucose (one of my favorite and reliable methods) 3. ML + 25-30% EG, maybe DMSO PEG400 is not likely to work here because its solubility in high sulfate solutions is limited. In 2 M AmSO4, you can only dissolve about 4% PEG-400. Typically flash-cooling increases mosaicity to some degree. If you have crystals to burn, you can try flash-annealing them in the cryostream by interrupting the flow with a credit card for about a 5-count. For some crystals this is magic in reducing mosaicity of flash-cooled crystals, but it *never* works for me :( Cheers. ycheng wrote: Hi, I am trying to find an appropriate cryo-condition for my protein crystals. The mother liquid is 2-2.5M Ammonium phosphate dibasic 100mM TrisHCL pH8. The room-temperature diffraction looks not bad (mosaicity 0.8, resolution 2.6) But the diffraction turned to be very mosaic if I freeze the crytals in the absence of cryos or in the presence of mother liquid plus different concentration of glycerol (5%,10%,15%.20%). I don't think the ice formation is the problem since I didn't see any ice by my eyes or ice diffraction in the presence or absence of cryos. Also, I didn't see any cracks on my crystals when I transfered them to the cryo conditions I have already tried. My question here is: 1)what's the role of cryo? I know it helps prevent ice formation. Based on my case, it looks like cryo might also help to keep the crytal packing good when frozed. 2) What do I need to do to find a good cryo? What in my mind is to try other cryos like sucrose, PEG400, ethylene glycol. Thanks a lot for your attention! Yuan -- Roger S. Rowlett 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
[ccp4bb] Questions about cryoprotectant
Hi, I am trying to find an appropriate cryo-condition for my protein crystals. The mother liquid is 2-2.5M Ammonium phosphate dibasic 100mM TrisHCL pH8. The room-temperature diffraction looks not bad (mosaicity 0.8, resolution 2.6) But the diffraction turned to be very mosaic if I freeze the crytals in the absence of cryos or in the presence of mother liquid plus different concentration of glycerol (5%,10%,15%.20%). I don't think the ice formation is the problem since I didn't see any ice by my eyes or ice diffraction in the presence or absence of cryos. Also, I didn't see any cracks on my crystals when I transfered them to the cryo conditions I have already tried. My question here is: 1)what's the role of cryo? I know it helps prevent ice formation. Based on my case, it looks like cryo might also help to keep the crytal packing good when frozed. 2) What do I need to do to find a good cryo? What in my mind is to try other cryos like sucrose, PEG400, ethylene glycol. Thanks a lot for your attention! Yuan
Re: [ccp4bb] Questions about Phaser
Peter Zwart wrote: Hi, This can might well be due to wrong space group. I suggest fixing the space groups by hand and running it in each possible space group that is possible. (P 31 2 1, P 32 2 1, P 3 2 1 are your options) Did you run xtriage already? P 2009/8/17 Yuan Cheng : Hey, I am trying to use phaser to solve a protein structure. There is predicted to be 8 mol/asu based on Matthew coefficient analysis.I am using a protein that shares about 35% identity with my protein as a search model. Phaser found the first five solutions and then failed to find the 6th. The LLG and Z-score are as following. The possible loop in the chainsawed model has been truncated. "SOLU SET RFZ=5.2 TFZ=7.1 PAK=0 LLG=44 RFZ=5.3 TFZ=13.8 PAK=0 LLG=169 RFZ=4.3 TFZ=69.6 PAK=0 LLG=1209 RFZ=4.6 TFZ=60.6 PAK=0 LLG=2200 RFZ=4.3 TFZ=5.7 PAK=0 LLG=2163 I used coot to check the difference map made with the model including the above five solutions.The first four solutions fit the density very well (didn't see many positive or negative densities). The 5th solution didn't fit the density at all. I saw many empty density in the map, indicating I still need to find more solutions.The space group I am using is P3 2 1. Could this be caused by a wrong space group? Could anyone give me some suggestions about this? Thanks a lot! Yuan Hi every, Thanks a lot for your reply! Actually I am pretty confusing here about the using of different space groups. The.mtz file I am using now as the input file for phaser is in space group P3. Phaser gave me the first four solutions like I mentioned in last email, but failed at the 5th one. Then I realized there might be something wrong with the space group or the data. I used Phenix.xtriage to re-analyze my data (P3 space group). Merohedral twinning and pseu-translational symmetry were found as following, Statistics depending on twin laws -- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | -- | -h,-k,l | M | 0.738 | -0.137| 0.000 | 0.022| | h,-h-k,-l | M | 0.047 | 0.390 | 0.429 | 0.478| | -k,-h,-l | M | 0.746 | -0.143| 0.000 | 0.022| -- "The analyses of the Patterson function reveals a significant off-origin peak that is 82.25 % of the origin peak, indicating pseudo translational symmetry." Also, The analysis indicates P 3 2 1 and its alternatives P31 2 1 and P32 2 1 might be the correct space group. Then I used the Sort/reindex MTZ fils module to change the space group to P 3 2 1. I forced Phaser to use P 3 2 1 as the space group to search for more solutions with the four solutions already found fixed. But I didn't get any better result. I used phenix.xtriage to analyze the data in P 3 2 1. It indicates there might be one twinning operator,but different tests gave different answers.But there is still a pseudo-translational symmetry. Statistics depending on twin laws - | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | - | -h,-k,l | M | 0.759 | -0.152| 0.000 | 0.022| - "The analyses of the Patterson function reveals a significant off-origin peak that is 82.17 % of the origin peak, indicating pseudo translational symmetry." I have a couple of question now 1)Do I need go back to HKL2000 and redo the index,integrate and scale. Since the .sca and .mtz I have now is in P3. I don't know whether the unit cell dimension is going to change if I redo it in P3 2 1. 2)what does the pseudo-translational symmetry actually means? I don't quite understand this concept and what should I do about it? This is a really long email. I appreciate your attention very much. Yuan
Re: [ccp4bb] Questions about Phaser
Hi, The drop in LLG for the 5th monomer could fit with your observation that it didn't look good in the e.d. map. There two things I can suggest (based on a similar experience with 6 mols. in the a.u.) 1) I would definitely try other possible s.g.'s (very easy in Phaser). 2) You might want to check the log file and see why other monomers weren't picked up. Is it because of packing problems ? It is possible that your model has extra loops that don't match your current protein and that the sequence alignment may show. In this case you could just chop them out and use the chopped model. If you have SAD/MAD data the 4 monomers Phaser found could be sufficient to bootstrap phasing. Cheers, Boaz - Original Message - From: Jiamu Du Date: Monday, August 17, 2009 8:22 Subject: Re: [ccp4bb] Questions about Phaser To: CCP4BB@JISCMAIL.AC.UK > Dear Yuan, > If the first solution is right by looking at the density. Then > you can > search the others by fixing the the first solution. This will > accelarateyour searching accuraty. Or you can first try to do > some limited model > building to the first solution and then use the refined model as > template to > search. > Best wishes. > > On Mon, Aug 17, 2009 at 11:42 AM, Yuan Cheng > wrote: > > > Hey, > > I am trying to use phaser to solve a protein structure. > There is predicted > > to be 8 mol/asu based on Matthew coefficient analysis.I am > using a protein > > that shares about 35% identity with my protein as a search > model. Phaser > > found the first five solutions and then failed to find the > 6th. The LLG and > > Z-score are as following. The possible loop in the chainsawed > model has been > > truncated. > > > > "SOLU SET > > RFZ=5.2 TFZ=7.1 PAK=0 LLG=44 > > RFZ=5.3 TFZ=13.8 PAK=0 LLG=169 > > RFZ=4.3 TFZ=69.6 PAK=0 LLG=1209 > > RFZ=4.6 TFZ=60.6 PAK=0 LLG=2200 > > RFZ=4.3 TFZ=5.7 PAK=0 LLG=2163 > > > > I used coot to check the difference map made with > the model including the > > above five solutions.The first four solutions fit the density > very well > > (didn't see many positive or negative densities). The 5th > solution didn't > > fit the density at all. I saw many empty density in the map, > indicating I > > still need to find more solutions.The space group I am using > is P3 2 1. > > Could this be caused by a wrong space group? > > Could anyone give me some suggestions about this? Thanks > a lot! > > > > Yuan > > > > > > -- > Jiamu Du, Ph.D. > State Key Laboratory of Molecular Biology > Institute of Biochemistry and Cell Biology > Shanghai Institutes for Biological Sciences > Chinese Academy of Sciences > 320 Yue-Yang Road > Shanghai 200031 > P. R. China > Tel: +86-21-5492-1117 > E-mail: jiam...@gmail.com > Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan
Re: [ccp4bb] Questions about Phaser
Dear Yuan, maybe you only have four - and a high solvent content. Why don't you refine the four for a while with strict NCS, and then use that improved model of one of them to do the original search again (to satisfy yourself there are only four, or to find additional ones)? On the other hand, while refining the four, additional density may show up around them. Mark Quoting Yuan Cheng : Hey, I am trying to use phaser to solve a protein structure. There is predicted to be 8 mol/asu based on Matthew coefficient analysis.I am using a protein that shares about 35% identity with my protein as a search model. Phaser found the first five solutions and then failed to find the 6th. The LLG and Z-score are as following. The possible loop in the chainsawed model has been truncated. "SOLU SET RFZ=5.2 TFZ=7.1 PAK=0 LLG=44 RFZ=5.3 TFZ=13.8 PAK=0 LLG=169 RFZ=4.3 TFZ=69.6 PAK=0 LLG=1209 RFZ=4.6 TFZ=60.6 PAK=0 LLG=2200 RFZ=4.3 TFZ=5.7 PAK=0 LLG=2163 I used coot to check the difference map made with the model including the above five solutions.The first four solutions fit the density very well (didn't see many positive or negative densities). The 5th solution didn't fit the density at all. I saw many empty density in the map, indicating I still need to find more solutions.The space group I am using is P3 2 1. Could this be caused by a wrong space group? Could anyone give me some suggestions about this? Thanks a lot! Yuan
Re: [ccp4bb] Questions about Phaser
Dear Yuan, If the first solution is right by looking at the density. Then you can search the others by fixing the the first solution. This will accelarate your searching accuraty. Or you can first try to do some limited model building to the first solution and then use the refined model as template to search. Best wishes. On Mon, Aug 17, 2009 at 11:42 AM, Yuan Cheng wrote: > Hey, > I am trying to use phaser to solve a protein structure. There is predicted > to be 8 mol/asu based on Matthew coefficient analysis.I am using a protein > that shares about 35% identity with my protein as a search model. Phaser > found the first five solutions and then failed to find the 6th. The LLG and > Z-score are as following. The possible loop in the chainsawed model has been > truncated. > > "SOLU SET > RFZ=5.2 TFZ=7.1 PAK=0 LLG=44 > RFZ=5.3 TFZ=13.8 PAK=0 LLG=169 > RFZ=4.3 TFZ=69.6 PAK=0 LLG=1209 > RFZ=4.6 TFZ=60.6 PAK=0 LLG=2200 > RFZ=4.3 TFZ=5.7 PAK=0 LLG=2163 > > I used coot to check the difference map made with the model including the > above five solutions.The first four solutions fit the density very well > (didn't see many positive or negative densities). The 5th solution didn't > fit the density at all. I saw many empty density in the map, indicating I > still need to find more solutions.The space group I am using is P3 2 1. > Could this be caused by a wrong space group? > Could anyone give me some suggestions about this? Thanks a lot! > > Yuan > -- Jiamu Du, Ph.D. State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 P. R. China Tel: +86-21-5492-1117 E-mail: jiam...@gmail.com
[ccp4bb] Questions about Phaser
Hey, I am trying to use phaser to solve a protein structure. There is predicted to be 8 mol/asu based on Matthew coefficient analysis.I am using a protein that shares about 35% identity with my protein as a search model. Phaser found the first five solutions and then failed to find the 6th. The LLG and Z-score are as following. The possible loop in the chainsawed model has been truncated. "SOLU SET RFZ=5.2 TFZ=7.1 PAK=0 LLG=44 RFZ=5.3 TFZ=13.8 PAK=0 LLG=169 RFZ=4.3 TFZ=69.6 PAK=0 LLG=1209 RFZ=4.6 TFZ=60.6 PAK=0 LLG=2200 RFZ=4.3 TFZ=5.7 PAK=0 LLG=2163 I used coot to check the difference map made with the model including the above five solutions.The first four solutions fit the density very well (didn't see many positive or negative densities). The 5th solution didn't fit the density at all. I saw many empty density in the map, indicating I still need to find more solutions.The space group I am using is P3 2 1. Could this be caused by a wrong space group? Could anyone give me some suggestions about this? Thanks a lot! Yuan
Re: [ccp4bb] Questions regarding Peptidoglycan-binding protein
Apologies for the slightly frivolous language - technically peptidoglycan is *an* endotoxin, as there's a separate entity also commonly referred to as endotoxin which is the LPS/LOS component of the cell wall. Peptidoglycan is also an endotoxin (or at least quite a few researchers consider it to be one) which is why I would like to correct my previous answer. You could try BacTx from Immunetics, for pure peptidoglycan assay. Not sure if it's fully commercial or not but they might send you a sample. Since endotoxin contamination and peptidoglycan contamination usually go hand in hand, the endotoxin assay should be indicative of PG presence as well. The crab blood assay (I am not kidding!) is probably too sensitive as it is designed to detect minuscule amounts of contamination. Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: Artem Evdokimov [mailto:ar...@xtals.org] Sent: Thursday, February 26, 2009 6:57 PM To: 'JOE CRYSTAL' Cc: 'CCP4BB@JISCMAIL.AC.UK' Subject: RE: [ccp4bb] Questions regarding Peptidoglycan-binding protein Look up a standard endotoxin assay (usually an immunoassay) because that's exactly what endotoxin is :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of JOE CRYSTAL Sent: Thursday, February 26, 2009 5:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions regarding Peptidoglycan-binding protein Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
Re: [ccp4bb] Questions regarding Peptidoglycan-binding protein
Look up a standard endotoxin assay (usually an immunoassay) because that's exactly what endotoxin is :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of JOE CRYSTAL Sent: Thursday, February 26, 2009 5:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions regarding Peptidoglycan-binding protein Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
[ccp4bb] Questions regarding Peptidoglycan-binding protein
Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: *how to detect PG fragments in my protein sample*? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
Re: [ccp4bb] Questions about (possibly) twinned data
Bert, Your self-Patterson peak may be real, i.e. you have pseudo translation, which can then make the statistics *look* like the crystal is twinned. Try a self-Patterson (perhaps sharpened) at somewhat lower resolution, e.g 6 A. Maybe the peak is real, but is only 6% of origin due to a slight mis-orientation of the molecules. Dave David Borhani, Ph.D. D. E. Shaw Research, LLC 120 West Forty-Fifth Street, 39th Floor New York, NY 10036 david.borh...@deshawresearch.com 212-478-0698 http://www.deshawresearch.com <http://www.deshawresearch.com/> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Van Den Berg, Bert Sent: Monday, February 16, 2009 9:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions about (possibly) twinned data Hello all, we have a dataset collected from multiple (2 or 3) parts of the same crystal with a microbeam (20 micron). The merged data scales OK (not great) in monoclinic (1-3% rejections). The resolution is 3.2-3.3 A, so the data is not fantastic. This is the cell (similar for other datasets): Cell: 70.012 126.449 107.98890.00089.94690.000 p21 Processing in orthorhombic makes the scaling a lot worse, so I'm assuming its monoclinic for now. Running xtriage gives the following summary: --- Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws - /^2 : 1.877 - ^2/ : 0.834 - <|E^2-1|> : 0.663 - <|L|>, : 0.411, 0.235 Multivariate Z score L-test: 6.737 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. Statistics depending on twin laws - | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | - | h,-k,-l | PM | 0.167 | 0.367 | 0.339 | 0.152 | - Patterson analyses - Largest peak height : 5.962 (corresponding p value : 0.72096) The largest off-origin peak in the Patterson function is 5.96% of the height of the origin peak. No significant pseudotranslation is detected. So, I'm assuming that these crystals are monoclinic and that they are pseudo-merohedrally twinned. Is this a reasonable assumption? I get a decent solution for the P21 data from molecular replacement with a 50% identical model (LLG 900, with the rotation Z-scores low (4-5), but the corresponding translation Z-scores high (8-20)). My questions are: what would be the best way to refine? More specifically, what twin fraction should be used as the different tests give different fractions. Is the twin fraction automatically determined in phenix.refine or does this need to be specified? Finally, can twinning be responsible for the fact that the data do not scale well (using data collected on different parts of the same crystal)? Any hints appreciated! Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm
Re: [ccp4bb] Questions about (possibly) twinned data
Hi Bert, It seems unikely you are experiencing merohedral twinning in your crystal since none of your unit cell dimensions are equal length or integer multiples. For your cell, you would expect to see multiple lattices. Is it possible you have a dimer in the asymmetric unit? Strong NCS parallel to a principle lattice direction can sometimes give "twin-like" statistics especially at lower resolutions. Hope this helps, Chris On Mon, 16 Feb 2009, Van Den Berg, Bert wrote: >>>Hello all, >>> >>>we have a dataset collected from multiple (2 or 3) parts of the same >>>crystal with a microbeam (20 micron). The merged data scales OK (not great) >>>in monoclinic (1-3% rejections). The resolution is 3.2-3.3 A, so the data is >>>not fantastic. This is the cell (similar for other datasets): >>> >>>Cell: 70.012 126.449 107.98890.00089.94690.000 p21 >>> >>>Processing in orthorhombic makes the scaling a lot worse, so I'm assuming >>>its monoclinic for now. Running xtriage gives the following summary: >>> >>>--- >>>Twinning and intensity statistics summary (acentric data): >>> >>>Statistics independent of twin laws >>> - /^2 : 1.877 >>> - ^2/ : 0.834 >>> - <|E^2-1|> : 0.663 >>> - <|L|>, : 0.411, 0.235 >>> Multivariate Z score L-test: 6.737 >>> The multivariate Z score is a quality measure of the given >>> spread in intensities. Good to reasonable data are expected >>> to have a Z score lower than 3.5. >>> Large values can indicate twinning, but small values do not >>> necessarily exclude it. >>> >>> >>>Statistics depending on twin laws >>>- >>>| Operator | type | R obs. | Britton alpha | H alpha | ML alpha | >>>- >>>| h,-k,-l | PM | 0.167 | 0.367 | 0.339 | 0.152| >>>- >>> >>>Patterson analyses >>> - Largest peak height : 5.962 >>> (corresponding p value : 0.72096) >>> >>> >>>The largest off-origin peak in the Patterson function is 5.96% of the >>>height of the origin peak. No significant pseudotranslation is detected. >>> >>>So, I'm assuming that these crystals are monoclinic and that they are >>>pseudo-merohedrally twinned. Is this a reasonable assumption? I get a decent >>>solution for the P21 data from molecular replacement with a 50% identical >>>model (LLG 900, with the rotation Z-scores low (4-5), but the corresponding >>>translation Z-scores high (8-20)). >>> >>>My questions are: what would be the best way to refine? More specifically, >>>what twin fraction should be used as the different tests give different >>>fractions. Is the twin fraction automatically determined in phenix.refine or >>>does this need to be specified? Finally, can twinning be responsible for the >>>fact that the data do not scale well (using data collected on different >>>parts of the same crystal)? >>> >>>Any hints appreciated! >>> >>>Cheers, Bert >>> >>> >>>Bert van den Berg >>>University of Massachusetts Medical School >>>Program in Molecular Medicine >>>Biotech II, 373 Plantation Street, Suite 115 >>>Worcester MA 01605 >>>Phone: 508 856 1201 (office); 508 856 1211 (lab) >>>e-mail: bert.vandenb...@umassmed.edu >>>http://www.umassmed.edu/pmm/faculty/vandenberg.cfm >>> >>> >>> Christopher L. Colbert, Ph.D. InstructorPhone: (214) 645 5944 University of Texas Southwestern Medical Center FAX: (214) 645 5945 6001 Forest Park Lane Dallas, TX 75390
[ccp4bb] Questions about (possibly) twinned data
Hello all, we have a dataset collected from multiple (2 or 3) parts of the same crystal with a microbeam (20 micron). The merged data scales OK (not great) in monoclinic (1-3% rejections). The resolution is 3.2-3.3 A, so the data is not fantastic. This is the cell (similar for other datasets): Cell: 70.012 126.449 107.98890.00089.94690.000 p21 Processing in orthorhombic makes the scaling a lot worse, so I'm assuming its monoclinic for now. Running xtriage gives the following summary: --- Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws - /^2 : 1.877 - ^2/ : 0.834 - <|E^2-1|> : 0.663 - <|L|>, : 0.411, 0.235 Multivariate Z score L-test: 6.737 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. Statistics depending on twin laws - | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | - | h,-k,-l | PM | 0.167 | 0.367 | 0.339 | 0.152| - Patterson analyses - Largest peak height : 5.962 (corresponding p value : 0.72096) The largest off-origin peak in the Patterson function is 5.96% of the height of the origin peak. No significant pseudotranslation is detected. So, I'm assuming that these crystals are monoclinic and that they are pseudo-merohedrally twinned. Is this a reasonable assumption? I get a decent solution for the P21 data from molecular replacement with a 50% identical model (LLG 900, with the rotation Z-scores low (4-5), but the corresponding translation Z-scores high (8-20)). My questions are: what would be the best way to refine? More specifically, what twin fraction should be used as the different tests give different fractions. Is the twin fraction automatically determined in phenix.refine or does this need to be specified? Finally, can twinning be responsible for the fact that the data do not scale well (using data collected on different parts of the same crystal)? Any hints appreciated! Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm
Re: [ccp4bb] Questions about diffraction
Good summary as expected from James. "Have you ever heard of photon-photon scattering?" Well yes! See for example http://2physics.blogspot.com/2006/03/photon-photon-scattering.html which says "according to Quantum Electrodynamics (QED), particles can still be created in this emptiness of vacuum through light-light interactions." There we don't need x-ray generators, synchrotrons etc. The vacuum will do it for us. Cheers Colin From: CCP4 bulletin board on behalf of James Holton Sent: Tue 28/08/2007 16:56 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Questions about diffraction For a full answer to all your questions, I refer you to the classic textbook of M. M. Woolfson "an introduction to x-ray crystallography" by Cambridge University Press. This book has been quite helpful to me of late. Unlike some similar texts I find it easy to read. There are even examples! With real numbers! Woolfson begins by describing scattering as it was originally derived by J. J. Thomson from classical mechanics of a charged mass (the electron) vibrating in an electric field (the photon), and takes you, step-by-step all the way to Bragg scattering from a rotating 3-D crystal. You have received many comments so far, so I will try not to repeat those in answering your questions here: 1) yes, x-ray sources are "non-coherent", but the photons ARE coherent on a short length scale. Specifically, this is about 0.7 micron if the x-rays are 1 A in wavelength and have a spectral dispersion of 1/7000, such as with a Si(111) monochromator. That is, after traveling 0.7 micron, two photons that were once in phase will no longer be, because they have different wavelengths. This is approximately the "coherence length" in the direction of propagation. The other two directions are ... more complicated. It is actually quite difficult to make an x-ray source with a very short spatial coherence. I've heard "talk" about shaping x-ray wavefronts in next-generation accelerators, such as the ERL planned at CHESS. Thus would have the advantage of changing the phase relationship between atoms as you described and (potentially) getting phase information directly. However, I don't think anybody has actually done this yet. 2) In general, photons do not interfere with each other. Have you ever heard of photon-photon scattering? Neither have I. However, there actually is a body of work on multi-photon correlations in scattering. Apparently, two photons can have correlated wave functions, and this leads to correlations in the arrival time of photons at the detector: something called the Hanbury-Brown and Twiss effect. The math behind it it a bit beyond me. However, theoretically, such correlated scattering events could be used to get phase information if you have a fast detector and a REALLY fast source. My colleague Ken Frankel <[EMAIL PROTECTED]> can tell you more about it if you are interested. 2b) WRT the "all electrons scattering together" question. Yes, they do all scatter "together" because they are all confined in the same atom. The total scattering is explained by integrating Thomson's scattering formula (including the phase shift) over all of the electron positions. Yes, a single photon can interact with more than one electron, just as a single photon can pass through two slits in the famous experiment by Thomas Young. For any given photon, the positions of each electron will (in the classical view) be at some well-defined position, but we experimentally integrate over a LOT of photons. If no two photons ever see a consistent arrangement of electron positions (such as when you shoot x-rays at a free electron beam) then the scattering is "incoherent" and the scattered intensity distribution you see simply follows Thomson's classical scattering formula for one electron, with the intensity multiplied by the number of electrons in the x-ray beam. However, if the "electron density" is not random but instead has some kind of consistent feature from photon to photon, then the interference of the scattered waves will also be consistent as it builds up on your detector. This "binding effect" is only significant on the length scale of the electron confinement (the size of an atom in this case), so it falls off with increasing scattering angle. Remember, the phase of the scattered wave depends on the total path length traversed by the incident and scattered photons. The phase shift between scattering at any two points in the sample will always become vanishingly small at a small scattering angle because the difference in path length from one "side" of the atom to the other "side" becomes smaller and smaller at low scattering angle. For larger scattering angles there is a larger phase shift, and the interfere
Re: [ccp4bb] Questions about diffraction
For a full answer to all your questions, I refer you to the classic
textbook of M. M. Woolfson "an introduction to x-ray crystallography" by
Cambridge University Press. This book has been quite helpful to me of
late. Unlike some similar texts I find it easy to read. There are even
examples! With real numbers! Woolfson begins by describing scattering
as it was originally derived by J. J. Thomson from classical mechanics
of a charged mass (the electron) vibrating in an electric field (the
photon), and takes you, step-by-step all the way to Bragg scattering
from a rotating 3-D crystal.
You have received many comments so far, so I will try not to repeat
those in answering your questions here:
1) yes, x-ray sources are "non-coherent", but the photons ARE coherent
on a short length scale. Specifically, this is about 0.7 micron if the
x-rays are 1 A in wavelength and have a spectral dispersion of 1/7000,
such as with a Si(111) monochromator. That is, after traveling 0.7
micron, two photons that were once in phase will no longer be, because
they have different wavelengths. This is approximately the "coherence
length" in the direction of propagation. The other two directions are
... more complicated. It is actually quite difficult to make an x-ray
source with a very short spatial coherence. I've heard "talk" about
shaping x-ray wavefronts in next-generation accelerators, such as the
ERL planned at CHESS. Thus would have the advantage of changing the
phase relationship between atoms as you described and (potentially)
getting phase information directly. However, I don't think anybody has
actually done this yet.
2) In general, photons do not interfere with each other. Have you ever
heard of photon-photon scattering? Neither have I. However, there
actually is a body of work on multi-photon correlations in scattering.
Apparently, two photons can have correlated wave functions, and this
leads to correlations in the arrival time of photons at the detector:
something called the Hanbury-Brown and Twiss effect. The math behind it
it a bit beyond me. However, theoretically, such correlated scattering
events could be used to get phase information if you have a fast
detector and a REALLY fast source. My colleague Ken Frankel
<[EMAIL PROTECTED]> can tell you more about it if you are interested.
2b) WRT the "all electrons scattering together" question. Yes, they do
all scatter "together" because they are all confined in the same atom.
The total scattering is explained by integrating Thomson's scattering
formula (including the phase shift) over all of the electron positions.
Yes, a single photon can interact with more than one electron, just as a
single photon can pass through two slits in the famous experiment by
Thomas Young. For any given photon, the positions of each electron will
(in the classical view) be at some well-defined position, but we
experimentally integrate over a LOT of photons.
If no two photons ever see a consistent arrangement of electron
positions (such as when you shoot x-rays at a free electron beam) then
the scattering is "incoherent" and the scattered intensity distribution
you see simply follows Thomson's classical scattering formula for one
electron, with the intensity multiplied by the number of electrons in
the x-ray beam.
However, if the "electron density" is not random but instead has
some kind of consistent feature from photon to photon, then the
interference of the scattered waves will also be consistent as it builds
up on your detector. This "binding effect" is only significant on the
length scale of the electron confinement (the size of an atom in this
case), so it falls off with increasing scattering angle. Remember, the
phase of the scattered wave depends on the total path length traversed
by the incident and scattered photons. The phase shift between
scattering at any two points in the sample will always become
vanishingly small at a small scattering angle because the difference in
path length from one "side" of the atom to the other "side" becomes
smaller and smaller at low scattering angle. For larger scattering
angles there is a larger phase shift, and the interference becomes more
destructive. The quantitative angular dependence of scattering from any
atom is called the "atomic form factor", and it is tabulated in
${CLIBD}/atomsf.lib. The "form factor" is why high-angle spots are
weaker than low-angle spots, even if all the B-factors are zero. Atomic
form factors are derived from scattering measurements on gasses (which
don't have B-factors), and theoretical electron-distribution
calculations have been found to be consistent with these observations.
The upper limit to constructive interference from a single atom is
if all of the electrons in the atom are scattering in phase. This will
always occur at vanishingly small scattering angles (forward
scattering). Since there is
Re: [ccp4bb] Questions about diffraction
> Something for you to chew on: how is it that the electrons of the protein, > which are presumably not in phase with each other nor in exactly the same > place in their orbitals from unit cell to unit cell (maybe they are?) when > they scatter the photons, they result in interference? What are the chances > that the scattering electrons are exactly in the same place as the > electrons in another unit cell, or of the same phase? And would they not > need to be in the same place to sub-angstrom precision to scatter > coherently? They do not need to be in the same place to sub-angstrom precision. The situation is easier to understand when you think about a crystal composed of small molecules or even better - atoms (e.g. a Silicon crystal). If all electrons (pointlike particles) were in the same place, atomic scattering factor would be a flat function against the scattering angle (the atomic scattering factor would be a constant function (putting aside polarization considerations)) and we would not observe a falloff of intensities for large scattering angles. By the way, thinking in terms of orbitals can be a bit misleading, because orbitals are like components of a vector: in different coordinate systems one gets different sets of components. What has a physical meaning is a wave function. The wave function can be decomposed into orbitals (localized, delocalized etc.) in many different ways. Andrzej __ Andrzej Olczak Institute of General and Ecological Chemistry Technical University of Lodz Zeromskiego 116 90-924 Lodz Poland __
Re: [ccp4bb] Questions about diffraction
The "Bragg planes" are a contrivance of our invention to make the math simpler and allow us to converse in shorthand terms like "Bragg's Law". The photon's wave function interacts with the wave functions of every electron it overlaps with, which is many unit cells because our photons have quite diffuse wave functions, reasonably approximated by plane waves. One writes out the integral over all these interactions to get the amount of net scattering in any direction, but the mathematicians have done this and told us that you can get the same result, for our case single wavelength illumination, by calculating a Fourier Transform to get the amplitudes and phases, while the reciprocal lattice construction along with Ewald's sphere will tell us the directions of the diffracted beams. Unfortunately text books usually start and end with Bragg Planes so their descriptions are confusing to people who start thinking about continuous electron density. The problem is that the real math is rather involved and the discussion requires knowledge of optics that is beyond, and probably uninteresting, to most of the people who want to solve protein structures. If your sample is not crystalline, the Fourier Transform and Ewald's sphere still works, but you then have a continuous function instead of spots and your life will be hard. The best book I've seen on this topic, but by no means have absorbed, is "The Optical Principles of the Diffraction of X-Rays", by R. James. Dale Tronrud Michel Fodje wrote: Would it be taking it too far to suggest that one could go all the way and consider that each electron diffracts not as groups in a plane but as individual electrons and a photon impinging on an electron with with a specific phase will be diffracted in a specific direction. However the lattice arrangement of the electrons will statistically accumulate photons which impinged on electrons on a specific family of planes in one direction at the detector. Such that the crystal is a phase sorter. In which case diffraction is not based on constructive or destructive interference but on conservation of some property of the photon, such as angular momentum? IANAM either. On Fri, 2007-08-24 at 15:36 -0700, James Stroud wrote: Without resorting to a circular argument? You are asking too much. However, this probability distribution is perfectly described by considering a component wave model wherein coherence of the component waves correlates with peaks in the probability distribution--i.e. Bragg's Law. IANAM (I am not a mathematician), but, if pressed, I would posit that one could decompose the fun description just a little bit and consider the lattice not as *groups* of reflecting planes, but as individual planes. In such a case, each single reflecting plane would contribute a probability distribution with an angular dependence. The total probability distribution would then be the sum of the probability distributions for every plane in the lattice. Your next question might be, "what's the probability distribution for a single plane". Well, I would imagine that it has a maximum where the angle of incidence equals the angle of reflection and that the phase of a component probability distributions is spatially (i.e. angularly) directly related to the phase of the originating photon. The sum distribution of the reflected photon takes into account the angular phase dependence of its components and so one gets positive and negative interference between component distributions. James Jacob Keller wrote: Yes, but why should the directions of diffraction conditions be most probable (one of your premises)? ==Original message text=== On Fri, 24 Aug 2007 4:54:53 pm CDT James Stroud wrote: Here's a fun way to think of it: A photon hits a crystal and will diffract off in a certain direction with the same energy as the original photon. The direction is subject to a probability distribution based on the lattice, with angles at the diffraction conditions being most likely and the broadness of the peaks in the distribution arising from imperfections in the lattice. The photon propagates as this probability distribution and then is forced to select from the distribution because we stuck a detector up. The diffraction pattern we observe is the sum of many such photons interacting with the crystal.
Re: [ccp4bb] Questions about diffraction
Would it be taking it too far to suggest that one could go all the way and consider that each electron diffracts not as groups in a plane but as individual electrons and a photon impinging on an electron with with a specific phase will be diffracted in a specific direction. However the lattice arrangement of the electrons will statistically accumulate photons which impinged on electrons on a specific family of planes in one direction at the detector. Such that the crystal is a phase sorter. In which case diffraction is not based on constructive or destructive interference but on conservation of some property of the photon, such as angular momentum? IANAM either. On Fri, 2007-08-24 at 15:36 -0700, James Stroud wrote: > Without resorting to a circular argument? You are asking too much. > > However, this probability distribution is perfectly described by > considering a component wave model wherein coherence of the component > waves correlates with peaks in the probability distribution--i.e. > Bragg's Law. > > IANAM (I am not a mathematician), but, if pressed, I would posit that > one could decompose the fun description just a little bit and consider > the lattice not as *groups* of reflecting planes, but as individual > planes. In such a case, each single reflecting plane would contribute a > probability distribution with an angular dependence. The total > probability distribution would then be the sum of the probability > distributions for every plane in the lattice. > > Your next question might be, "what's the probability distribution for a > single plane". Well, I would imagine that it has a maximum where the > angle of incidence equals the angle of reflection and that the phase of > a component probability distributions is spatially (i.e. angularly) > directly related to the phase of the originating photon. > > The sum distribution of the reflected photon takes into account the > angular phase dependence of its components and so one gets positive and > negative interference between component distributions. > > James > > Jacob Keller wrote: > > Yes, but why should the directions of diffraction conditions be most > > probable (one of your premises)? > > > > ==Original message text=== > > On Fri, 24 Aug 2007 4:54:53 pm CDT James Stroud wrote: > > > > Here's a fun way to think of it: > > > > A photon hits a crystal and will diffract off in a certain direction > > with the same energy as the original photon. The direction is subject to > > a probability distribution based on the lattice, with angles at the > > diffraction conditions being most likely and the broadness of the peaks > > in the distribution arising from imperfections in the lattice. The > > photon propagates as this probability distribution and then is forced to > > select from the distribution because we stuck a detector up. The > > diffraction pattern we observe is the sum of many such photons > > interacting with the crystal. > >
Re: [ccp4bb] Questions about diffraction
Without resorting to a circular argument? You are asking too much. However, this probability distribution is perfectly described by considering a component wave model wherein coherence of the component waves correlates with peaks in the probability distribution--i.e. Bragg's Law. IANAM (I am not a mathematician), but, if pressed, I would posit that one could decompose the fun description just a little bit and consider the lattice not as *groups* of reflecting planes, but as individual planes. In such a case, each single reflecting plane would contribute a probability distribution with an angular dependence. The total probability distribution would then be the sum of the probability distributions for every plane in the lattice. Your next question might be, "what's the probability distribution for a single plane". Well, I would imagine that it has a maximum where the angle of incidence equals the angle of reflection and that the phase of a component probability distributions is spatially (i.e. angularly) directly related to the phase of the originating photon. The sum distribution of the reflected photon takes into account the angular phase dependence of its components and so one gets positive and negative interference between component distributions. James Jacob Keller wrote: Yes, but why should the directions of diffraction conditions be most probable (one of your premises)? ==Original message text=== On Fri, 24 Aug 2007 4:54:53 pm CDT James Stroud wrote: Here's a fun way to think of it: A photon hits a crystal and will diffract off in a certain direction with the same energy as the original photon. The direction is subject to a probability distribution based on the lattice, with angles at the diffraction conditions being most likely and the broadness of the peaks in the distribution arising from imperfections in the lattice. The photon propagates as this probability distribution and then is forced to select from the distribution because we stuck a detector up. The diffraction pattern we observe is the sum of many such photons interacting with the crystal. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com/
Re: [ccp4bb] Questions about diffraction
Here's a fun way to think of it: A photon hits a crystal and will diffract off in a certain direction with the same energy as the original photon. The direction is subject to a probability distribution based on the lattice, with angles at the diffraction conditions being most likely and the broadness of the peaks in the distribution arising from imperfections in the lattice. The photon propagates as this probability distribution and then is forced to select from the distribution because we stuck a detector up. The diffraction pattern we observe is the sum of many such photons interacting with the crystal. I think this is consistent with the math. James Jacob Keller wrote: For the total integrated energy to be conserved, energy will have to be created in certain directions to compensate for the loss in other directions. So in a direction in which the condition is met, the total will have to be more than the sum of the waves in that direction. How about considering the possibility that all photons coming into the sample are diffracted -- just in different directions. So that what is happening is not constructive and destructive interference but a kind sorting of the photons based on a certain property of the photons, maybe the phase. * I think of it that each photon that happens to be perturb an electron, i.e., Thomson scattering, sends out a spherical wave, which has anisotropy to it, i.e., the wave front is more concentrated in the forward direction. These spherical waves interfere with each other, making the diffraction pattern. Something for you to chew on: how is it that the electrons of the protein, which are presumably not in phase with each other nor in exactly the same place in their orbitals from unit cell to unit cell (maybe they are?) when they scatter the photons, they result in interference? What are the chances that the scattering electrons are exactly in the same place as the electrons in another unit cell, or of the same phase? And would they not need to be in the same place to sub-angstrom precision to scatter coherently? I would suggest two possible answers, neither of which am I entirely satisfied: 1. Something about the crystalline state induces the protein molecules' molecular orbitals to be totally in synch with each other. This seems too miraculous to be true, in a way. Nevertheless, it would account for the data, I think. 2. The scattering electrons are elusive probablistic entities which are really no place at all. This, however, does not solve the problem of the phases (not in the usual sense of finding fourier phases) which is that it seems unlikely that electrons in multiple unit cells should be exactly in phase with each other, something which it seems would be necessary to produce interference. NB this issue came up in a crystallography class several years ago, and I have been ruminating on it, on and off, since then. JPK *** Jacob Keller Northwestern University 6541 N. Francisco #3 Chicago IL 60645 (847)467-4049 [EMAIL PROTECTED] *** -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com/
Re: [ccp4bb] Questions about diffraction
Michel Fodje wrote: For every direction where there is destructive interference and a loss of energy there is a direction where there is constructive interference that piles up energy. If you integrate over all directions energy is conserved. For the total integrated energy to be conserved, energy will have to be created in certain directions to compensate for the loss in other directions. So in a direction in which the condition is met, the total will have to be more than the sum of the waves in that direction. How about considering the possibility that all photons coming into the sample are diffracted -- just in different directions. So that what is happening is not constructive and destructive interference but a kind sorting of the photons based on a certain property of the photons, maybe the phase. You seem to be operating under the impression that there are two diffracting waves that later destructively interfere. All constructive and destructive interference occurs at the point of scattering. There is no energy that heads off in a direction that later disappears - Nothing ever went in that direction. You have the same problem with your idea of two waves, out of phase but identical in wavelength, that scatter off an electron. The two waves, if they are coherent, would interfere with each other long before they reach the electron and become a single wave. If they are not coherent they will interact with the scatter independently and produce incoherent diffraction waves, which will sum by intensity independent of phase. I can get into deep trouble with this next point so I hope a physicist jumps on me where I'm wrong. All light sources are coherent to a degree. A laser is pretty much 100% coherent and my pocket flashlight is hardly coherent at all. I seem to recall that there is a parameter called the coherence length that measures the distance within a beam that the light is coherent. The coherence length of a rotating anode X-ray generator is small but unit cells are smaller so there are plenty of unit cells to form a nice diffraction pattern. Your second paragraph is just the Copenhagen Interpretation of the wave function. If you want to think of photons then the diffraction wave we are talking about is the wave function and that function maps the probability of finding a photon. Wave/particle duality says we can look at the experiment either way. Dale Tronrud
Re: [ccp4bb] Questions about diffraction
On Fri, 24 Aug 2007 14:40:13 -0600 Michel Fodje <[EMAIL PROTECTED]> wrote: The mathematics works but doesn't necessarily mean the current interpretation of the mathematics has any resemblance to what actually happens in reality. Sure, it does. Crystallography is traditionally derived using classical wave mechanics, but you can take a quantum approach, using the First Born approximation (a single photon scatters elastically from a point source exactly once). If you want to speak about what a single photon does, then you have to resort to that approach. Except in rare instances, the photon interferes only with itself, regardless of how many or how few are present. The particle is the photon, and the wave is the propensity for the photon to appear at a given position on the detector. QM teaches us that the entire experiment, in this case the crystal lattice, has to be taken into account, (which simply means you have to add amplitudes rather than intensities). It is the same thing with a single particle going through a double slit. BOTH slits must be taken into account, as both are possible paths. A crystal is simply a near-infinite diffraction grating in three dimensions, but the physical interpretation is identical. Feynman developed the most intuitive way of looking at this, which is to sum over all possible paths before squaring the wave. Unfortunately, the accompanying mathematical treatment is a bit hairy. Bill
Re: [ccp4bb] Questions about diffraction
>For the total integrated energy to be conserved, energy will have to be >created in certain directions to compensate for the loss in other >directions. So in a direction in which the condition is met, the total >will have to be more than the sum of the waves in that direction. >How about considering the possibility that all photons coming into the >sample are diffracted -- just in different directions. So that what is >happening is not constructive and destructive interference but a kind >sorting of the photons based on a certain property of the photons, maybe >the phase. * I think of it that each photon that happens to be perturb an electron, i.e., Thomson scattering, sends out a spherical wave, which has anisotropy to it, i.e., the wave front is more concentrated in the forward direction. These spherical waves interfere with each other, making the diffraction pattern. Something for you to chew on: how is it that the electrons of the protein, which are presumably not in phase with each other nor in exactly the same place in their orbitals from unit cell to unit cell (maybe they are?) when they scatter the photons, they result in interference? What are the chances that the scattering electrons are exactly in the same place as the electrons in another unit cell, or of the same phase? And would they not need to be in the same place to sub-angstrom precision to scatter coherently? I would suggest two possible answers, neither of which am I entirely satisfied: 1. Something about the crystalline state induces the protein molecules' molecular orbitals to be totally in synch with each other. This seems too miraculous to be true, in a way. Nevertheless, it would account for the data, I think. 2. The scattering electrons are elusive probablistic entities which are really no place at all. This, however, does not solve the problem of the phases (not in the usual sense of finding fourier phases) which is that it seems unlikely that electrons in multiple unit cells should be exactly in phase with each other, something which it seems would be necessary to produce interference. NB this issue came up in a crystallography class several years ago, and I have been ruminating on it, on and off, since then. JPK *** Jacob Keller Northwestern University 6541 N. Francisco #3 Chicago IL 60645 (847)467-4049 [EMAIL PROTECTED] ***
Re: [ccp4bb] Questions about diffraction
> You are just using the coherent fraction of the beam. My point is that Braggs' law as currently understood does not preclude the diffraction from waves which were non-coherent before hitting the sample > It is not clear at all how you arrive to that condition. By definition, if > two waves are non coherent, you cannot define a "phase difference". The > phase difference is continuosly changing at random with non coherent waves. if the phase difference between two waves is y, the extra distance the second wave has to travel to again be in phase is y*lambda/2pi if this distance is the same as the 2dsin(theta), the diffraction condition will also be met. My understanding is that coherence has to do with the phase not the wavelength. Only when the wavelengths are also different will the phase difference be changing continuously. No? > You do not annihilate energy with interferences, you just spread it > differently. Apart for conservation of energy, Thermodynamics is seldom > involved in these considerations. My point is that destructive interference implies that energy is destroyed which does not make sense. If as you say the energy is simply spread differently, what would be the mechanism/geometry of this spreading? > A complete lack of coherence leads simply to adding intensities of the two > waves. On the contrary. complete lack of coherence leads to destructive interference. No? Perfect coherence leads to the amplitudes adding up.
Re: [ccp4bb] Questions about diffraction
> For every direction where there is destructive interference and a > loss of energy there is a direction where there is constructive > interference that piles up energy. If you integrate over all directions > energy is conserved. For the total integrated energy to be conserved, energy will have to be created in certain directions to compensate for the loss in other directions. So in a direction in which the condition is met, the total will have to be more than the sum of the waves in that direction. How about considering the possibility that all photons coming into the sample are diffracted -- just in different directions. So that what is happening is not constructive and destructive interference but a kind sorting of the photons based on a certain property of the photons, maybe the phase.
Re: [ccp4bb] Questions about diffraction
> > 1. In every description of Braggs' law I've seen, the in-coming waves > > have to be in phase. Why is that? Given that the sources used for > > diffraction studies are mostly non-coherent. > > Think of Bragg's Law as explaining what happens to a single photon > that is probabilistically scattered by every atom in the lattice. > It's perfectly coherent with itself. Why does the photon not diffract at all angles then, since one 'part' of the photon will never have a different phase from another 'part'? If you are correct that it is a single photon diffracting, it would suggest that at some point the photon is in-coherent with itself thus the need to have a diffraction condition for specific angles. It would make more sense to me in such a case, if we interpreted Braggs' law as the condition under which the photon keeps it's 'internal coherence but instead we talk of constructive and destructive interference! > This idea should be no stranger than textbook illustrations of the > result of sending a single particle through a narrow slit or pinhole. > The interference effects follow the expected predictions even for > illumination by one particle at a time. The mathematics works but doesn't necessarily mean the current interpretation of the mathematics has any resemblance to what actually happens in reality.
Re: [ccp4bb] Questions about diffraction
Michel Fodje wrote: Dear Crystallographers, Here are a few paradoxes about diffraction I would like to get some answers about: ... 3. What happens to the photon energy when waves destructively interfere as mentioned in the text books. Doesn't 'destructive interference' appear to violate the first and second laws of thermodynamics? Besides, since the sources are non-coherent, how come the photon 'waves' don't annihilate each other before reaching the sample? If they were coherent, would we just end up with a single wave any how? With what will it interfere to cause diffraction? For every direction where there is destructive interference and a loss of energy there is a direction where there is constructive interference that piles up energy. If you integrate over all directions energy is conserved. I'm not sure what your concern is about the second law. The radiation is spreading out into space and so entropy increases. Dale Tronrud
Re: [ccp4bb] Questions about diffraction
On Friday 24 August 2007 12:22, Michel Fodje wrote: > 1. In every description of Braggs' law I've seen, the in-coming waves > have to be in phase. Why is that? Given that the sources used for > diffraction studies are mostly non-coherent. Think of Bragg's Law as explaining what happens to a single photon that is probabilistically scattered by every atom in the lattice. It's perfectly coherent with itself. This idea should be no stranger than textbook illustrations of the result of sending a single particle through a narrow slit or pinhole. The interference effects follow the expected predictions even for illumination by one particle at a time. -- Ethan A Merritt
[ccp4bb] Questions about diffraction
Dear Crystallographers, Here are a few paradoxes about diffraction I would like to get some answers about: 1. In every description of Braggs' law I've seen, the in-coming waves have to be in phase. Why is that? Given that the sources used for diffraction studies are mostly non-coherent. 2. Trying to derive the diffraction condition for a pair of non-coherent waves with a phase difference of 'y' where 0 < y < 2pi, I obtain the following diffraction condition y * (lambda/2pi) = 2d sin (theta) i.e. the phase difference y = 4pi * sin(theta) * d / lambda This seems to imply that diffraction will occur if the incident waves are not in phase but the phase difference still satisfies the above condition. One may be able to envision a case where for a given distance d, the diffracting condition will be met for various angles depending on the phase shift of the waves diffracting. Does this make sense? Has anyone looked at the significance of this relationship before? Any pointers will be welcome. 3. What happens to the photon energy when waves destructively interfere as mentioned in the text books. Doesn't 'destructive interference' appear to violate the first and second laws of thermodynamics? Besides, since the sources are non-coherent, how come the photon 'waves' don't annihilate each other before reaching the sample? If they were coherent, would we just end up with a single wave any how? With what will it interfere to cause diffraction? I'm sure some of these may have some really obvious answers I may be missing. Thanks, Michel
Re: [ccp4bb] questions about CNS
Well, I guess you can mess with the "overall B-factor correction" in the anneal.inp and rigid.inp files and say, turn them off. NOT a good way to proceed. That is just to let you know where to find it. If you haven't already done so, do the following after the simulated annealing step. After annealing, run minimization (minimize.inp) and bgroup refinement (bgroup.inp). The grouped B refinement will refine the B factor. Make sure that you keep an eye on how many cycles of refinement you run for each refinement protocol by correlating it with the drop in Rfactor and Rfree. YOu might have to cut back on the no. of refinement cycles in the later stages. Hope that helps. Raji -Included Message-- >Hi everyone, >I have some basic questions about CNS. First, I am wondering if there is >anywhere I can set my initial B-factor during my early refinement. When I generate my initial model in generate.inp, I can set the B-factore. However, after I did the rigid.inp and anneal.inp, the B-factor just go up to more than 100 by itself. I look through the input files, and couldn't find where to set the B- factor. Do I have to set the B-factor by hand each time? Or will the crazy B-factor affect my refinement? Second, I am refining a new protein/DNA complex. I started with the DNA alone. After rigid body refinement I got ~1% decrease of Rf. But when I tried anneal after that, the Rf just went up much higher than where I started? If you work on a protein/DNA complex, what is your stategy? Will the anneal help refine the DNA alone? How much decrease of Rf usually you can get from refining just DNA? Third, I was wondering if I want to extend my resolution, do I have to make sure my cv file was made with the highest resolution? Thank you for your suggestion! >Cortney >_ >Don't get caught with egg on your face. Play Chicktionary! >http://club.live.com/chicktionary.aspx?icid=chick_wlmailtextlink > -End of Included Message--
[ccp4bb] questions about CNS
Hi everyone, I have some basic questions about CNS. First, I am wondering if there is anywhere I can set my initial B-factor during my early refinement. When I generate my initial model in generate.inp, I can set the B-factore. However, after I did the rigid.inp and anneal.inp, the B-factor just go up to more than 100 by itself. I look through the input files, and couldn't find where to set the B-factor. Do I have to set the B-factor by hand each time? Or will the crazy B-factor affect my refinement? Second, I am refining a new protein/DNA complex. I started with the DNA alone. After rigid body refinement I got ~1% decrease of Rf. But when I tried anneal after that, the Rf just went up much higher than where I started? If you work on a protein/DNA complex, what is your stategy? Will the anneal help refine the DNA alone? How much decrease of Rf usually you can get from refining just DNA? Third, I was wondering if I want to extend my resolution, do I have to make sure my cv file was made with the highest resolution? Thank you for your suggestion! Cortney _ Don't get caught with egg on your face. Play Chicktionary! http://club.live.com/chicktionary.aspx?icid=chick_wlmailtextlink
Re: [ccp4bb] questions about hydrophobic core
Hi, There exists a formal analytical way in doing this using the "survol" command in BRUGEL package. In short, this command define the accessible surface (external and cavities) to the probe you choose and creates separate masks (ensembles) of all atoms/residues that define these surfaces. The way to go, then, would be to create a collection mask of all accessible atoms/residues and subtract this from your protein mask to be left with the mask that contains the core residues. You could also use a cutoff of 5-10% ASA max (there are different ways of calculating these !). On the other hand, taking away even residues that display very little ASA (< 5%) would certainly leave you with genuine core residues. Hope this helps. Greetings, Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 UHP - Nancy 1, School of Medicine 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] Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Sebastiano Pasqualato wrote: Hi all, a few days ago I sent a post in which I was asking if anybody knew a program to automatically define the hydrophobic core of a protein, given the pdb. Unfortunately I got no answers, and indeed a more thorough googling around revealed that such a program might not exist. So it seems I have to define my hydrophobic core residues by hand... So now my question would be: how to define the hydrophobic core residues? I would tend to say that those that bury more than ## % (say 70%, 80% ??) of their otherwise solvent accessible surface area could be defined as such, but how can I get such a /per residue/ percentage? (NB: this is not the asa buried upon interaction, so I don't know how to get the asa of the "free" amino acid) Alternatively, are there other simple and defined rules to state which are the hydrophobic core residues? Any help appreciated, thanks in advance, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 Milano Italy tel +39 02 9437 5094 fax +39 02 574 303 310 No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.9.0/853 - Release Date: 6/18/2007 3:02 PM
Re: [ccp4bb] questions about hydrophobic core
while evaluating it, you might want to check the importance of buried residues by looking at how conserved they are in homologs. tommi Quoting Eleanor Dodson <[EMAIL PROTECTED]>: > I dont know the answer but have you looked at the PISA site at the eBI - > > there is extensive documentation addressing these sorts of questions. > http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html > Eleanor > Sebastiano Pasqualato wrote: > > > > Hi all, > > a few days ago I sent a post in which I was asking if anybody knew a > > program to automatically define the hydrophobic core of a protein, > > given the pdb. > > Unfortunately I got no answers, and indeed a more thorough googling > > around revealed that such a program might not exist. > > So it seems I have to define my hydrophobic core residues by hand... > > So now my question would be: how to define the hydrophobic core > residues? > > I would tend to say that those that bury more than ## % (say 70%, 80% > > ??) of their otherwise solvent accessible surface area could be > > defined as such, but how can I get such a /per residue/ percentage? > > (NB: this is not the asa buried upon interaction, so I don't know how > > to get the asa of the "free" amino acid) > > Alternatively, are there other simple and defined rules to state which > > > are the hydrophobic core residues? > > Any help appreciated, > > thanks in advance, > > ciao > > s > > > > > > > > -- > > Sebastiano Pasqualato, PhD > > IFOM-IEO Campus > > Dipartimento di Oncologia Sperimentale > > Istituto Europeo di Oncologia > > via Adamello, 16 > > 20139 Milano > > Italy > > > > tel +39 02 9437 5094 > > fax +39 02 574 303 310 > > > > > > > > > > > > > No virus found in this outgoing message. > > Checked by AVG Free Edition. > > Version: 7.5.472 / Virus Database: 269.9.0/853 - Release Date: > 6/18/2007 3:02 PM > > > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] questions about hydrophobic core
Sebastiano, I can suggests one approach to finding core residues.. CCP4mg will calculate the total asa buried per residue and residues can be selected by the criteria of their buried area. One way to define the protein core would be to select residues with, say, <5.0A*2 asa. You can easily see what has been selected in the molecular graphics and try varying the criteria if necessary. If you need to do this for many structures we could write a script. Liz On Tuesday 19 June 2007 09:55, Sebastiano Pasqualato wrote: > Hi all, > a few days ago I sent a post in which I was asking if anybody knew a > program to automatically define the hydrophobic core of a protein, > given the pdb. > Unfortunately I got no answers, and indeed a more thorough googling > around revealed that such a program might not exist. > So it seems I have to define my hydrophobic core residues by hand... > So now my question would be: how to define the hydrophobic core residues? > I would tend to say that those that bury more than ## % (say 70%, 80% > ??) of their otherwise solvent accessible surface area could be > defined as such, but how can I get such a per residue percentage? > (NB: this is not the asa buried upon interaction, so I don't know how > to get the asa of the "free" amino acid) > Alternatively, are there other simple and defined rules to state > which are the hydrophobic core residues? > Any help appreciated, > thanks in advance, > ciao > s > > > > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 Milano > Italy > > tel +39 02 9437 5094 > fax +39 02 574 303 310
Re: [ccp4bb] questions about hydrophobic core
I dont know the answer but have you looked at the PISA site at the eBI - there is extensive documentation addressing these sorts of questions. http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html Eleanor Sebastiano Pasqualato wrote: Hi all, a few days ago I sent a post in which I was asking if anybody knew a program to automatically define the hydrophobic core of a protein, given the pdb. Unfortunately I got no answers, and indeed a more thorough googling around revealed that such a program might not exist. So it seems I have to define my hydrophobic core residues by hand... So now my question would be: how to define the hydrophobic core residues? I would tend to say that those that bury more than ## % (say 70%, 80% ??) of their otherwise solvent accessible surface area could be defined as such, but how can I get such a /per residue/ percentage? (NB: this is not the asa buried upon interaction, so I don't know how to get the asa of the "free" amino acid) Alternatively, are there other simple and defined rules to state which are the hydrophobic core residues? Any help appreciated, thanks in advance, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 Milano Italy tel +39 02 9437 5094 fax +39 02 574 303 310 No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.9.0/853 - Release Date: 6/18/2007 3:02 PM
[ccp4bb] questions about hydrophobic core
Hi all, a few days ago I sent a post in which I was asking if anybody knew a program to automatically define the hydrophobic core of a protein, given the pdb. Unfortunately I got no answers, and indeed a more thorough googling around revealed that such a program might not exist. So it seems I have to define my hydrophobic core residues by hand... So now my question would be: how to define the hydrophobic core residues? I would tend to say that those that bury more than ## % (say 70%, 80% ??) of their otherwise solvent accessible surface area could be defined as such, but how can I get such a per residue percentage? (NB: this is not the asa buried upon interaction, so I don't know how to get the asa of the "free" amino acid) Alternatively, are there other simple and defined rules to state which are the hydrophobic core residues? Any help appreciated, thanks in advance, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 Milano Italy tel +39 02 9437 5094 fax +39 02 574 303 310 No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.9.0/853 - Release Date: 6/18/2007 3:02 PM
Re: [ccp4bb] questions related to refmac refinement and map-fitting
If the atoms belong to hydroxyl-, sulfhydryl- or carboxygroups, it could be a sign of radiation damage. In this case, I would model these with occupancy < 1. -- Ist Ihr Browser Vista-kompatibel? Jetzt die neuesten Browser-Versionen downloaden: http://www.gmx.net/de/go/browser
Re: [ccp4bb] questions related to refmac refinement and map-fitting
Hi, You could turn down the weight on the B-factor restraints (in geometric parameters). It seems like the restraints are two tight and do not allow the B-factor to increase enough to fit the density. ~L~ ___ Lari Lehtiö Structural Genomics Consortium Medical Biochemistry & Biophysics Dept. Karolinska Institute Stockholm, Sweden ___ - Original Message - From: lithomas <[EMAIL PROTECTED]> Date: Monday, March 19, 2007 7:47 am Subject: [ccp4bb] questions related to refmac refinement and map-fitting To: CCP4BB@JISCMAIL.AC.UK > Hello, > I am not sure if I can clearly explain the problem , but .. > > The protein structure that I tried to solve is 407 a.a. in length. > The crystal diffracts to 1.9 Ang and there is one molecule per asym > unit. The program that I used is Molrep. Currently I have put in > most of the residues and the R is 22.8% and the Rfree is 28.7% (no > water added yet) after refmac (v. 5.2) refinement. > > I had a hard time trying to finish the refinemnt task because I > noticed about ten residues' sidechains persistently had negative > density. Typically what I would do is to put these a.a. back to ala > and re-check the delFWT map in the next refinement cycle. However, > the refmac delFWT map in the next refinement cycle would show > strong positive (at least 2.5sigma) values. These amino acids don't > seem to have alternate conformations and the Rfactor increase <1% > and Rfree increase by 1%. Now, I seems to fall into this trap of > endless loop and trying to find a way out. > > Since this is the first time that I used refmac, I am not sure if I > did something wrong. Everything seems quite straightfoward from > the pull-down menu and a couple main parameters that I selected are > "restraint refinement", "no prior phase information", "anisotropic" > and "babinet scaling". I also tried CNS (v. 1.1) and the results > are pretty much the same. > > I know I should not let these residues bearing a large blob of > negative density on the very end of the side chains. But did anyone > have similar probelm and how to resolve this? > > Thanks for the help. > > Thomas >
[ccp4bb] questions related to refmac refinement and map-fitting
Hello, I am not sure if I can clearly explain the problem , but .. The protein structure that I tried to solve is 407 a.a. in length. The crystal diffracts to 1.9 Ang and there is one molecule per asym unit. The program that I used is Molrep. Currently I have put in most of the residues and the R is 22.8% and the Rfree is 28.7% (no water added yet) after refmac (v. 5.2) refinement. I had a hard time trying to finish the refinemnt task because I noticed about ten residues' sidechains persistently had negative density. Typically what I would do is to put these a.a. back to ala and re-check the delFWT map in the next refinement cycle. However, the refmac delFWT map in the next refinement cycle would show strong positive (at least 2.5sigma) values. These amino acids don't seem to have alternate conformations and the Rfactor increase <1% and Rfree increase by 1%. Now, I seems to fall into this trap of endless loop and trying to find a way out. Since this is the first time that I used refmac, I am not sure if I did something wrong. Everything seems quite straightfoward from the pull-down menu and a couple main parameters that I selected are "restraint refinement", "no prior phase information", "anisotropic" and "babinet scaling". I also tried CNS (v. 1.1) and the results are pretty much the same. I know I should not let these residues bearing a large blob of negative density on the very end of the side chains. But did anyone have similar probelm and how to resolve this? Thanks for the help. Thomas
[ccp4bb] questions on SF likelihood
Hi, I've managed to do a pretty good job of confusing myself in my latest attempt to understand the likelihood stuff, and was hoping that I could get some pointers as to what I'm misunderstanding. 1. How does one determine the amplitude and phase to use from a given likelihood surface? Some of the papers I've read refer to using the centroid; others seem to be talking about using the location of the maxima. Is there any guidance for when you'd use one instead of the other, or is this one of those "try both and see which works best" situations? 2. How do you get the HL coefficients out of a likelihood surface? The only way I could think of to do this would be to pick up the likelihood values over the full phaser circle for a constant amplitude, and fit a 2-term fourier series to the ln of those values. But this approach feels more like a work-around than anything else (and would lead to the same point in complex space having two difference likelihoods for a centric reflection), so I'm fairly sure there's a better way to do this (although I don't have any ideas what that would be). SigmaA weights might be a possibility, but as far as I know they wouldn't work for all cases (MAD and SAS don't have native amplitude measurements). Thanks in advance for any help, Pete Pete Meyer Fu Lab BMCB grad student Cornell University
