Re: [ccp4bb] Data Processing questions

[Tom Peat]

Hello Marco,

There are schools that will teach you about the software (and theory) used in 
crystallography and they tend to be very good, so one suggestion is to enroll 
in one of these 1-2 week courses to learn everything properly.

You should process all of your data sets- it is only after having processed 
everything you will be able to look at the log files and determine what frames/ 
images you may want to discard.  These log files should also give you an 
estimate as to whether you have enough selenium signal to solve the structure.
For the CCP4 suite, Aimless is the program that will give you the merging 
statistics. Pointless is run just before Aimless and will give you its best 
guess as to the space group (and yes, sometimes the space group will be 
ambiguous at this point in the process). Pointless/Aimless also allow you to 
merge multiple data sets from the same crystal. As to whether you can merge 
data sets from different crystals- you will need to process each one 
independently first and then determine whether they are isomorphous to each 
other- if they are, you can merge them if you think it will be helpful.

Hopefully that is a starting point for you.
Best regards, tom

From: CCP4 bulletin board  on behalf of Marco Bravo 

Sent: Thursday, May 23, 2024 11:01 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Data Processing questions

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Hi All,
I just collected a lot of data for native crystals and Seleno-methionine 
derivative crystals. I have a couple of questions
1. How do I know which images to process and which ones to exclude which might 
be radiation damaged and not be good to further process? I have been told it is 
possible to cutoff the damaged images, how do I know where to cut it off?
2. I also collected MAD data at two wavelengths (Remote, and at the arithmetic 
mean of the peak and inflection) and three for some crystals (remote, peak, and 
inflection), How do I proceed about solving the structure with this data and 
how should I process them? Is there an advantage to 3 wavelengths over 2 for 
MAD?
3. For some crystals I collected multiple datasets from the same crystal. I 
have been told I can merge the datasets (images) into a single, larger dataset. 
How do I do that? Can I combine datasets from different crystals grown under 
the same conditions with the same material? Or only the same individual 
crystal? Is it advantageous to merge datasets like this?
4. When Elves indexes the crystal initially, it always thinks the space group 
is P4. When I finish data collection it thinks P21 21 2. Is this normal? Is 
elves just guessing wrong initially? Why would my space group be ambiguous? Our 
resolution is around 3 angstroms.

Thank You!



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Re: [ccp4bb] Experimental phasing Selenomethionine data collection etc. tips

[Tom Peat]

I agree with Harry, I suspect that embarrassment has no definition/ meaning in 
the context of Nature.

From: CCP4 bulletin board  on behalf of Harry Powell 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
Sent: Tuesday, May 14, 2024 6:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Experimental phasing Selenomethionine data collection 
etc. tips

Hi

With this in mind, I have found the easiest way to generate AlphaFold models 
(for those not in the DB) is actually in CCP4 Cloud-remote - makes MR using AF 
models a real doddle.

See the Tutorials (in particular on MR) available with CCP4 Cloud.

Harry

> On 14 May 2024, at 09:20, Randy John Read  wrote:
>
> Dear Marco,
>
> You don’t mention here or in the earlier thread whether you tried AlphaFold 
> models and, if you did, how you prepared them for MR. I’m happy to hear of 
> any case where we still need experimental phasing methods to solve new 
> protein structures, but we’ve seen very few examples where AlphaFold models 
> didn’t work!
>
> I’m probably not the only one who would be delighted to take a look at the 
> problem to see what we can learn from it, if AlphaFold models really aren’t 
> working.
>
> Now, back to your question: to prepare for a SAD phasing experiments one 
> place I would look would be Tom Terwilliger’s recent papers on planning and 
> analysing SAD experiments (https://doi.org/10.1107/S2059798315019269, 
> https://doi.org/10.1107/S2059798315019403) as well as other information on 
> this from the Phenix website, including the YouTube tutorials.
>
> Best wishes,
>
> Randy Read
>
>> On 14 May 2024, at 01:17, Marco Bravo 
>>  wrote:
>>
>> Hello all,
>> I have a data collection trip next week and plan to collect data on 
>> selenomethionine derivative crystals at the al831 beamline. Are there any 
>> resources, tips, tutorials, literature etc. That you can recommend to help 
>> me prepare for these experiments. Also is there a way to plug in the 
>> experimental data into ccp4 cloud to do the automatic structure solution? Do 
>> I need native and derivative data to solve the structure? Last trip I 
>> collected a seemingly 2.8 angstrom resolution data on a crystal of the 
>> native protein but could not get a solution depsite extensive molecular 
>> replacement attempts. It seems that assigning a space group for the crystals 
>> has been troublesome as well. here is my last thread I posted about the 
>> issue for reference.
>>
>> https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=ind2402=CCP4BB=D=CCE6DFA19FA3D40346=mbrav005%40ucr.edu=112302
>>
>> 
>>
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>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research Tel: +44 1223 336500
> The Keith Peters Building
> Hills Road   E-mail: 
> rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.  
> www-structmed.cimr.cam.ac.uk
>
>
> 
>
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Re: [ccp4bb] Ubuntu and Coot

[Tom Peat]

And yet another quick update- I rebooted the machine again and now the ccp4 
version of Coot is working, but the standalone version is not (and the Phenix 
version continues to work). Just thought I would throw this out there for 
others...
cheers, tom

From: CCP4 bulletin board  on behalf of Tom Peat 
<b7e4a7a8af49-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, May 9, 2024 9:29 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Ubuntu and Coot

You don't often get email from b7e4a7a8af49-dmarc-requ...@jiscmail.ac.uk. 
Learn why this is important<https://aka.ms/LearnAboutSenderIdentification>
Just an update- I tested using Coot through the Phenix package and that seems 
to work (version 1.21-5207-000 and version 0.9.8.93 of Coot). Maybe a version 
issue? Or Phenix just comes with libraries that ccp4 doesn't? Not sure yet.
cheers, tom

From: CCP4 bulletin board  on behalf of Martin Malý 

Sent: Wednesday, May 8, 2024 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Ubuntu and Coot

Dear Tom,
I am using basically the same setup (Linux Mint 21.3 Xfce based on Ubuntu 
22.04, CCP4 8.0.019 including Coot 0.9.8.93). Everything works well on my 
computer. I did not have any issues with Coot even in CCP4 8.0.017. I am sorry 
I don't know the reason of the error...
Cheers,
Martin

On 08/05/2024 06:59, Tom Peat wrote:
Hello All,

I'm not sure if this is specific to me or whether I missed the fix, but I'm 
currently running Ubuntu 22.04.1 and updated to the latest ccp4 version 8.0.019 
and Coot crashes. I tested to see whether it was just the ccp4 version by 
downloading coot separately into my bin directory 
(coot-Linux-x86_64-ubuntu-20.04.3-pre-release-gtk2-python as this was the 
latest version I could find) and that gave the same outcome: core: #f
No core file found. No debugging

Coot crashed in the previous version of ccp4 8.0.017, which is one reason I 
upgraded to 8.0.019. I'm not sure if there was an update to Ubuntu that may 
have started this whole debacle, but everything seemed to work last month...

Has anyone else encountered this? Was there a fix? I googled several times and 
got some potential issues with libraries (libssl, etc), but none of these 
potential fixes helped.
cheers, tom



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Re: [ccp4bb] Ubuntu and Coot

[Tom Peat]

Just an update- I tested using Coot through the Phenix package and that seems 
to work (version 1.21-5207-000 and version 0.9.8.93 of Coot). Maybe a version 
issue? Or Phenix just comes with libraries that ccp4 doesn't? Not sure yet.
cheers, tom

From: CCP4 bulletin board  on behalf of Martin Malý 

Sent: Wednesday, May 8, 2024 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Ubuntu and Coot

Dear Tom,
I am using basically the same setup (Linux Mint 21.3 Xfce based on Ubuntu 
22.04, CCP4 8.0.019 including Coot 0.9.8.93). Everything works well on my 
computer. I did not have any issues with Coot even in CCP4 8.0.017. I am sorry 
I don't know the reason of the error...
Cheers,
Martin

On 08/05/2024 06:59, Tom Peat wrote:
Hello All,

I'm not sure if this is specific to me or whether I missed the fix, but I'm 
currently running Ubuntu 22.04.1 and updated to the latest ccp4 version 8.0.019 
and Coot crashes. I tested to see whether it was just the ccp4 version by 
downloading coot separately into my bin directory 
(coot-Linux-x86_64-ubuntu-20.04.3-pre-release-gtk2-python as this was the 
latest version I could find) and that gave the same outcome: core: #f
No core file found. No debugging

Coot crashed in the previous version of ccp4 8.0.017, which is one reason I 
upgraded to 8.0.019. I'm not sure if there was an update to Ubuntu that may 
have started this whole debacle, but everything seemed to work last month...

Has anyone else encountered this? Was there a fix? I googled several times and 
got some potential issues with libraries (libssl, etc), but none of these 
potential fixes helped.
cheers, tom



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[ccp4bb] Ubuntu and Coot

[Tom Peat]

Hello All,

I'm not sure if this is specific to me or whether I missed the fix, but I'm 
currently running Ubuntu 22.04.1 and updated to the latest ccp4 version 8.0.019 
and Coot crashes. I tested to see whether it was just the ccp4 version by 
downloading coot separately into my bin directory 
(coot-Linux-x86_64-ubuntu-20.04.3-pre-release-gtk2-python as this was the 
latest version I could find) and that gave the same outcome: core: #f
No core file found. No debugging

Coot crashed in the previous version of ccp4 8.0.017, which is one reason I 
upgraded to 8.0.019. I'm not sure if there was an update to Ubuntu that may 
have started this whole debacle, but everything seemed to work last month...

Has anyone else encountered this? Was there a fix? I googled several times and 
got some potential issues with libraries (libssl, etc), but none of these 
potential fixes helped.
cheers, tom



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Re: [ccp4bb] Crystallographic data table

[Tom Peat]

Hello Marco,

You can find all of that information in the Aimless log file.
Best regards, Tom

Get Outlook for iOS

From: CCP4 bulletin board  on behalf of Marco 

Sent: Monday, April 1, 2024 2:40:06 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Crystallographic data table

[You don't often get email from mbrav...@ucr.edu. Learn why this is important 
at https://aka.ms/LearnAboutSenderIdentification ]

I am trying to put together a crystallographic data table for my data. Where 
can I get the Rsym value for my table? Also where can I get the unique 
reflections and total reflections value for my table? I also need the I/sigma 
for the low and high resolution shells.

Thank you for your help

Marco



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Re: [ccp4bb] Difficult Molecular replacement

[Tom Peat]

Hello Marco,

This may seem a little obvious, but did you check the sequence of your protein 
from the gel/ crystals?
I've found mass spec to be very useful in this regard as we've had at least two 
instances of having a protein that looked to be the right size/ correct 
protein, but wasn't after mass spec analysis.
Good luck, tom

From: CCP4 bulletin board  on behalf of Marco Bravo 

Sent: Tuesday, February 20, 2024 9:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Difficult Molecular replacement

[You don't often get email from mbrav...@ucr.edu. Learn why this is important 
at https://aka.ms/LearnAboutSenderIdentification ]

Hello all,
I recently collected data on some plate crystals for a previously 
uncharacterized protein at the ALS light source. The XDS auto-data processing 
log output indicates that my resolution is 2.8 angstroms. The protein is a 
helicase with homologs already in the protein data bank making it a suitable 
target for molecular replacement which I thought initially. However after 
trying molecular replacements with all known homologs in the protein data bank 
the R values remain high after MR >0.5. After an initial round of Rigid body or 
restrained refinement. The R values still remain very high at around >.5. I 
have tried MR with Rosetta and alphaphold models but the problem of high R 
values persists. The best solution I get is from the CCP4 cloud automatic 
molecular replacement and model building pipeline which gives me a free R value 
of 0.46.. However the solution is only for residues ~100-326 out of a 543 amino 
acid long protein. And even then the model still has a lot of missing residues 
and truncated sidechains and overall fits the map quite poorly. Does anyone 
have any suggestions about how I can solve my structure if at all possible at 
this point? I ran the crystals and pre-crystalized samples on a gel and it 
appears that the protein remains stable during crystallization as the molecular 
weight did not change or any degradation does not appear.



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Re: [ccp4bb] Automated refinement convergence

[Tom Peat]

That is an interesting point, for those using big clusters to do extensive 
computing work.
I assume universities are 'going green' but that it will take a while for this 
to happen.
I just use my home computer and have my own solar panels and battery to keep 
things running.
cheers, tom


From: CCP4 bulletin board  on behalf of Guillaume 
Gaullier 
Sent: Friday, January 19, 2024 9:58 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Automated refinement convergence

You don't often get email from guillaume.gaull...@kemi.uu.se. Learn why this is 
important

Hello all,


While there is definitely scientific value in reaching perfect convergence by 
"cooking" refinement jobs "à point", in this day and age I think we should all 
reflect on whether this value is worth it, given that computing is also 
contributing to cooking the planet.

I am not advocating for poorly refined models, but for finding a reasonable 
baseline with no more cooking than is necessary to achieve satisfactory models. 
I know, define "necessary" and "satisfactory"... they are not the same if you 
want a reasonable model to answer a biological question or if you want to 
benchmark refinement strategies...

Here is an interesting resource if you worry about the sustainability of your 
computing: https://www.green-algorithms.org/
The calculator is nice too: https://calculator.green-algorithms.org/

Cheers,


Guillaume


---

Guillaume Gaullier, PhD
Researcher, Blikstad group
Molecular Biomimetics / Microbial Chemistry
Department of Chemistry - Ångström
Uppsala University
Lägerhyddsvägen 1
752 37 Uppsala
Sweden


From: CCP4 bulletin board  on behalf of Nigel Moriarty 

Sent: Friday, January 19, 2024 2:10:42 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Automated refinement convergence

I, too, love the smell of cooking (or cooked?) jobs in the morning.

Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Email : nwmoria...@lbl.gov
Web  : CCI.LBL.gov
ORCID : orcid.org/-0001-8857-9464


On Thu, Jan 18, 2024 at 1:16 PM James Holton 
mailto:jmhol...@lbl.gov>> wrote:
Hey there Robert,

Refmac has a keyword called "kill" that I think is what you are looking
for.  It is documented here:
https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html

   You can specify a conditional exit based on R factor, etc. Or you can
just create a specified file containing "stop Y" from an external
process.  I use it when running refmac on a cluster that has run time
limits but difficult-to-predict CPU speeds.

Phenix, I don't think has a checkpointing feature. Not that I know of.

Amber does support checkpointing and now counts as a refinement program
since support for structure factor restraints was added in 22.

Personally, when I do refinements I do dozens to hundreds of
macro-macro-cycles. As in, take the pdb file output by one run and feed
it into another run. There is an instantiation overhead to doing this,
as you note, but I like my models to be super converged. I define
convergence as the x,y,z,B and occ values in the pdb file are not
changed by the refinement program. This does not happen quickly, but it
does eventually happen. Yes, you can get oscillations, but one way to
deal with those is to add a bit more damping, or to adjust the x-ray
weight down and then up and then back to auto again. This "weight snap"
tends to take things that were dangling from a cliff in the energy
landscape and knock them to the ground. After that, the oscillations are
less common.

  And like an equilibrated chromatography column, an xyz-converged model
is the best way to know that when you edit and re-refine, everything you
see is due to the edit, and not some other process that just wasn't
finished yet.

That's what I do. Maybe I just want to feel like I've got something
cooking while I sleep...

Cheers,

-James Holton
MAD Scientist

On 1/18/2024 3:04 AM, Robert Oeffner wrote:
> Hi,
>
> I am wondering if authors of refinement programs would like to consider 
> putting on their users wish list the ability of refinement programs to 
> automatically terminate once the refinement has reached convergence. Various 
> refinement metrics such as R factors, CC or RMS values typically will reach a 
> plateau once the refinement of a macromolecular structure with X-ray or 
> EM-data has converged and further macro-cycles of refinement will no longer 
> improve the structure. The default number of macro-cycles in programs such as 
> Phenix-refine and Refmac are probably sensible for most cases but in some 
> cases it would be nice if the programs automatically extended the number of 
> macro-cycles as needed (or decreased the number).
>
> The user can of course examine 

Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[Tom Peat]

ffect of the extra parameters so their effective weight is maximum 3/5, 
but probably less. The weight of the restraints must be somewhere between 0 
(e.g when the B-factor restraint weight in Refmac is set to zero) and 0.6 (the 
restraint weight is huge). Assuming that the restraints do something, the 
degrees of freedom go up with less than 3 for our glycol refinement. How much 
less is related to the restraint weight we set in Refinement.

Cheers,
Robbie




On 6 Jan 2024 23:26, Tom Peat mailto:t.p...@unsw.edu.au>> 
wrote:
Hello Robbie,

Thanks for the stats and description of what has been done.
I think this puts us back into the realm of restraints versus constraints and 
what is possible when trying to reduce the number of parameters to be refined. 
Although grouped B-factors don't capture the reality of side chains being more 
mobile, it is a constraint that reduces the number of parameters being refined 
and helps highlight regions of the structure which are more mobile (which a 
flat or average B-factor would not do).
Making tighter restraints on the system as a whole doesn't change the 
data/parameter ratio, which can lead to its own issues, but is certainly better 
than just letting things go wild.
As is often the outcome, we state 'it depends on your individual situation' and 
generally suggest looking at various possibilities until one finds some 
compromise which works. Not as intellectually gratifying as having a cut and 
dry answer to these questions that come up rather frequently.
Thanks again for the stats and description.
cheers, tom


From: Robbie Joosten 
mailto:robbie_joos...@hotmail.com>>
Sent: Sunday, January 7, 2024 8:24 AM
To: Tom Peat mailto:t.p...@unsw.edu.au>>; 
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: RE: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[You don't often get email from 
robbie_joos...@hotmail.com<mailto:robbie_joos...@hotmail.com>. Learn why this 
is important at https://aka.ms/LearnAboutSenderIdentification ]

Hi Tom,

At 3A the median number of reflections per atom is 3.4 which is indeed lower 
than 4. So in unrestrained refinement the data/parameter ratio is indeed worse 
than 1. This is where the effect of the restraints really matter and starting 
from a flat B-factor model is interesting. This is what pdb-redo does if there 
are fewer than 4 reflections per atom. In such cases first TLS model are 
refined, one-group-per-chain (yes, that has room for improvement) plus any 
user-provided grouping. The TLS model that performs best in refinement is then 
kept (or no TLS model at all if they don't work). Given this TLS model, the 
structure model is refined with isotropic B-factors and flat B-factors. Both 
refinement results are then tested by a program called "bselect" that performs 
the Hamilton test plus some fallback test. The "best" model is then chosen. If 
this involves isotropic B-factors, the B-factor restraint weight is then 
optimised.

Some stats:
Of the 1958 cases in the databank, 573 are refined with a flat B-factor model 
(2.9 reflections/atom on average), 520 with isotropic B-factors and 
tighter-than-default restraint weights (3.8 reflections/atom on average), 612 
with isotropic B-factors and looser-than-default weights (4.1 reflections/atom 
on average), the rest is isotropic with default weights (3.8 ref/atom).

So there is a trend given the number of reflections per atom but it is not that 
strong for individual cases. Testing is needed. I won't claim that this is the 
best protocol for each of the cases, but I guess they are decent starting 
points for most.

Cheers,
Robbie

> -Original Message-
> From: Tom Peat mailto:t.p...@unsw.edu.au>>
> Sent: Saturday, January 6, 2024 21:42
> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>; Robbie Joosten
> mailto:robbie_joos...@hotmail.com>>
> Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by
> group?
>
> It appears that Zhonghao might be worried about his data to parameter ratio.
> At 3 A, one can easily be in a situation where one has fewer reflections than
> four times the number of atoms (X, Y, Z plus B).
> I like the idea of starting out with the average B (or even Wilson B) and then
> doing TLS as that should reduce the number of parameters being refined.
> Best regards, tom
>
> 
>
> From: CCP4 bulletin board 
> mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Robbie
> Joosten mailto:robbie_joos...@hotmail.com>>
> Sent: Saturday, January 6, 2024 8:13 PM
> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
> mailto:CCP4BB@JISCMAIL.AC.UK>>
> Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by
> group?
>
>   You don't often get email f

Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[Tom Peat]

Hello Robbie,

Thanks for the stats and description of what has been done.
I think this puts us back into the realm of restraints versus constraints and 
what is possible when trying to reduce the number of parameters to be refined. 
Although grouped B-factors don't capture the reality of side chains being more 
mobile, it is a constraint that reduces the number of parameters being refined 
and helps highlight regions of the structure which are more mobile (which a 
flat or average B-factor would not do).
Making tighter restraints on the system as a whole doesn't change the 
data/parameter ratio, which can lead to its own issues, but is certainly better 
than just letting things go wild.
As is often the outcome, we state 'it depends on your individual situation' and 
generally suggest looking at various possibilities until one finds some 
compromise which works. Not as intellectually gratifying as having a cut and 
dry answer to these questions that come up rather frequently.
Thanks again for the stats and description.
cheers, tom


From: Robbie Joosten 
Sent: Sunday, January 7, 2024 8:24 AM
To: Tom Peat ; CCP4BB@JISCMAIL.AC.UK 
Subject: RE: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[You don't often get email from robbie_joos...@hotmail.com. Learn why this is 
important at https://aka.ms/LearnAboutSenderIdentification ]

Hi Tom,

At 3A the median number of reflections per atom is 3.4 which is indeed lower 
than 4. So in unrestrained refinement the data/parameter ratio is indeed worse 
than 1. This is where the effect of the restraints really matter and starting 
from a flat B-factor model is interesting. This is what pdb-redo does if there 
are fewer than 4 reflections per atom. In such cases first TLS model are 
refined, one-group-per-chain (yes, that has room for improvement) plus any 
user-provided grouping. The TLS model that performs best in refinement is then 
kept (or no TLS model at all if they don't work). Given this TLS model, the 
structure model is refined with isotropic B-factors and flat B-factors. Both 
refinement results are then tested by a program called "bselect" that performs 
the Hamilton test plus some fallback test. The "best" model is then chosen. If 
this involves isotropic B-factors, the B-factor restraint weight is then 
optimised.

Some stats:
Of the 1958 cases in the databank, 573 are refined with a flat B-factor model 
(2.9 reflections/atom on average), 520 with isotropic B-factors and 
tighter-than-default restraint weights (3.8 reflections/atom on average), 612 
with isotropic B-factors and looser-than-default weights (4.1 reflections/atom 
on average), the rest is isotropic with default weights (3.8 ref/atom).

So there is a trend given the number of reflections per atom but it is not that 
strong for individual cases. Testing is needed. I won't claim that this is the 
best protocol for each of the cases, but I guess they are decent starting 
points for most.

Cheers,
Robbie

> -Original Message-
> From: Tom Peat 
> Sent: Saturday, January 6, 2024 21:42
> To: CCP4BB@JISCMAIL.AC.UK; Robbie Joosten
> 
> Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by
> group?
>
> It appears that Zhonghao might be worried about his data to parameter ratio.
> At 3 A, one can easily be in a situation where one has fewer reflections than
> four times the number of atoms (X, Y, Z plus B).
> I like the idea of starting out with the average B (or even Wilson B) and then
> doing TLS as that should reduce the number of parameters being refined.
> Best regards, tom
>
> 
>
> From: CCP4 bulletin board  on behalf of Robbie
> Joosten 
> Sent: Saturday, January 6, 2024 8:13 PM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by
> group?
>
>   You don't often get email from robbie_joos...@hotmail.com. Learn
> why this is important <https://aka.ms/LearnAboutSenderIdentification>
>
>
> One wonders who those "many people" are. You may not want to use them
> as your go-to reference for refinement techniques.
>
> Anyway, Refmac cannot do grouped B-factor refinement, but you are not
> missing out on anything. As Eleanor implied, one-per-residue B-factors give
> unrealistic results. You are much better off using isotropic B-factors with 
> tight
> restraints (Refmac's default is already quite tight). Add TLS in your 
> refinement
> to see if that helps.
> If you have a really poor data/parameter ratio you could go for a flat 
> B-factor
> model and try to capture most of the B-factor in the TLS model. This is
> typically not needed at 3A, but there are exceptions (low solvent -more
> atoms-  or low completeness -fewer reflections- are factors to consider). If
> you do go for a fl

Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

[Tom Peat]

It appears that Zhonghao might be worried about his data to parameter ratio. At 
3 A, one can easily be in a situation where one has fewer reflections than four 
times the number of atoms (X, Y, Z plus B).
I like the idea of starting out with the average B (or even Wilson B) and then 
doing TLS as that should reduce the number of parameters being refined.
Best regards, tom


From: CCP4 bulletin board  on behalf of Robbie Joosten 

Sent: Saturday, January 6, 2024 8:13 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

You don't often get email from robbie_joos...@hotmail.com. Learn why this is 
important

One wonders who those "many people" are. You may not want to use them as your 
go-to reference for refinement techniques.

Anyway, Refmac cannot do grouped B-factor refinement, but you are not missing 
out on anything. As Eleanor implied, one-per-residue B-factors give unrealistic 
results. You are much better off using isotropic B-factors with tight 
restraints (Refmac's default is already quite tight). Add TLS in your 
refinement to see if that helps.
If you have a really poor data/parameter ratio you could go for a flat B-factor 
model and try to capture most of the B-factor in the TLS model. This is 
typically not needed at 3A, but there are exceptions (low solvent -more atoms-  
or low completeness -fewer reflections- are factors to consider). If you do go 
for a flat B-factor model, you need to define sensible TLS groups. This takes 
some trial and error.

pdb-redo has decent algorithms to select the B-factor model and weight for 
Refmac. You could use that as a starting point for your model.

HTH,
Robbie



On 6 Jan 2024 03:13, "chenzhonghao...@163.com"  wrote:

Dear Prof. Dr. Dodson and all CCP4 community,



  Thanks for your reply.



 Just now, I used baverage. I found that it can average the B factor but not 
refine it.

  This function does not fit my requirement, because my resolution is low as 3 
A.

 Many people said that Refmac5 overrefines the structure if I used isotropic 
temperature refinement.



Did refmac5 or other programs in CCP4 have similar functions like 
one_adp_group_per_residue or two_adp_groups_per_residue in Phenix?



 Any help would be highly appreciated!




chenzhonghao...@163.com

From: Eleanor Dodson
Date: 2024-01-05 23:48
To: CCP4BB
Subject: Re: [ccp4bb] Can Refmac5 refine temperature factor residue by group?

Hmmm -  I am not sure about the value of this - one expects the longer floppier 
side chains to have very different B values for the CB than the OE2..

The program BAVERAGE gives you a plot of mean B value residue by residue..



baverage - averages B over main and side chain atoms

SYNOPSIS¶
baverage XYZIN foo_in.pdb RMSTAB foo_out1.tab XYZOUT foo_out2.pdb
[Keyworded input]
DESCRIPTION¶

A very simple minded program to read a PDB file, tabulate to RMSTAB the average 
B values residue by residue (main chain and side chain separately) and the RMS 
deviation of the B values from this mean. It also outputs a PDB file with 
outlying B factors reset to lie within the given range.

On Fri, 5 Jan 2024 at 03:08, 
chenzhonghao...@163.com 
mailto:chenzhonghao...@163.com>> wrote:
Dear CCP4 community,

 I found that Refmac5 refined the temperature factor only by four modes (see 
the bottom of the attached figure). However, no
grouped B-factor (one or two per residue instead of one per atom) was found.

 Actually, PHENIX and CNS can do it. But we are not familiar with both 
software. I want to know whether Refmac5 refines one or
two group B per residue (for side and main chains) grouped temperature factor?

 Any help would be highly appreciated

 Thanks in advance.

best,


 Zhonghao Chen










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Re: [ccp4bb] Query on density fitting to phosphate

[Tom Peat]

Hello Dale,

Thank you for the correction/ clarification.
I think this is still a tricky question, as in solution, this is an average 
state and one doesn't have a stable hydrogen (or two) sitting discretely on the 
phosphate. More specifically, the hydrogens are coming off and popping back on 
constantly (just the ratios change depending on the pH). It is likely that the 
phosphate is also moving in and out of the binding site of a protein in 
solution. What state is captured in a crystal structure and is that consistent 
across all of the proteins in that crystal?
As you say, one needs very high resolution to determine the bond length 
difference between those oxygens with and without a potential hydrogen attached 
to orient a phosphate correctly in a structure, assuming that there is only a 
single preferred orientation to start with.
I believe the original question was whether in fact the density supported a 
phosphate ion, and I still believe that looking for some anomalous signal may 
be a good way to approach that question.
Nonetheless, I stand corrected and there is likely to be some hydrogen on 
phosphate ions found in crystal structures.
Happy holidays to all, tom

From: Dale Tronrud 
Sent: Monday, December 25, 2023 8:52 PM
To: Tom Peat ; CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Query on density fitting to phosphate

[You don't often get email from de...@daletronrud.com. Learn why this is 
important at https://aka.ms/LearnAboutSenderIdentification ]

Hi,

I wanted to correct a statement by Prof. Peat about the ionic state
of phosphate in solution.  Phosphate has four states differing by the
number of attached hydrogen atoms.  The number of hydrogen atoms depends
on the pH, or maybe it is the other way around since phosphate is used
as a buffer.  I've attached a plot of the fraction of each species as a
function of pH (Citation: "By Clarolux - Own work, CC BY-SA 4.0,
https://commons.wikimedia.org/w/index.php?curid=90586171;).  There you
can see that for all pH's usually seen in mother liquors the solution is
almost completely either HPO4(-2) and H2PO4(-1).  A binding site may, of
course, prefer a species that is present in low concentration but such a
protein will be fighting entropy to fill its pocket.

Unless your mother liquor has an extreme pH you should expect that
the phosphate species you are seeing in your crystal has either one or
two hydrogen atoms attached.  Their presence will affect both the nature
of the hydrogen bonding of the protein to the phosphate and will change
the length of the P-O bonds (with the P-O-H bond being about 0.05 A
longer than the P=O bond).  The two lengths will only be distinguishable
given very high resolution diffraction data but there are examples in
the PDB where the differences are clear.  You can determine the presence
of an hydrogen atom at much lower resolution if the hydrogen bond is
made with an obligate hydrogen bond acceptor.

The inappropriate identification of an ion as PO4(-3) will
significantly degrade the quality of any electrostatic potential one
calculates from the model.

I did a quick-and-dirty search of the PDB for the various species of
phosphates in PDB entries.  While I found 5979 models with PO4(-3) (ID:
PO4) I only found 42 with HPO4(-2) (ID: PI) and 27 with H2PO4(-1) (ID:
2HP).  I didn't find any H3PO4 and could not find an ID code for that
molecule.  (This search was done quite a while ago.)   I believe
depositors are mostly assuming the ID PO4 indicates any protonation of a
phosphate ion but that is not correct.  I am unaware of any ID that is
defined as a phosphate ion with unknown protonation state.  To conform
to the wwPDB standards a depositor must do their best, using the limited
data available to them, to choose one species of phosphate when they
identify the presence of one, but almost certainly that choice should
not be PO4.

As usual, just causing trouble,
Dale E. Tronrud

On 12/17/2023 1:05 PM, Tom Peat wrote:
> Dear Arpita,
>
> The hydrogens on phosphate, just like sodium and potassium, will come
> off the oxygens in water.
> To be more explicit, you don't have mono- or di-hydrogen phosphate in
> water (except transiently), you just have phosphate, depending somewhat
> on the pH of course. At 2.5 Angstrom resolution, there is no way to
> 'see' hydrogens with X-rays.
> Depending on the wavelength you used for your data collection, you could
> try doing an anomalous map and see if you have any anomalous signal at
> this position, which may help in identifying what the density is.
> Best of luck, tom
>
> 
> *From:* CCP4 bulletin board  on behalf of Arpita
> Goswami 
> *Sent:* Sunday, December 17, 2023 9:46 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Query on density fitting to phosphate
>
> You don't often get email fro

Re: [ccp4bb] what is isomorphous?

[Tom Peat]

Hello All,

I think Randy makes a very good point here- it depends on what you are trying 
to do with your data sets.
If you are trying to merge them, 'isomorphous' is important for this to work. 
If you are using them for cross crystal averaging, being less isomorphous is 
better (more signal).

James Holton has a story of Louise Johnson collecting data on lysozyme (back in 
the 60's?) where she looked at one specific reflection to determine whether the 
data sets she was collecting would be isomorphous and scale. It turns out that 
although the cell was very similar, the dehydration state of the crystal was 
very important for two lysozyme data sets to scale together. The Rmerge for the 
two dehydration states was something crazy large, like 44%, even though under 
the standard 'rules' (more rules of thumb), one would have believed that these 
data sets should have been 'isomorphous'. For the data sets that had the same 
dehydration state, the data merged with 'typical' statistics of lysozyme (like 
3-4%).

James will have the details that I do not.
cheers, tom

From: CCP4 bulletin board  on behalf of Randy John Read 

Sent: Thursday, December 21, 2023 10:53 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] what is isomorphous?

[You don't often get email from rj...@cam.ac.uk. Learn why this is important at 
https://aka.ms/LearnAboutSenderIdentification ]

I think we’ve strayed a bit from Doeke’s original question involving crystals 
A, B and C, where I think the consensus opinion would be that we would refer to 
crystal C as not being isomorphous to either A or B.

On the question of what “isomorphous” means in the context of related crystals, 
I’m not sure we have complete consensus. I would tend to say that any two 
crystals are isomorphous if they have related unit cells and similar fractional 
coordinates of the atoms, so that (operationally) their diffraction patterns 
are correlated. However, there might be differences of opinion on whether two 
crystals can be considered isomorphous if one has exact crystallographic 
symmetry and the other has pseudosymmetry. (I would probably be on the more 
permissive side here.)

In principle, I suppose being isomorphous (“same shape”) should be a binary 
decision, but in practice we’re interested in the implications of the degree to 
which perfect isomorphism is violated. So I would tend to use the term “poorly 
isomorphous” for a pair where the correlation between the diffraction patterns 
drops off well before the resolution limit. Crick was focused on percentage 
change in cell dimensions, but Bernhard is right that what matters is the ratio 
between the difference in cell lengths and the resolution of the data. It’s a 
bit counter-intuitive, but the effect of the difference between cell edges of 
20 and 25 is the same as for cell edges of 200 and 205! By the way, the first 
time I learned this was from K. Cowtan and I hadn’t realised it’s also in Jan 
Drenth’s book.

For isomorphous replacement (something some of us dimly remember from the days 
before AlphaFold), being poorly isomorphous is bad, but for cross-crystal 
averaging the more poorly isomorphous the better, because the molecular 
transform is being sampled in different places in reciprocal space.

Best wishes,

Randy Read

> On 21 Dec 2023, at 10:53, Jon Cooper 
> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> Hello Harry,
>
> I think this is the paper you mean:
> https://scripts.iucr.org/cgi-bin/paper?S0365110X56002552
>
> They gave depressingly low estimates of how much the cell dimensions could 
> change in order for isomorphous replacement to still work. In reality, unit 
> cells can shrink and swell, but the fractional atomic coordinates remain 
> relatively unchanged (right?) so bigger unit cell differences still allow the 
> method to work.
>
> Best wishes, Jon Cooper. jon.b.coo...@protonmail.com
>
> Sent from Proton Mail mobile
>
>
>
>  Original Message 
> On 21 Dec 2023, 09:07, Harry Powell < 
> 193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:
> Hi Didn’t Francis Crick have something to say about this in the early 1950s? 
> I’m sure it was published but off the top of my mind I can’t think where (one 
> of the more “established” members of this community will be able to give 
> chapter and verse)! If you want to read something a little more detailed than 
> people have mentioned here, there’s a “Methods in Enzymology” chapter by 
> Charlie Carter (?) et al from the early part of this century on the subject - 
> again, I can’t remember exactly who or when. Have a good break (which reminds 
> me to register for the CCP4 Study Weekend)! Harry > On 21 Dec 2023, at 08:04, 
> Tim Gruene wrote: > > Hi Doeke, > > you can take the coordinates of B and do 
> a rigid body refinement > against the data from A. If this map is sufficient 
> to reproduce model A > (including model building and more refinement cycles), 
> then B is > 

Re: [ccp4bb] Query on density fitting to phosphate

[Tom Peat]

Hello Arpita,

If you look up the f" values of various elements ( 
http://www.bmsc.washington.edu/ ) you will see that there are elements which 
will give you more or less signal at your wavelength of 1.5412 eV. For example, 
phosphate can be seen to have an f" value of about 0.43 e at that wavelength/ 
energy. As a counter example, Na only has ~0.1 e at that energy. Assuming you 
have reasonable completeness and multiplicity in your data, you should be able 
to see that kind of difference (if it is there).
Best regards, tom

From: CCP4 bulletin board  on behalf of Arpita Goswami 

Sent: Wednesday, December 20, 2023 3:53 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Query on density fitting to phosphate

You don't often get email from bt.arp...@gmail.com. Learn why this is 
important
Hi,

Thank you all for the replies.

As suggested, this is the residual positive difference density after putting 
water and refinement in coot-refmac:
https://i.postimg.cc/DfbrbDZm/Screenshot-from-2023-12-19-16-39-38.png

Does this look like any of the three phosphate ions? Two sides are similar, 
while one side is a little longer than others. H2PO4- has single negative 
charged Oxygen.

The alternate conformation of the said arginine may not be possible as it is 
situated quite far from aspartate.

As suggested both H2PO4- and HPO42- should dissociate to phosphate ions. Is it 
possible some H2PO4- will remain undissociated at pH 6.2 at low temperature 
like 4 degree centigrade (crystallization condition)? The pKa of H2PO4- 
dissociation is 7.2.

Thank you for the suggestion of an anomalous map. We used 1.5412 A wavelength 
for data collection. So for an anomalous map for Phosphorus we do not have the 
data for now.

I tried sodium and potassium ions. Sodium gives residual positive difference 
density as seen in water, for potassium also likewise. Also presence of lysine 
at close distance is not helpful for them.


Waiting eagerly for your reply.

--

Thank you.

Best Regards,

Arpita


On Mon, Dec 18, 2023 at 7:01 AM Joel Tyndall 
mailto:joel.tynd...@otago.ac.nz>> wrote:

Have you tried sodium?



From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Arpita Goswami
Sent: Sunday, December 17, 2023 11:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Query on density fitting to phosphate



Dear All,



Hope you all are doing well.



The density in the image (in link below)  is fitted with PO4 ion, although the 
crystallization condition has both mono and dihydrogen phosphate which is not 
fitting without hydrogen. But the resolution is 2.5 A, so hydrogen may not be 
put in, or is there any way to do so? Otherwise placing water is the final 
option.



https://i.postimg.cc/4N7q2K0p/Screenshot-from-2023-12-17-16-07-07.png



Also the density is quite close to Aspartate, so PO4 may not be right. Can it 
be dihydrogen phosphate as two positively charged residues (Specially the 
lysine) are also nearby to neutralize positive charge? Other ions in the 
crystallization condition are Cl-, K+ and Na+. These are not put as both 
aspartate and lysine are at comparable distances from the density. The pH is 
6.2 in which dihydrogen phosphate is reported to interact with aspartate 
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855859/).



Waiting eagerly for your reply.

--

Thanks and Merry Christmas in advance.

Best Regards,

Arpita





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Re: [ccp4bb] Query on density fitting to phosphate

[Tom Peat]

Dear Arpita,

The hydrogens on phosphate, just like sodium and potassium, will come off the 
oxygens in water.
To be more explicit, you don't have mono- or di-hydrogen phosphate in water 
(except transiently), you just have phosphate, depending somewhat on the pH of 
course. At 2.5 Angstrom resolution, there is no way to 'see' hydrogens with 
X-rays.
Depending on the wavelength you used for your data collection, you could try 
doing an anomalous map and see if you have any anomalous signal at this 
position, which may help in identifying what the density is.
Best of luck, tom


From: CCP4 bulletin board  on behalf of Arpita Goswami 

Sent: Sunday, December 17, 2023 9:46 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Query on density fitting to phosphate

You don't often get email from bt.arp...@gmail.com. Learn why this is 
important
Dear All,

Hope you all are doing well.

The density in the image (in link below)  is fitted with PO4 ion, although the 
crystallization condition has both mono and dihydrogen phosphate which is not 
fitting without hydrogen. But the resolution is 2.5 A, so hydrogen may not be 
put in, or is there any way to do so? Otherwise placing water is the final 
option.

https://i.postimg.cc/4N7q2K0p/Screenshot-from-2023-12-17-16-07-07.png

Also the density is quite close to Aspartate, so PO4 may not be right. Can it 
be dihydrogen phosphate as two positively charged residues (Specially the 
lysine) are also nearby to neutralize positive charge? Other ions in the 
crystallization condition are Cl-, K+ and Na+. These are not put as both 
aspartate and lysine are at comparable distances from the density. The pH is 
6.2 in which dihydrogen phosphate is reported to interact with aspartate 
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855859/).

Waiting eagerly for your reply.
--
Thanks and Merry Christmas in advance.
Best Regards,
Arpita



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Re: [ccp4bb] Coot query

[Tom Peat]

Just in case something like this happens to someone else:
I am running the Gnome display server and I did another restart and I can now 
run Coot.
The magic of rebooting...
cheers, tom

From: Paul Emsley 
Sent: Friday, July 7, 2023 2:09 AM
To: Tom Peat ; CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Coot query



On 06/07/2023 08:51, Tom Peat wrote:
Hello All,

I just had a very strange experience- I rebooted my linux machine (Ubuntu jammy 
7.81.0) and tried to start up Coot and received the following error message:


It is my understanding that jammy jellyfish, by default, uses Wayland as the 
display server. It seems to me then that running an X11-based application will 
have to go via XWayland, and who knows what goodies that could bring. So my 
suggestion to you is to try rebooting and using an X11-based display manager.


I will note here that a person such as yourself Tom could compile the gtk4 
branch of Coot, which can now run on Wayland directly.


Paul.




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[ccp4bb] Coot query

[Tom Peat]

Hello All,

I just had a very strange experience- I rebooted my linux machine (Ubuntu jammy 
7.81.0) and tried to start up Coot and received the following error message:

The program 'coot-bin' received an X Window System error.
This probably reflects a bug in the program.
The error was 'BadValue (integer parameter out of range for operation)'.
  (Details: serial 351 error_code 2 request_code 152 minor_code 3)
  (Note to programmers: normally, X errors are reported asynchronously;
   that is, you will receive the error a while after causing it.
   To debug your program, run it with the --sync command line
   option to change this behavior. You can then get a meaningful
   backtrace from your debugger if you break on the gdk_x_error() function.)
. -- Coot crashed - trying to diagnose -
ldd -r on guile gives:
  linux-vdso.so.1 (0x7ffd6d0b9000)
  libguile.so.17 => 
/opt/xtal/ccp4-8.0/coot_py2/libexec/../lib/libguile.so.17 (0x7fd11380)
  libgmp.so.10 => /opt/xtal/ccp4-8.0/coot_py2/libexec/../lib/libgmp.so.10 
(0x7fd11340)
  libcrypt.so.1 => /lib/x86_64-linux-gnu/libcrypt.so.1 (0x7fd113c6b000)
  libm.so.6 => /lib/x86_64-linux-gnu/libm.so.6 (0x7fd113b84000)
  libltdl.so.7 => /opt/xtal/ccp4-8.0/coot_py2/libexec/../lib/libltdl.so.7 
(0x7fd11300)
  libpthread.so.0 => /lib/x86_64-linux-gnu/libpthread.so.0 
(0x7fd113b7d000)
  libc.so.6 => /lib/x86_64-linux-gnu/libc.so.6 (0x7fd112c0)
  /lib64/ld-linux-x86-64.so.2 (0x7fd113cbc000)
  libdl.so.2 => /lib/x86_64-linux-gnu/libdl.so.2 (0x7fd113b78000)
Guile 1.8.8
Copyright (c) 1995, 1996, 1997, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 
2008 Free Software Foundation
Guile may be distributed under the terms of the GNU General Public Licence;
certain other uses are permitted as well.  For details, see the file
`COPYING', which is included in the Guile distribution.I
There is no warranty, to the extent permitted by law.
catching the crash log:
Gtk-Message: Failed to load module "gail"
Gtk-Message: Failed to load module "atk-bridge"
Gtk-Message: Failed to load module "canberra-gtk-module"
coot-exe: "/opt/xtal/ccp4-8.0/coot_py2/libexec/coot-bin"
/usr/bin/ls
-rwxr-xr-x 1 tom tom 9256240 Jun 21 12:54 
/opt/xtal/ccp4-8.0/coot_py2/libexec/coot-bin
coot-version:
/opt/xtal/ccp4-8.0/coot_py2/libexec/coot-bin
Builder_info: CCP4, Oxfordshire

I tried this both as command line (coot xxx.pdb) and through the CCP4i window 
and I get the same error message. I also tried with more than one coordinate 
file (in different directories).
I find this strange as I was just finishing up some structures with Coot/ 
Refmac (everything working fine) and had turned off my machine to take a break 
for a day, so things were working very recently. I don't remember doing any 
major updates either...
Has anyone else seen this behaviour?
cheers, tom



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[ccp4bb] Just a reminder

[Tom Peat]

Hello All,

The early bird registration comes to an end for the IUCr 2023 meeting this year 
in Melbourne at the end of March (very soon). For those that would like the 
discounted rate for registration, it is time to take the plunge.
https://www.iucr.org/iucr/cong/2023-iucr-xxvi
https://iucr2023.org/

cheers, tom

Tom Peat, PhD
one of many on the committee...



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Re: [ccp4bb] To Trim or Not to To Trim

[Tom Peat]

Gotta love the extra wood on the fire.
In the bad old days, I agree, we had no real idea.
But these days I usually have mass spec done on most everything and 
additionally look at different crystal forms. Often I find that in one space 
group more will be seen than in another (and that one isn't necessarily the 
highest resolution) and these are even often from the same plate/ screen. 
Additionally, many proteins form oligomers or are found in multiple copies in 
the asymmetric unit, and these again vary as to how much of that mystical 
N-terminus or C-terminus one sees. I would be pretty confident that it isn't 
just one protomer in a trimer/ tetramer that has that extension and the rest of 
have selectively proteolysed...
Good point though. cheers, tom

From: CCP4 bulletin board  on behalf of Jurgen Bosch 

Sent: Saturday, March 11, 2023 8:37 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] To Trim or Not to To Trim

You don't often get email from jxb...@case.edu. Learn why this is 
important<https://aka.ms/LearnAboutSenderIdentification>
Well Tom,

Your missing N-terminus might just be degraded same with the C-terminus two do 
you know it is actually there waving at you? Do you have mass spec evidence for 
the full existence of your protein? You might just have crystalized that 
magical fragment thinking that your full length construct is actually 
crystallized.

Just throwing some wood into the fire …

Jürgen

On Mar 10, 2023, at 4:01 PM, Debanu Das  wrote:

Hi Tom,

-"so no matter how much we try to educate people, the vast (vast, vast) 
majority of people will take the models as the 'truth'
-"So if we don't see something, the conservative approach is to probably avoid 
putting it in, otherwise it will get propagated forever"

I absolutely agree. I have a recent anecdote about this to share. I was at a 
workshop where the majority of attendees were medicinal chemists, comp 
chemists, biologists. I found out that some (many?) were even performing comp 
chem studies (where ligands and waters were important) on structures downloaded 
straight from the PDB without even checking associated electron density for 
such ligands/waters or even realizing potential side chain issues (not even 
including about any potential main chain or other quality/validation issues).

Best regards,
Debanu
--
Debanu Das,
https://bio.site/debanu_das

On Fri, Mar 10, 2023 at 12:41 PM Tom Peat 
mailto:t.p...@unsw.edu.au>> wrote:
Hello All,

I agree with Dale that we don't have a good way to model these things and it 
has been a discussion for a very long time with no proper answer.

Two more small points- we (or at least I do this all the time) don't model the 
N- or C-terminal residues that I don't see. They are most likely there 
somewhere (waving goodbye in the solvent mask) and that seems to be 
(relatively) standard practice, so I'm not sure how different this is to not 
putting in side chains that can't be seen? We don't replace with Ala, so people 
should know what the actual sequence is, but there is no evidence for a side 
chain. A multi-model is likely the better way to go, but isn't as feasible 
currently as the single model we normally deposit. As mentioned by others, we 
shouldn't try to put in ligands or co-factors that we don't see either...

One other point- although we should educate, the PDB has estimated that 99% of 
the users are not structural biologists (nor modellers or others with 
training), so no matter how much we try to educate people, the vast (vast, 
vast) majority of people will take the models as the 'truth'. So if we don't 
see something, the conservative approach is to probably avoid putting it in, 
otherwise it will get propagated forever.

My two cents. cheers, tom

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Dale Tronrud mailto:de...@daletronrud.com>>
Sent: Saturday, March 11, 2023 7:15 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] To Trim or Not to To Trim

Hi

As a frequent contributor to prior discussions on this same topic I
would like to broaden the discussion a bit.  I'm sorry to say that most
of the comments on this threat are exactly the same positions that have
been expressed many times over the years.  I don't want to spent time,
again, retyping my opinions on how I prefer to torment the parameters of
my models to express what I believe is going on inside my crystals.

The fundamental problem is that the parameters we are forced to use,
in our PDB depositions and in the refinement and model building programs
available to us, are wholly inadequate.  We cannot accurately (or
precisely) describe what we are are envisioning for the surface side
chains, and sometimes entire stretches of main chain, in our proteins.
We can continue to argue with each other year after year, 

Re: [ccp4bb] To Trim or Not to To Trim

[Tom Peat]

Hello All,

I agree with Dale that we don't have a good way to model these things and it 
has been a discussion for a very long time with no proper answer.

Two more small points- we (or at least I do this all the time) don't model the 
N- or C-terminal residues that I don't see. They are most likely there 
somewhere (waving goodbye in the solvent mask) and that seems to be 
(relatively) standard practice, so I'm not sure how different this is to not 
putting in side chains that can't be seen? We don't replace with Ala, so people 
should know what the actual sequence is, but there is no evidence for a side 
chain. A multi-model is likely the better way to go, but isn't as feasible 
currently as the single model we normally deposit. As mentioned by others, we 
shouldn't try to put in ligands or co-factors that we don't see either...

One other point- although we should educate, the PDB has estimated that 99% of 
the users are not structural biologists (nor modellers or others with 
training), so no matter how much we try to educate people, the vast (vast, 
vast) majority of people will take the models as the 'truth'. So if we don't 
see something, the conservative approach is to probably avoid putting it in, 
otherwise it will get propagated forever.

My two cents. cheers, tom

From: CCP4 bulletin board  on behalf of Dale Tronrud 

Sent: Saturday, March 11, 2023 7:15 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] To Trim or Not to To Trim

Hi

As a frequent contributor to prior discussions on this same topic I
would like to broaden the discussion a bit.  I'm sorry to say that most
of the comments on this threat are exactly the same positions that have
been expressed many times over the years.  I don't want to spent time,
again, retyping my opinions on how I prefer to torment the parameters of
my models to express what I believe is going on inside my crystals.

The fundamental problem is that the parameters we are forced to use,
in our PDB depositions and in the refinement and model building programs
available to us, are wholly inadequate.  We cannot accurately (or
precisely) describe what we are are envisioning for the surface side
chains, and sometimes entire stretches of main chain, in our proteins.
We can continue to argue with each other year after year, but there is
no solution to this problem other than changing the nature of PDB models
and allowing a reasonable description of multi-conformation models.

I believe it is fair to say that the consensus after a previous
round of this discussion was that, at the very least, we need a flag for
each atom which indicates whether that atom was placed based on electron
density or simply to make a chemically complete set of atoms for that
type of monomer.  I haven't looked but I think that was about five or
ten years ago.  Since then the PDB has made major changes to the
structure of PDB entries that will require most software for analysis of
macromolecular models be rewritten and right now that organization is
making a major push to get us to virtually attend a workshop to help us
make this transition.  And yet I don't think there is anything in this
new data dictionary to help us with this important but intractable
problem.

Unless the PDB gives us the parameters we need to properly describe
a macromolecular model, and the refinement/model building developers
give us the tools to make use of them, we will be back here again, every
five years or so, rehashing this debate over exactly the same,
irreconcilably poor, solutions to this problem.

Dale E. Tronrud


On 3/10/2023 1:05 AM, Julia Griese wrote:
> Hi all,
>
> My impression has been that the most common approach these days is to
> “let the B-factors take care of it”, but I might be wrong. Maybe it’s
> time to run another poll?
>
> Personally, I call any other approach R-factor cosmetics. The goal in
> model building is not to achieve the lowest possible R-factors, it’s to
> build the most physically meaningful, most likely to be correct, model.
> So if you know that the side chain is part of the protein, you should
> model it the best way you can. If it’s there, just disordered, then the
> most correct way to model it is to let it have high B-factors. Most
> molecular graphics programs don’t flag zero-occupancy atoms, so the user
> might never notice. Truncation of a side chain, unless there is evidence
> that it really physically isn’t there, is also misleading, in my
> opinion. I don’t believe that it is more helpful to the non-expert user
> than high B-factors either.
>
> If people who are not structural biologists themselves don’t know how to
> use a structure, then we need to educate them better. It is very
> straightforward these days to look at electron density in the PDB
> viewer. It used to be difficult, but nowadays there’s no excuse for not
> checking the electron density. The PDB validation flags RSRZ outliers.
> You can easily colour a 

[ccp4bb] a reminder

[Tom Peat]

Hello All,

We have less than one week before the abstract deadline comes to an end for the 
IUCr 2023 meeting this year in Melbourne.
This is a second, and possibly last reminder...
For those that would like to present their work at the meeting, it is time to 
pull everything together.
https://www.iucr.org/iucr/cong/2023-iucr-xxvi
https://iucr2023.org/

cheers, tom

Tom Peat, PhD
one of many on the committee...



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[ccp4bb] Just a reminder

[Tom Peat]

Hello All,

We have about two weeks before the abstract deadline comes to an end for the 
IUCr meeting this year in Melbourne.
For those that would like to present their work at the meeting, it is time to 
pull everything together.
https://www.iucr.org/iucr/cong/2023-iucr-xxvi
https://iucr2023.org/

cheers, tom

Tom Peat, PhD
one of many on the committee...



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Re: [ccp4bb] Interesting Density

[Tom Peat]

I second glycerol.
Best regards, tom

Get Outlook for iOS

From: CCP4 bulletin board  on behalf of kotping 

Sent: Tuesday, December 6, 2022 6:02:00 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Interesting Density

You don't often get email from kotp...@gate.sinica.edu.tw. Learn why this is 
important
Glycerol

-Original message-
From:John Smith
To:CCP4BB
Date: Tue, 06 Dec 2022 14:05:35
Subject: [ccp4bb] Interesting Density
Dear all!

I noticed some interesting density at the surface of my protein.
It looks like a distorted T - when I change the Fo-Fc to 5.5 sigma one arm is 
still visible, one weakly and the other one disappeared.  Crystallisation 
condition has Hepes, NaCl, NaSCN, TCEP and PEG3350. Any ideas what this could 
be?

I appreciate any help you can provide. Best John



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Tzu-Ping  Ko,  PhD
Staff  Research  Assistant
Institute  of  Biological  Chemistry
Academia  Sinica,  Taipei,  Taiwan


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[ccp4bb] IUCr in 2023

[Tom Peat]

Hello All,

Some of you may have noticed that the deadline for abstract submission for the 
IUCr meeting in Melbourne, Australia passed just recently. There are reasons 
for the early deadline, but please forgive us for that. There is an extension/ 
second call for abstracts which will be advertised in the near future. In the 
meantime, the portal is still open and you are welcomed and encouraged to 
continue to submit and register for this conference (where we hope to see many 
of you).

This link should work to get you there: 
https://icmsaust.eventsair.com/iucr-2023/portal-2

Best regards, Tom



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Re: [ccp4bb] Lower b-factors with increasing T

[Tom Peat]

I think the basic question being asked is why are the B-factors going the 
'wrong' way?
That is, as the temperature increases, one might expect higher B-factors (at 
least that is what we are taught) whereas what Matt is seeing is the opposite- 
decreasing B-factors as one goes up in temperature (which I also think is a 
little strange and I don't have an explanation).
cheers, tom

From: CCP4 bulletin board  on behalf of Phoebe A. Rice 

Sent: Thursday, September 8, 2022 10:48 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Lower b-factors with increasing T


I guess the big question is what is the question that you’re trying to address 
from those numbers?   I’d be nervous about making conclusions about trends in B 
factors from just 1 data set per temperature.  As you probably know, the B 
factors will reflect static differences in atomic position across asymmetric 
units as well as thermal motion, and it can be difficult to control variables 
such as exactly how fast a crystal freezes or how much trauma it experiences in 
its journey from sitting happily in a drop to the frozen state.



From: CCP4 bulletin board  on behalf of Matt McLeod 

Date: Wednesday, September 7, 2022 at 1:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Lower b-factors with increasing T

Hi everyone,

I have a series of datasets at 253K (~2.0A), 273K (2.0A), 293K (2.0A), 313K 
(2.2A) and I am curious as to the details in determining B-factors.

I have treated these datasets more-or-less identically for comparison's sake.  
I used DIALS to index, integrate, and scale the data.  I scaled the data to a 
~0.6 CC1/2 cutoff.

After fully refining the datasets, there is an odd trend with respect to 
temperature (from what has been previously published) and I assume that this is 
because of "behind-the-scenes" computation rather than a biophysical 
observation.  The B-factors slightly decrease from 252-293K, and then 
significantly drop at 313K.  The maps look pretty well identical across the 
datasets.

253K - 53.8 A^2
273K - 48.4 A^2
293K - 45.5 A^2
313K - 18.6 A^2

I compared the wilson intensity plots from DIALS scaling for 273K and 313K and 
they are very comparable.

I am looking for suggestions as to where to look at how these b-factors are 
selected or how to validate that these B-factor are or are not accurate.  Also, 
any relevant literature would be welcomed.  From what I have read, there is a 
general trend that as T increase, the atoms have more thermal energy which 
raises the b-factors and this trend is universal when comparing datasets from 
different temperatures.

Thank you and happy to supply more information if that is helpful,
Matt



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[ccp4bb] coot crashing

[Tom Peat]

Hello All, 

Just wondering if anyone else is having issues with Coot crashing (regularly, 
not just once in a while). 
I get the following error message: 

/home/pea246/bin/ccp4-7.0/bin/coot: line 318:  2025 Segmentation fault  
(core dumped) $coot_bin "$@"
guile (GNU Guile) 2.2.3
Packaged by Debian (2.2.3-deb+1-3ubuntu0.1)
Copyright (C) 2017 Free Software Foundation, Inc.

License LGPLv3+: GNU LGPL 3 or later .
This is free software: you are free to change and redistribute it.
There is NO WARRANTY, to the extent permitted by law.
catching the crash log:
Gtk-Message: Failed to load module "gail"
Gtk-Message: Failed to load module "atk-bridge"
Gtk-Message: Failed to load module "canberra-gtk-module"
coot-exe: "/home/pea246/bin/ccp4-7.0/libexec/coot-bin"
/bin/ls
coot-version: 
/home/pea246/bin/ccp4-7.0/libexec/coot-bin
platform: 
/bin/uname
core: #f
No core file found.  No debugging


I'm currently using Ubuntu 18.04.2 LTS and I think I'm reasonably up to date 
wrt updates... 

Any help would be appreciated if someone has resolved any similar issue. 

cheers, tom 



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Re: [ccp4bb] Non-Cysteine modification with heavy atom

[Tom Peat]

Hello Chandra,

Soaking heavy atoms into crystals has a long and successful history (I use it 
pretty often and it works much of the time). If you need phase information and 
you have some crystals, I would certainly give it a try. There are many papers 
and books to give you methods to do this (good old Blundell and Johnson for 
one, Methods in Enzymology for another).
No reason to waste good native crystals if you still have some ;-)
Cheers, tom

Sent remotely

On 3 Jan 2019, at 6:43 am, Chandramohan Kattamuri 
<1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk>
 wrote:


Dear CCP4 members, Can someone suggest to me a methodology to covalently modify 
(non-cysteine) on component of a multicomponent complex with heavy atoms in 
order to achieve better phase determination?

Thanks in advance


Chandra
kattamuricha...@yahoo.com






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Re: [ccp4bb] Assumptions on protein activity (again)

[Tom Peat]

Hello Markus, 

As has been mentioned by others, there are many ways to inactivate or inhibit a 
protein's activity. 
Why do you assume your protein is unfolded? From the information you have 
given, the protein is folded, just inactive or has low activity under several 
buffer conditions other than the one 'optimal' condition. 

Best regards, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Markus Heckmann 

Sent: Friday, October 12, 2018 10:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Assumptions on protein activity (again)

Dear all
Thanks to for all the responses. I would continue my question for
getting more advice.

I expressed a dimeric multi-protein (600 KDa).  This protein was
purified (affinity chromatography) in 4 different common buffers and
run with the same SEC-buffer. We observe clear single size-exclusion
peak referring to the dimer. We then measure activity of these 4
different protein yields in a well established assay.

What we observe: Only one out of 4 has high activity and even one
sample has NO activity at all. In all cases, the HPLC-SEC-MALS signal
using 'protein sample from activity assay' confirms unequivocally  a
dimeric state.

The observation of dimeric peak without activity means that  the
protein have a state that is either mis-folded - either locally or
partially. Are there any *sensitive* methods to detect this behaviour
- minor structural changes?

Can circular-dichroism detect these changes? or any other methods for
a large multi-domain protein?

Many thanks,
Markus



Previous responses:
---
I would not say *active* in the case of an enzyme, but probably
*folded*. An enzyme may have many conformational states, some of which
may represent inactive states, which will not be distinguished with
gel filtration (because their hydrodynamic radii will be roughly the
same), unless the inactivation involved unfolding and aggregation of
the protein.

Is your enzyme pH sensitive? For example, if it has a histidine in the
active site and most of the buffer conditions you are testing are
below pH 6, you may be looking at a well folded protein that just
isn't active because you've protonated the active site residue. Or it
could be that the buffers you are testing are binding to your protein
and sterically interfering with your substrate? It doesn't mean that
your protein isn't folded or even inactive if you have just blocked
the binding site, merely inhibited. There could be all kinds of
reasons that changing buffers could change the activity of the protein
without unfolding the protein itself. Another example is that people
often use phosphate buffer in purification, but if the enzyme requires
a Mg, you could be inadvertently pulling that out of the enzyme by
using phosphate buffer (or using sulfate with an enzyme that requires
Ca, etc).
I'm sure it is possible that there are many enzymes in the PDB that
are clearly well folded (have good structures) that are not in their
fully active states due to the crystallisation conditions used to
obtain the crystals. We are usually capturing a single state of a
protein which usually has to be mobile to perform its enzymatic
function.
---
You can speak for yourself, but not for me. I do not assume activity
from a gel; that's what assays are for.Different buffers: it could be
you have a cofactor, perhaps a metal. The best practice is to document
what you do in your publications to the extent that a reader could
duplicate your results.
---
There are lots of examples in the PDB of incorrect structures. And a
single peak on SE doesnt guaruntee correctly folded protein. What were
the differences between the buffers? pH, ionic strength and additives
all matter for enzyme activity, and many buffers do bind to active
sites thus affecting activity (despite the general attempt to use
large molecules which are unlikely to bind in the cases of the Good
buffers). All that being said, the idea of a single, correctly folded
conformation of an enzyme/protein is an oversimplification used in
textbooks rather than the more complicated picture held by experts in
the field.
--
I believe the strong assumption in the community is that a clear
single peak of appropriate Mw is a clear indication of pure protein,
worthy intensive crystallization efforts. Whether it is active is
another question and this should be measured.For your analysis, it is
not important in which buffers the protein is not active, but whether
the protein you purified is active in the buffer (maybe without
precipitant) you used for crystallization.A single apo structure is
usually not enough to determine the catalytic mechanism of an enzyme,
you usually need some substrate-, transition state

Re: [ccp4bb] Protein is dimmer in solution, hard to build two chains in crystal structure

[Tom Peat]

?Hello Zhu,


The implication being that one can have half of a dimer in the asymmetric unit 
without any issues (biological or crystallographic). And having a R/Rfree after 
a bit of refinement of the values you report for a 3 A data set is not that far 
off from being appropriate.


Best of luck, tom


Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, August 20, 2018 7:55 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein is dimmer in solution, hard to build two chains 
in crystal structure

75% solvent is not uncommon, and such crystals often only diffract to 3A.
Eleanor

On 20 August 2018 at 10:24, SUBSCRIBE CCP4BB Zhu Qiao 
mailto:jasonqia...@gmail.com>> wrote:
Hi All

My protein is dimer both in protein buffer and crystallisation reservoir, which 
is confirmed by calibration column Supderdex 200 10/300 increase.

The crystal can diffract to 3 and the space group was determined to be P212121.

The solvent content and Matthews coefficient shows
1 copy, solvent content is 74.5% and Matthews coeff is 4.82
2 cpoies, solvent content is 49% and Matthews coeff is 2.41 .

I did MR. I can only get one molecule in ASU and the R work / R free is 
0.31/0.35 after several cycles of refinement. There are some uncontinued 
electron density, which indicating may be one more chain is there.

I tried to search the other one by disallow the packing test or increase the 
packing cut off value in phaser, I can get two molecules, but only one has good 
fit to the electron density map. The other chain hardly have any electron 
density.

Does anyone have any experience regarding this situation? We appreciate your 
help.



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Re: [ccp4bb] crystals that dont diffract :( :(

[Tom Peat]

?We have some crystals that diffract well when fresh (less than one week old) 
but lose almost all diffraction by the end of 2 weeks, so age can matter. One 
crystals are in liquid nitrogen, they should be safe from further degradation, 
but may suffer from ice contamination.

cheers, tom


Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board  on behalf of Careina Edgooms 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: Tuesday, August 14, 2018 7:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystals that dont diffract :( :(

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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Re: [ccp4bb] Acceptable range of CC1/2

[Tom Peat]

I think all of those numbers would be pretty acceptable to almost all referees 
;-)
Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Deepali 
Verma
Sent: Tuesday, 5 June 2018 7:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Acceptable range of CC1/2

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




   Overall  InnerShell  OuterShell

  Low resolution limit   74.67 74.67  2.95

  High resolution limit   2.80  8.85  2.80



  Rmerge 0.067 0.042 0.964

  Rmerge in top intensity bin0.043- -

  Rmeas (within I+/I-)   0.072 0.046 1.032

  Rmeas (all I+ & I-)0.072 0.046 1.032

  Rpim (within I+/I-)0.026 0.018 0.368

  Rpim (all I+ & I-) 0.026 0.018 0.368

  Fractional partial bias   -0.025-0.019-0.071

  Total number of observations  289022  8892 42020

  Total number unique37156  1218  5376

  Mean((I)/sd(I)) 16.5  39.0   2.1

  Mn(I) half-set correlation CC(1/2) 0.999 0.998 0.771

  Completeness99.3  98.7  99.0

  Multiplicity 7.8   7.3   7.8



  Anomalous completeness  99.3  98.5  98.9

  Anomalous multiplicity   4.0   3.9   4.0

  DelAnom correlation between half-sets -0.042-0.036 0.015

  Mid-Slope of Anom Normal Probability   0.937   - -

--
Regards,
Deepali Verma
Junior Research Fellow
Jaypee Institute of Information Technology
[Image removed by sender.]



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Re: [ccp4bb] suggestion of crystallization optimization

[Tom Peat]

Just a quick note- even if the crystals appear the same (i.e. the same 
morphology in a light microscope), that doesn't necessarily mean that they 
diffract the same. Did you try putting some of these optimised crystals into an 
x-ray beam? 
Or as previously suggested, try them at room temperature? 
Best of luck, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Liuqing Chen 
<519198...@163.com>
Sent: Monday, June 4, 2018 8:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestion of crystallization optimization

Hello everyone!

I get a crystal several months ago, but the crystals diffraction very low 
resolution (around 8A)  or no diffraction.
  My sample buffer is 20mm Hepes ph7.0,  50mm NaCl,   my protein pI is 5.
  the codition grow crytal  is : 30% PEG400, 100mm hepes 7.5,  200mm MgCl2.

I also tried  additive screen,  all the crystals appear the same apparence,
even i seeding optimization,  have no improve.
the  attach is  my crystals.

what should   i  do next?

thanks in advance.
sincerely
Liuqing chen



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Re: [ccp4bb] What's happened over the last five years with high-throughput protein crystallization screening?

[Tom Peat]

Unfortunately it is unlikely that the costs for robotic equipment (at least the 
larger scale equipment) will come down much.
It is effectively all ‘bespoke’ equipment and will never benefit from high 
volume manufacturing (nothing like phones or cars).
How many crystallisation centres are needed around the world? Maybe 100? 
Nothing like the tens of millions of cars, phones, etc.
Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Daniel M. 
Himmel, Ph. D.
Sent: Friday, 27 April 2018 8:14 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] What's happened over the last five years with 
high-throughput protein crystallization screening?

I skimmed your paper, and overall it looks like a good overview
of high-throughput protein crystallization.  However, I was surprised
that no mention was made of Formulatrix Rock Maker software,
which is an excellent computer-aided graphical tool for designing
crystallization screens rapidly.  This software works either as a standalone
or in conjunction with the Formulatrix Formulator (which the paper DOES 
mention),
for preparing crystallization solutions, and/or an integrated system for
storing crystal drops and automatically photographing them under a microscope.

A great deal of experience and knowhow on high-throughput
protein crystallization was accumulated by
such researchers as Wladek Minor, Steve Almo, Jeff Bonanno,
and others, with whom I had the privilege of working during the
waning years of the "Protein Structure Initiative" and "Enzyme
Function Initiative".  The controversy of these projects stemmed
from the high expense of the robotic equipment that made them
possible, but the methodologies developed and lessons learned
may be useful for high-throughput protein crystallization both
in academia and industry.  Hopefully the cost of the robotic equipment
will come down.

-Daniel


On Thu, Apr 26, 2018 at 11:43 AM, Yibin Lin 
> wrote:
Dear CCP4BB Community,

I would like to comment you guy a review of the last five years of
high-throughput protein crystallization screening  that would be a
magnificent help for all scientists that struggle with macromolecular
crystallization.

Please see attachment, as well as below:


https://www.tandfonline.com/doi/full/10.1080/17460441.2018.1465924

Thanks.

Kind regards,

Frank Lin

Re: [ccp4bb] new ContaMiner features

[Tom Peat]

I agree with Tristan, it can be quite easy to crystallise a contaminant even 
when one is trying to be careful during the purification process. 
Before everyone had a mass spec, looking at gels didn't tell you as much as you 
needed to know, as many proteins don't stain well, so are hard to see on the 
gel (and as mentioned, can be in very small quantities and still crystallise). 

Cheers, tom

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
r...@mrc-lmb.cam.ac.uk
Sent: Friday, 24 November 2017 6:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] new ContaMiner features

Dear Stefan,

Just a couple of thoughts:

- first of all I think that Gerard is absolutely right, it would have been nice 
to raise such issues first with the developers. In my experience, Staraniso 
does a fantastic job if used correctly.

- but if you're OK with public trials, may I ask: why on Earth would anybody 
need ContaMiner? Are you trying to offer some sort of computational cure for 
sloppy biochemistry? There is zero point in crystallizing crap samples, sorry 
to say this. In my 17 or so years in Strubi I've never heard of anybody 
crystallizing a "contaminant", being it a purification tag or whatever.

I suppose this might have happened to somebody you know, hence the motivation 
to spend time on the bizarre ContaMiner. Which is a pity, a silly outcome would 
only teach people to do their job (or train their robots) properly.

Best wishes,

Radu

--
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu

> Dear Stefan,
>
>  Regarding your final paragraph: your server carries a warning 
> with the exact wording:
>
>  "Submitting StarAniso files can give you suspicious results. Use 
> with care!"
>
>  It seems rather regrettable that you are posting such a public 
> warning without ever having contacted the STARANISO developers about 
> your observations, nor giving any information about what you call 
> "suspicious" or what the "care" you recommend would consist of.
>
>  We have taken a great deal of care ourselves in developing the 
> program and offering it to the community through a server, and the 
> least we would have expected is that any pattern of "suspicious"
> results would be referred to us so that we could investigate them.
> There may be some assumptions made in MoRDa that we are not aware of, 
> that might be incompatible with assumptions made in STARANISO - who 
> knows? Or it could be that some particularly badly collected datasets 
> are made to look worse after their anisotropy analysis.
>
>  Could we discuss your observations, and what it is exactly that 
> you call "suspicious", before they end up being referred to in such an 
> uninformative manner as some sort of "Government Health Warning"?
>
>  I think that would be nice :-) and we would be only too keen to 
> take whatever extra "care" is needed ourselves. We would all learn 
> something.
>
>
>  With best wishes,
>
>   Gerard.
>
> (on behalf of the STARANISO developers)
>
> --
> On Thu, Nov 23, 2017 at 05:02:39PM +0100, Stefan Arold wrote:
>> Dear Community,
>>
>> A quick message to announce the following two new features on our 
>> ContaMiner web server for the automated detection of unwantedly 
>> crystallised contaminants (
>> https://strube.cbrc.kaust.edu.sa/contaminer/submit)
>>
>> 1) online visualisation of 2FoFc and FoFc maps. In cases of positive 
>> results, the ‘UglyMol’ tab allows to inspect 2FoFc and FoFc maps 
>> directly in the web browser. Thi
>>
>> 2) life-update. Previously, results were sent to you once all ~2000 
>> MR jobs were finished. Now, the individual results for each potential 
>> contaminant will appear as soon as they are finished. This feature 
>> should substantially shorten the time for identifying positive 
>> results (i.e. contaminant detected), which are terminated faster than 
>> negative ones.
>>
>> 3) custom contaminants. In the ‘Advanced’ tab, users can upload own 
>> PDB files (more than one is possible) to be included as search 
>> models. This feature can be used to include PDB files from your lab 
>> bench neighbour’s project to test for potential lab internal 
>> contaminations (through bacterial contamination or through mix-up of 
>> plasmids or glycerol stocks).
>> This feature could also be ‘abused’ as a means to use the MoRDa 
>> pipeline to run molecular replacements with template structures that 
>> are not yet deposited in the PDB; for example to run molecular 
>> replacement and initial refinement for liganded or complexed versions 
>> of an unpublished structure.
>> This might be particularly interesting for crystallographers away 
>> from their usual home software environment (e.g. at the beamline).
>>
>> Finally, a word of 

Re: [ccp4bb] Large scale mammalian expression system recommendation?

[Tom Peat]

Hello All, 

I would second the notion that transient systems are quite good for testing 
expression levels on the small scale and can be used for scale up (to a certain 
degree at least). It is probably the fastest way to go to screen a number of 
constructs. 

Cheers, tom

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
r...@mrc-lmb.cam.ac.uk
Sent: Sunday, 22 October 2017 10:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Large scale mammalian expression system recommendation?

Hi Nate,

There can be no consensus I guess, most importantly because transmembrane 
proteins are so diverse :-)

We typically use transient transfection at the screening stage, and often for 
large-scale expression (e.g. PMID 24909990). Bac-Mam has some advantages 
(nicely described in 25299155, 27041595), but it's obviously more time 
consuming. Lentiviral systems (we use Clontech) are also great.

But again the target often determines the choice of system. Whether it contains 
multiple subunits, whether overexpression has a negative impact on cell health 
(a tetO switch can be helpful, 12370422). Also, I'd use different systems 
depending whether the recombinant protein is intended for functional, 
fluorescence microscopy, structural analyses (say X-ray vs cryo-EM).

Best wishes,

radu
--
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu


> Hello everyone,
>
> I wonder if there is a consensus for what is currently the best system to
> express transmembrane proteins in mammalian cells?
>
> I think that baculovirus transduction ("Bacmam" anf the likes) has been
> used historically more (?) but would like to know if the modern adenovirus
> systems offer any advantages in terms of expression levels. I used both in
> the past but not for transmembrane proteins and never compared them back to
> back for the same protein.
>
> Has anyone attempted a direct comparison? (It has to be mammalian cells, so
> baculovirus/insect cells won't do).
>
> Thanks for any comments/insights,
>
> Nate
>

Re: [ccp4bb] Primer design

[Tom Peat]

A lot of plant genomes are big- wheat for example is 12 Gb, so it may not be 
quite as trivial as one might expect. 
cheers, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org>
Sent: Tuesday, July 25, 2017 1:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Primer design

>No need of the whole exome. Sequencing The second PCR product will do the job 
>I guess. Second PCR (from the cDNA pool) with specific forward primer and and 
>oligodA reverse primer. Surely a matter of less than $3

$3 is a major understimation, but I see your point. On the other hand, it is 
important to consider new technologies, and it would be a service to the 
scientific community to publish the exome somewhere, so other researchers would 
not have to spend their $3.

JPK





Best,

DKG


- Original Message -
From: "Jacob Keller" <kell...@janelia.hhmi.org>
To: "Debasish Kumar Ghosh" <dkgh...@cdfd.org.in>, CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 6:48:25 PM
Subject: RE: Primer design

Or sequence the whole exome for what, $500-1000?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish 
Kumar Ghosh
Sent: Monday, July 24, 2017 7:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Primer design

Dear Syed,

The process is very trivial to clone your gene of interest. Assuming your gene 
is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse 
primer. First isolate the total RNA from the tissue or cells and do the cDNA 
synthesis using oligodT primer followed by gene specific PCR with forward 
primer and oligodA primer from the cDNA pool. Sequence the PCR product to get 
to know the first stop codon in the ORF. Making the specific reverse primer 
from the sequence is then just matter of time.
Hope this helps.

Best wishes,

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group Centre for DNA Fingerprinting and 
Diagnostics Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: 
http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 4:56:00 PM
Subject: [ccp4bb] Primer design

Hello All

I am interested in cloning a gene from a plant. I searched the database only 
partial sequence is available, ie: for 160 residues only. The full length of 
the protein is around 570 residues. I designed forward primer and I have no 
clue to design reverse primer.

Any help

Thank you

Syed

[ccp4bb] postdoctoral position available

[Tom Peat]

Dear Board Readers,

For anyone who might be interested, or know of interested persons, there is a 
postdoctoral position available at CSIRO:

https://career10.successfactors.com/sfcareer/jobreqcareer?jobId=39270=CSIRO

Best regards,  tom

Re: [ccp4bb] intermolecular dissulphides

[Tom Peat]

Hello Eleanor,


We found some intermolecular vicinal disulfides recently that we think are 
'real'. This class of proteins forms tetramers and in one version we find these 
intermolecular disulfides across molecules. We did some tests and found that 
oxidation or reduction has an effect on the stability of the protein. ?We also 
saw this was consistent across multiple space groups. If you would like to have 
a look, they were just released: 5HY0, 5HY2, 5HY4.

As a comparison to another protein in this fold class that doesn't have the 
disulfide is 5HWE.


cheers, tom


Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Eleanor Dodson 
<eleanor.dod...@york.ac.uk>
Sent: Thursday, February 2, 2017 2:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] intermolecular dissulphides

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15

We seem to have one but it would have to form after crystalisation?

Eleanor

Re: [ccp4bb] just out of totally idle curiosity ...

[Tom Peat]

I don't know about Europe, but it is very tight Down Under... 


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of William 
G. Scott
Sent: Wednesday, 9 November 2016 4:38 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] just out of totally idle curiosity ...

What’s the job situation in Europe looking like for refugee scientists these 
days?



William G. Scott
Director, Program in Biochemistry and Molecular Biology Professor, Department 
of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 
University of California at Santa Cruz Santa Cruz, California 95064 USA

http://scottlab.ucsc.edu

Re: [ccp4bb] Interesting DNA contamination

[Tom Peat]

I think the more interesting questions are: should one want to disrupt such a 
tight interaction?
Wouldn’t the structure of the protein bound to DNA be more interesting than the 
protein alone?
Why not try to crystallize the complex and show how the protein binds?
Sometimes you should just run with what Nature gives you.
Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bonsor, 
Daniel
Sent: Friday, 26 June 2015 11:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Interesting DNA contamination

Several different approaches may help you to separate DNA from protein 
including;

1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 
1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt 
protein-DNA interactions.
2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to 
partial unfold (increase breathing) to disrupt the interaction.
3) Combine salt with low concentrations of denaturants.
4) Try a couple of different restriction enzymes such as DpnI or FatI to see if 
you can break it into smaller fragments. If you can, you maybe able to clone 
into a cloning vector with compatable ends/blunt ligation to sequence and 
identify the region of host DNA that is causing the problem.

Dan




Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar 
[pramod...@gmail.com]
Sent: Thursday, June 25, 2015 5:23 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Interesting DNA contamination
Dear all

Sorry for off topic and lengthy post, but I came across a very unique DNA 
contamination during one membrane protein purification (a microbial external 
environment sensor/response protein)

Already done

* DNAse used as stranded protocol during cell break.
* Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl
* Fluorescence size exclusion done with 0.1M NaCl
* Well stable peak and pure protein profile observed
* But final purified protein inherently contains specific 0.5 Kb DNA stretch 
visible through out purification (observed by running Agarose gel of protien 
sample @ each step)


Approach failed

* Buffer switched from HEPES to Na-PO4
* O.N. dialysis in presence of DNAse
* Using Heparin Column purification between Ni-Aff and SE (failed and protein 
starts deterioration)
* Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion)

Now... I need help for

* How to get rid of DNA without loosing active protein?
* What are best lipids to dope as -ve DNA replacement?
* Since protein is pure and ample, how likely I can get crystal hits with such 
big DNA attached?
* What longest possible DNA can be crystallize/ed with protein?
* How exclusive are some mem-spanign-proteins to provide anchorage of 
prokaryotic genomic stretch?



​Thanks in advance​ :)

Pramod Kumar

Re: [ccp4bb] [RANT] Reject Papers describing non-open source software

[Tom Peat]

In practice, for me to be able to replicate and verify your computational 
analysis and results, I will need to be able to see your source code, compile 
it myself, and potentially modify it.

Why? In this case to verify something you need to have the same output for a 
given input, this does not require any of the above (source code, compilation 
or modification), you just require a working version of the program/software. 

I like the open source model, but there are problems with this model along with 
the others. One major problem is that the authors are expected to support their 
software after others have gone in and modified it, which is unrealistic.  

I just think it is unrealistic to expect the source code just because someone 
wrote a paper (again, I like open source, but not everything is destined to be 
open source). 

cheers, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Douglas Theobald 
[dtheob...@brandeis.edu]
Sent: Wednesday, May 13, 2015 6:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [RANT] Reject Papers describing non-open source software

On May 12, 2015, at 3:19 PM, Robbie Joosten robbie_joos...@hotmail.com wrote:

 I strongly disagree with rejecting paper for any other reasons than
 scientific ones.

I agree, but … one of the foundations of science is independent replicability 
and verifiability.  In practice, for me to be able to replicate and verify your 
computational analysis and results, I will need to be able to see your source 
code, compile it myself, and potentially modify it.  These requirements in 
effect necessitate some sort of open source model, in the broadest sense of the 
term.  To take one of your examples, the Ms-RSL license — I can’t effectively 
replicate and verify your results if I’m legally prohibited from compiling and 
modifying your source code, so the Ms-RSL is out.

 A paper describing software should properly describe the
 algorithms to ensure the reproducibility.

*Should*.  In practice, we all know (those programmers among us do, anyway) 
that descriptions of source code do not suffice.

 The source should be available for
 inspection to ensure the program does what was claimed, for all I care this
 can be under the Ms-RSL license or just under good-old copyright. The
 program should preferably be available free for academic users, but if the
 paper is good you should be able to re-implement the tool if it is too
 expensive or doesn't exactly do what you want so it isn't entirely
 necessary.

 Making the software open source (in an OSS sense) does not solve any
 problems that a good description of the algorithms doesn't do well already.

This is just wildly wrong.  It’s basically impossible to ensure and verify that 
a “good description of the algorithm actually corresponds to the source code 
without seeing, using, and modifying the source.  To take an experimental 
analogy — my lab has endured several cases where we read a “good published 
description of the subcloning and sequencing of some vector, only to find that 
the detailed published description is wrong when we are given the chance to 
analyze the vector ourselves.  It happens all the time, and computer code is no 
different in this respect.

 OSS does not guarantee long-term availability, a paper will like outlive the
 software repository. OSS licenses (not the BSD license) can be so
 restrictive that you end up having to re-implement the algorithms anyway. So
 not having an OSS license should not be a reason to reject the paper about
 the software.

 Cheers,
 Robbie

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 James Stroud
 Sent: Tuesday, May 12, 2015 20:40
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] [RANT] Reject Papers describing non-open source
 software

 On May 12, 2015, at 12:29 PM, Roger Rowlett rrowl...@colgate.edu
 wrote:

 Was the research publicly funded? If you receive funds from NSF, for
 example,
 you are expected to share and make widely available and usable software
 and inventions created under a grant (section VI.D.4. of the Award and
 administration guide). I don't know how enforceable that clause is,
 however.

 The funding shouldn't matter. I suggest that a publication that has the
 purpose
 of describing non-open source software should be summarily rejected by
 referees. In other words, the power is in our hands, not the NSF's.

[ccp4bb] question on charge charge interactions

[Tom Peat]

Hello All, 

I am appealing to the community as I don't seem to be able to find through 
Google what I am looking for, and I just don't have the ability to look through 
every structure in the PDB to find this. 
I have what I think is an interesting case: a two domain protein structure with 
a mostly hydrophobic interface between the two domains- the kicker is that I 
have two charged residues buried in this domain and they are identical. 
That is, I'm looking for an Arg-Arg or Asp-Asp type interaction (not Arg-Asp) 
where these residues are less than 4 Angstroms apart. So I was wondering if 
anyone had seen this in any other structure(s)?  
The really interesting bit is that this interaction actually regulates the 
activity of the enzyme domain although the domain interface isn't that close to 
the catalytic site.  

Thanks in advance to all those who can find a reference to this kind of 
interaction. 
Cheers, tom

Re: [ccp4bb] question on charge charge interactions

[Tom Peat]

Hello Joel, 

I like the example of HIV protease, but in this case these Asp residues are 
found in the active site of the protein, and unless there is substrate (or 
inhibitor) in the active site, these would be solvent exposed (unless I'm 
looking at the wrong pair of Asp residues). In the particular case I'm looking 
at, I have a buried pair with no other charged residues around- no waters/ 
metals, just Phe, Leu, etc. Which is why I think it might be slightly more rare 
than most of the examples I've heard about so far. 

Thanks for the help. 
Cheers, tom

-Original Message-
From: Joel Tyndall [mailto:joel.tynd...@otago.ac.nz] 
Sent: Friday, 28 March 2014 12:02 PM
To: Peat, Tom (CMSE, Parkville); CCP4BB@JISCMAIL.AC.UK
Subject: RE: question on charge charge interactions

Tom,

I would think the case can be made for sharing a proton (one ionised and one 
not) in either case but more so for acidic residues. See HIV protease Asp-Asp 
as a well-established example

Hope this helps

J

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tom Peat
Sent: Friday, 28 March 2014 12:12 p.m.
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] question on charge charge interactions

Hello All, 

I am appealing to the community as I don't seem to be able to find through 
Google what I am looking for, and I just don't have the ability to look through 
every structure in the PDB to find this. 
I have what I think is an interesting case: a two domain protein structure with 
a mostly hydrophobic interface between the two domains- the kicker is that I 
have two charged residues buried in this domain and they are identical. 
That is, I'm looking for an Arg-Arg or Asp-Asp type interaction (not Arg-Asp) 
where these residues are less than 4 Angstroms apart. So I was wondering if 
anyone had seen this in any other structure(s)?  
The really interesting bit is that this interaction actually regulates the 
activity of the enzyme domain although the domain interface isn't that close to 
the catalytic site.  

Thanks in advance to all those who can find a reference to this kind of 
interaction. 
Cheers, tom

Re: [ccp4bb] Biacore/SPR

[Tom Peat]

As Juergen has mentioned, both the T200 and the BioRad ProteOn (XPR36) are good 
options (we have both).
The XPR36 is a higher throughput machine and can also be used for small 
molecule work (not quite as sensitive as the T200, but more sensitive than we 
had anticipated and certainly higher throughput).
Good luck,  tom

Tom Peat
Biophysics Group
CSIRO, CMSE
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bosch, Juergen 
[jubo...@jhsph.edu]
Sent: Tuesday, October 29, 2013 2:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Biacore/SPR

BiaCore 3000 or if you can afford the T200.
Proteon XPR36 would be a cheaper Option and should work as well.
Jürgen

On Oct 28, 2013, at 10:54 AM, Gang Dong wrote:

We mainly measure protein-protein interactions (sometimes protein-small 
molecules). Thanks! _Gang

From: Bosch, Juergen [mailto:jubo...@jhsph.edu]
Sent: Monday, October 28, 2013 3:17 PM
To: Gang Dong
Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Biacore/SPR

Protein-protein interaction?
protein-small molecule interactions ?
Epitope mapping of mABs ?
Could you specify what you would like to do, as different models are good for 
different things.
Jürgen

On Oct 28, 2013, at 10:08 AM, Gang Dong wrote:


Dear all,

Could anyone tell me your experience with Biacore (any models) or a similar 
SPR-based technology for measuring interaction kinetics? I also want to know 
the price ranges to discuss with our department/facility managers.

Thanks,
Gang



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] questions

[Tom Peat]

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] statistical or systematic? bias or noise?

[Tom Peat]

Slightly off the topic, but still potentially relevant in terms of realistic 
experimental error: when dealing with the small volumes typically used in 
crystallization (say 1 uL + 1 uL drops), and using a 10 uL pipette, the errors 
are fairly high (more like 30% than 5-10%), leading to a lot of 
non-reproducibility in the experiment- even when setting up the same exact 
solution many times.  Going to robotics helps with the reproducibility in 
liquid transfer, but doesn't necessarily help with the reproducibility of 
crystallization (an example of this can be found in: 
http://journals.iucr.org/d/issues/2007/07/00/bw5202/ ).

Cheers,  tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
mjvdwo...@netscape.net
Sent: Thursday, 14 March 2013 12:07 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] statistical or systematic? bias or noise?

I think that in statistics you can build a model that describes (and predicts) 
the uncertainty. So if you have done similar (!) replicate experiments, from 
which you can build the model, you can apply it to a single observation and 
provide a reasonably good guess for the value that you were measuring and its 
variance. Of course that guess would not be as good as the average value and 
variance from true replicates.

With protein crystals (or solutions for that matter), the sample is often too 
precious to redo the experiment and it is worth thinking about doing replicate 
experiments with a cheap one, build the model, and then apply it to single 
expensive observations. That would be statistically justified (provided that 
the model is valid for all sets of experiments). I have not built such models, 
but we know that pipetting isn't really as good as we believe. If you randomly 
dial to a particular value on your pipetteman (say 5 uL), you will get a 
certain pattern of errors (which is really not a good word for it), while if 
you consistently dial either from a low (1uL) or a high (10uL) value towards 
the value you want, you will get another pattern. Those two patterns are not 
representative of each other, I don't think, and you would need to understand 
how to do experiments consistently to stay within your error-model (bad word).

Among many other things, statisticians try to come up with models that explain 
the uncertainty so that you know what to think, even if your set of observation 
is too small to say for sure, with n=1 being the ultimate too small. (Maybe not 
ultimate, n=0 is really too small.)

Mark



-Original Message-
From: Alexander Aleshin 
aales...@sanfordburnham.orgmailto:aales...@sanfordburnham.org
To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Sent: Wed, Mar 13, 2013 3:05 pm
Subject: Re: [ccp4bb] statistical or systematic? bias or noise?

On Mar 13, 2013, at 1:36 PM, Ed Pozharski wrote:


But what if I only have one measurement worth of sample?

Is it proper to use statistical analysis for a single measurement? I thought 
statistics, by definition, means multiple measurements.

Alex

Re: [ccp4bb] archival memory?

[Tom Peat]

A chip in my brain to remember all of the things I should know/remember would 
be very convenient if we are really talking about having a good memory system.  
It means it would also be easier to extract that personal perspective and pass 
it on.
Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem 
Evdokimov
Sent: Thursday, 13 December 2012 9:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] archival memory?

encoding into DNA and transforming some long lived critter is the best solution 
for me. My mind's eye is watering with glee when I imagine how TurtleBank might 
look -- fields of green grass, populated by herds of gentle grazing turtles, 
each encoding some priceless tidbit of information.

Of course, worrying about access to USB formatted memory 100 years from now is 
likely not necessary. We should be able to read these chips directly using a 
miniaturized form of scanning microscopy or somesuch (probably non-invasive, 
unlike SEM). Charge-state of a floating gate transistor can be derived by e.g. 
a properly designed SEM tip right now (this is one of the methods used to steal 
data from protected flash drives by the way), but i am SURE that in a century 
the technology to read the state of matter should be advanced to a level of 
household implementation.

Anyway.
On Wed, Dec 12, 2012 at 4:35 PM, Artem Evdokimov 
artem.evdoki...@gmail.commailto:artem.evdoki...@gmail.com wrote:
Given that it's basically a solid state tiny capacitor, temperature should 
indeed be a huge factor :) I am actually considering storing some flash sticks 
in a freezer, to see what happens. And in LN2 as well...

Artem
On Wed, Dec 12, 2012 at 4:14 PM, Richard Gillilan 
r...@cornell.edumailto:r...@cornell.edu wrote:
I don't think memory sticks have any internal electrolytics or power supplies. 
Both USB and FAT32 are widely documented standards in this era, so while they 
might no longer be supported (FAT32 is already very old), information on how to 
communicate and decode data will still likely be available. RS232, for example, 
is now 50 years old and one can still find adapters and software.

Richard

On Dec 12, 2012, at 4:45 PM, Roger Rowlett wrote:


Maybe the memory chips will retain their bits for 100 years, but what about the 
driver hardware or internal power supply? Anyone had an electrolytic capacitor 
last for 100 years? Just sayin...

I like the image of the USB sticks in the -80 freezer, though. :)
___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245tel:%28315%29-228-7245
ofc: (315)-228-7395tel:%28315%29-228-7395
fax: (315)-228-7935tel:%28315%29-228-7935
email: rrowl...@colgate.edumailto:rrowl...@colgate.edu

On 12/12/2012 4:38 PM, Artem Evdokimov wrote:
Or... (gasp) store a regular USB drive in a freezer, yes? If the relationship 
between data decay rate and temperature indeed follows the same good old 
Arrhenius formula then any old USB drive is virtually endless at -80C and safe 
for human life span at -20 (i.e. kitchen freezer, sans defrost cycles (so pack 
your USB in some ice packs so defrost doesn't kill it).

If this works, feel free to send me money, SanDisk...

Artem
On Wed, Dec 12, 2012 at 3:02 PM, Richard Gillilan 
r...@cornell.edumailto:r...@cornell.edu wrote:
SanDisk advertises a Memory Vault disk for archival storage of photos that 
they claim will last 100 years.

(note: they do have a scheme for estimating lifetime of the memory, Arrhenius 
Equation ... interesting. Check it out: 
www.sandisk.com/products/usb/memory-vault/http://www.sandisk.com/products/usb/memory-vault/
 and click the Chronolock tab.).

Has anyone here looked into this or seen similar products?

Richard Gillilan
MacCHESS

Re: [ccp4bb] Reservoir buffer

[Tom Peat]

The following could be of use:

Newman, J. Expanding screening space through the use of alternative reservoirs 
in vapor diffusion (2005) Acta Cryst D61(4), 490-493

Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Felix 
Frolow
Sent: Tuesday, 13 November 2012 5:17 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Reservoir buffer

You should and can try, but I guess 5 M NaCl will dry your drop quite fast, 
unless in your drop NaCl concentration is ~2,5 M :-) That is why first mixture 
of well and protein solution is 1:1 v/v.
Other variations such as 1:9, 9:1 etc sometimes help.

Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

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

On Nov 13, 2012, at 08:03 , Theresa Hsu 
theresah...@live.commailto:theresah...@live.com wrote:


Dear all

In *initial screening* using vapor diffusion crystallization, does it matter 
whether the reservoir buffer is also the precipitant in the drop or just a high 
salt solution like 5 M NaCl?

Thank you.

Theresa

Re: [ccp4bb] Who is using 64-bit Linux?

[Tom Peat]

Hello Tim,

I believe the notion comes about as one can thread 64 instead of 32 addresses 
concurrently, thereby boosting performance.  If it has no performance boost, 
why would they bother? 

Cheers, tom

-Original Message-
From: Tim Gruene [mailto:t...@shelx.uni-ac.gwdg.de] 
Sent: Wednesday, 4 April 2012 6:43 PM
To: Peat, Tom (CMSE, Parkville)
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Who is using 64-bit Linux?

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Tom,

64-bit is about memory addressing - why would you expect a performance
boost? I have wondered where this notion originated from.

Cheers,
Tim

On 04/03/12 22:07, Tom Peat wrote:
 We use the 64 bit Centos (Red Hat) distro and CCP4, Coot, etc seem to work 
 fine on this. 
 I can't say I notice a big performance boost from the 64 bit side of things. 
 Maybe I'm just impatient. 
 cheers, tom
 
 
 Tom Peat
 Biophysics Group
 CSIRO, CMSE
 343 Royal Parade
 Parkville, VIC, 3052
 +613 9662 7304
 +614 57 539 419
 tom.p...@csiro.au
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett 
 [rrowl...@colgate.edu]
 Sent: Wednesday, April 04, 2012 5:57 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Who is using 64-bit Linux?
 
 The time has come for me to upgrade my Linux OS to something more recent
 for me and my student workstations. A 32-bit distro is certainly
 conservative and compatible with CCP4 and Coot, but it seems like that
 solution hobbles my hardware and puts some limitations on available
 memory, even with PAE enabled. So who is using a 64-bit distro these
 days, and are there lingering issues of compatibility and dependency
 hell with commonly used XRD software, like CCP4, Coot, iMOSFLM etc.?
 
 Ubuntu 12.04 LTS (beta) actually works OK with one simple workaround for
 the global menu for CCP4 and Coot, and wine compatibility is fine for
 running CrysalisPro in the same environment, so it's really comes down
 to whether or not the extra performance of a 64-bit OS is worth the pain
 of compatibility issues for XRD software. Any thoughts?
 
 Cheers,
 
 ___
 Roger S. Rowlett
 Gordon  Dorothy Kline Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346
 
 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu
 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPfAmoUxlJ7aRr7hoRArGeAKDgsoIKEADDo6ycaJBpLf6W9tnCFACeOSM6
1gZUOKKWkQ6Ioo+pQkPtw4Y=
=DdSc
-END PGP SIGNATURE-

Re: [ccp4bb] Who is using 64-bit Linux?

[Tom Peat]

We use the 64 bit Centos (Red Hat) distro and CCP4, Coot, etc seem to work fine 
on this. 
I can't say I notice a big performance boost from the 64 bit side of things. 
Maybe I'm just impatient. 
cheers, tom


Tom Peat
Biophysics Group
CSIRO, CMSE
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: Wednesday, April 04, 2012 5:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Who is using 64-bit Linux?

The time has come for me to upgrade my Linux OS to something more recent
for me and my student workstations. A 32-bit distro is certainly
conservative and compatible with CCP4 and Coot, but it seems like that
solution hobbles my hardware and puts some limitations on available
memory, even with PAE enabled. So who is using a 64-bit distro these
days, and are there lingering issues of compatibility and dependency
hell with commonly used XRD software, like CCP4, Coot, iMOSFLM etc.?

Ubuntu 12.04 LTS (beta) actually works OK with one simple workaround for
the global menu for CCP4 and Coot, and wine compatibility is fine for
running CrysalisPro in the same environment, so it's really comes down
to whether or not the extra performance of a 64-bit OS is worth the pain
of compatibility issues for XRD software. Any thoughts?

Cheers,

___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Tom Peat]

I agree with Herbert that a pre-print setup is one way to establish priority 
and get useful comments for an author. 
And I know this has been discussed before, but another way is to remove the 
anonymous aspect of the review, as this would achieve the same as the community 
pre-print distribution (at least in many ways). 
I would be happy to give my name when reviewing, as I feel it is my job to 
improve the paper, and I can still face my colleagues after the exercise. 
cheers, tom


Tom Peat
Biophysics Group
CSIRO, CMSE
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Herbert J. 
Bernstein [y...@bernstein-plus-sons.com]
Sent: Wednesday, April 04, 2012 4:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] very informative - Trends in Data Fabrication

Dear Colleagues,

   One thing that would help is avoiding misappropriated priority of
research
results would be to join the math and physics community in their robust
use of open-access
preprints in arXiv.  Such public preprints establish reliable timelines
for research credit
and help to ensure timely access to new results by the entire community.
Fully peer-reviewed publications in real journals are still desirable,
but to make
this work, our journals would have to be willing to accept papers for
which such
a preprint system has been used.  To understand the complexity of the issue,
see

http://nanoscale.blogspot.com/2008/01/arxiv-and-publishing.html

I believe the IUCr is willing to accept papers that are posted on a
preprint server (somebody
correct me if I am wrong).

   It works for the math and physics community.  Perhaps it would work
for the
crystallographic community.


On 4/3/12 1:28 PM, Mark J van Raaij wrote:
 In fact, I would put it even stronger, if we know a referee is being 
 dishonest, it is our duty to make sure he is removed from science, 
 blacklisted from the journal etc.

 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij



 On 3 Apr 2012, at 19:13, Maria Sola i Vilarrubias wrote:


 Mark,

 I know some stories (which of course I'll not post here)  from the 
 Crystallography field and from other fields where reviewers profit from the 
 fact that suddenly they have new, interpreted data which fits very well with 
 their own results. Stories like to block a manuscript or ask for more 
 results for the reviewer to be able to submit its own paper (with new 
 ideas) in time, or copy a structure from the figures, or ask for experiments 
 that only the reviewer can do so he/she is included in the paper, or submit 
 as fast as possible in another journal with an extremely short delay of 
 acceptance (e.g. 10 days,  without revision?, talking to the editorial 
 board?) things like this. Well, it is not question of making a full list, 
 here!. The whole problem comes from publishing first, from competition.

 The hope with fraud with X-ray data is that it seems to be detectable, 
 thanks to valuable people that develop methods to detect it. But it is very 
 difficult to demonstrate that your work, ideas or results have been copied. 
 How do you defend from this? And how after giving to them the valuable PDB?

 Finally, how many crystallographers are in the world? 5000?  The concept of 
 ethics can change from one place to another and, more than this, there is 
 the fact that the reviewer is anonymous.

 I try to respond to my reviewers the best I can and I really trust their 
 criteria, sometimes a bit too much, indeed. I think they all have done a 
 very nice job. But some of the stories from above happened to me or close to 
 me and I feel really insecure with the idea of sending a manuscript, the 
 X-ray data and the PDB, altogether, to a reviewer shielded by anonymity. 
 It's too risky: with an easy molecular replacement someone can solve a 
 difficult structure and publish it first. And then the only thing left to 
 the bad reviewer is to change the author's list! (and for the true 
 author what is left is to feel like an idiot).

 In my humble opinion, we must be strict but not kill ourselves. Trust 
 authors as we trust reviewers. Otherwise, the whole effort might be useless.

 Maria

 Dep. Structural Biology
 IBMB-CSIC
 Baldiri Reixach 10-12
 08028 BARCELONA
 Spain
 Tel: (+34) 93 403 4950
 Fax: (+34) 93 403 4979
 e-mail: maria.s...@ibmb.csic.es

 On 3 April 2012 16:58, Mark J van Raaijmjvanra...@cnb.csic.es  wrote:
 The remedy for the fact that some reviewers act unethically is not 
 withholding coordinates and structure factors, but a more active role for 
 the authors to denounce these possible violations and more effective 
 investigations by the journals whose reviewers are suspected by the authors 
 of committing these violations.
 I

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Tom Peat]

Bernard went to a lot of work to verify that this structure was wrong, so we 
should also thank him for his efforts. 
It is good to see someone who has a hunch follow that up and let the rest of us 
know about it. 
Thanks Bernard! 


Tom Peat
Biophysics Group
CSIRO, CMSE
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis 
Perrakis [a.perra...@nki.nl]
Sent: Sunday, April 01, 2012 7:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] very informative - Trends in Data Fabrication

Reading the paper from Dr. Hofkristallrat a.D. and the editorial in ActaF, I 
must say that besides the rather reasonable demand for journals to include 
crystallography experts as referees, Table 1 would have fooled me as referee. 
A validation report of the VTF style might not had helped either in refereeing 
- in this case. Alarm bells could had rung possibly if the PDB was re-refining 
all submitted structures and look for 'too good to be true' improvements (sorry 
Robbie ... we are not there yet to improve things SO much!). Saving the images 
in a repository would had been equally unlikely to have helped (they would had 
submitted some data ... unless these were systematically validated and 
cross-matched to the CRYST data cards no alarm bells either - even if running 
PDB_REDO in all submissions appears a tad unrealistic, re-processing all images 
and matching them to CRYST records seems more troublesome at the present 
moment).

A thing that could had helped, would had been if our biology colleagues who 
want a structure for their story would had valued more the structural 
contribution by scrutinising the data (a corresponding author must scrutinise 
all data before accepting responsibility - and not when questioned throw the 
hands up waving 'it was not me ...'). Maybe ourselves as a community could also 
help by making our colleagues aware that crystallographic work is a tad more 
than 'and the author in the middle of the paper just contributed a structure' 
and explain them that if they want to be using structures for their 
publications they should be always prepared to engage in close and real 
collaborations where both sides accept responsibility for the data of each 
other, as it happens in many fruitful collaborations between biologists and 
crystallographers (such as these I had the privilege to engage with 
collaborators that criticised my data, as I did theirs ...).

regards to all -

Tassos

(and please, no 1st April joke with fraud cases !)

Re: [ccp4bb] pH optimisation for crystallisation

[Tom Peat]

If you find yourself in the situation where the buffer you started with is out 
of range of the pH you would 
like to attain, there are sets of buffers you can use that contain most of the 
standard buffers that will give 
you a fairly linear response across ~4-10, as described by Newman, Acta Cryst 
D, 2004, v60, pp 610-612. 

cheers, tom

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu 
[uppsala@hotmail.com]
Sent: Wednesday, February 08, 2012 8:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] pH optimisation for crystallisation

Hello Sreetama:

I think for crystallization, everything is hard to say. But if you find your 
crystal is sensitive to the pH, you certainly can optimize the pH value but it 
is better not to deviate a lot. For example you can make 0.2 unit interval (for 
example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH 
value ).
For the buffer, you can change or not.  Another thing is that, you can also 
incorporate bis-tris in your last purification, since you find your crystal in 
this buffer.
When you do additive screen, the drops which is clear, also can give you 
important information. You might find a compont which can inhibit crystal 
formation. You can use it to slow down the crystal formation to get a big or 
single crystal.
However, you see, sometimes, this optimization is time consuming. I suggest you 
to try seeding. It can give you a big surprise, sometimes.

Yu Xiaodi


Date: Wed, 8 Feb 2012 11:56:30 +0530
From: somon_...@yahoo.co.in
Subject: [ccp4bb] pH optimisation for crystallisation
To: CCP4BB@JISCMAIL.AC.UK

Dear all,
   I have a 17 KDa protein that gives crystals in a condition that has 
0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not 
diffract. I have tried additives, but they haven't improved the crystals. I 
intend to vary the pH of the condition.
   My questions are-
1. should the buffer be kept the same or can it also be changed (as long as the 
desired pH is within the range of both the buffers)?
2. in case of a different buffer, should its molarity be the same as that of 
the original one in the crystallization condition?

regards,
sreetama

Re: [ccp4bb] unusual bond lengths in PRODRG cif file

[Tom Peat]

To elaborate further on software packages available that do a reasonable job of 
fitting small molecules and outputting reasonable dictionaries, one could 
consider Afitt from OpenEye Scientific Software. They have academic as well as 
commercial licenses to their software. 

Cheers, tom

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle
Sent: Monday, 9 January 2012 11:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual bond lengths in PRODRG cif file

On 9 January 2012 06:13, Shveta Bisht shv...@mbu.iisc.ernet.in wrote:
 Dear all

 I have generated a refmac cif file for a molecule using PRODRG. I used
 JME editor to draw the molecule and ran PRODRG online with the
 options: Chirality-Yes, Charges-Reduced and EM-Yes. Please check the
 attachments for the molecule drawing and the expected bond lengths as
 listed in the PRODRG generated cif file. There are some unusual
 values. I have listed them below along with the likely explanations
 (just guesses).

 DRG      CAR    CAK       single      1.390    0.025
 DRG      CAQ    CAB       single      1.390    0.025
 DRG      CAT    CAI       single      1.390    0.020
 All three are C-C single bonds where one of the carbons belong to
 aromatic ring. This might lead to a short bond, although 1.39 seems to
 be too short for this.

 DRG      CAI    NAL       double      1.340    0.022
 DRG      CAP    NAL       single      1.340    0.022
 Here N and both the carbons are sp2 hybridization, so there can be
 delocalization of electrons. Thus both bonds (single and double) are
 of similar length.

 DRG      CAP    CAA       double      1.530    0.025
 As mentioned above, if pi (unhybridized) electrons of CAP are involved
 in CAP NAL bonding, then CAP CAA double bond essentially becomes a
 single bond with 1.53 bond length.

 I need advise on the way I have run prodrg and the explanations for the 
 results.
 Is it common to observe such values? Or it is due to the alternating single 
 and
 double bonds in this structure.

Dear Shveta

I agree completely with your assessment, there are certainly some
unusual values, but I think this goes beyond mistakes.  I have run
your structure through our own dictionary auto-generation software
which essentially takes suitable fragments from the CSD and stitches
them together (similar to CCDC-MOGUL but it doesn't use MOGUL and the
pieces are smaller).

Here are the values I get for comparison:

 DRG  CARCAK   single  1.3900.025 1.512(10)
 DRG  CAQCAB   single  1.3900.025 1.498(7)
 DRG  CATCAIsingle  1.3900.020  1.449(24)
 DRG  CAI NAL   double 1.3400.022  1.273(13)
 DRG  CAPNAL   single  1.3400.022  1.350(28)
 DRG  CAPCAA   double1.5300.025  1.329(21)

so essentially backing up what you said.

My suspicion is that it's not generally recognised the central role
played by bond length  angle restraints in MX refinement (after all
if they weren't important then we wouldn't need to use them!), and as
a consequence some people seem to think that their exact values are
unimportant and anything will do.  Their importance soon becomes
obvious (particularly of course at medium-low resolution) if you try
to do unrestrained refinement: then you can easily get deviations 10
times greater than you get with restraints.  So personally I believe
in using the most accurate restraints possible and anything will NOT
do.

My main concern would be that grossly inaccurate values such as these
find their way into the PDB and then people base their restraints on
these structures (though it's not a great idea to use the PDB
co-ordinates as a source of restraints!).

Before you ask, unfortunately our software is not available for
distribution but I can recommend GRADE from Global Phasing which gives
very high quality restraints; there's also of course JLigand from
CCP4, though I haven't used it personally and so cannot attest for the
quality.

Cheers

-- Ian

Re: [ccp4bb] spherulites and PEG3350

[Tom Peat]

You might to consider that PEG 3350 has phosphate contamination, so playing 
around with small amounts of phosphate (or removing it) might be worthwhile.
Cheers, tom

From: Regina Kettering [mailto:reginaketter...@yahoo.com]
Sent: Thursday, August 25, 2011 04:46 AM
To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] spherulites and PEG3350

Something to consider is the quality of the PEG 3350.  We have found that 
different qualities of PEG 3350 can give different results, depending on the 
type and amount of contaminants.  What used to be the Fluka PEG 3350 is now the 
pharm grade of PEG 3350 (aka Miralax).  We use high quality PEG 3350 for normal 
screening, but switch to the highest quality grade we can get for optimizing.

Regina


From: Jan van Agthoven janc...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350

Dear all,

I recently obtained some spherulites while trying to crystallize my protein. 
The spherulites are manually reproducible, but changing pH, protein 
concentration, and salt concentration does not result in crystal formation. 
Microseeding with crushed spherulites isn't a solution either as it only yields 
new spherulites. Next stepp is the use of an optimization kit but I have a 
limited amount of material, and I start doubting that these are protein 
spherulites, as the spherulites are not particularly soft. The condition 
contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms 
easily spherulites around that concentration?


Thanks,

[ccp4bb] CCP4 6.2.0 reading mtz files

[Tom Peat]

I took a quick look via Google and in the FAQ to see if there was anything on 
this topic, but didn't find the clue I was looking for. 
I just installed version 6.2.0 on a linux box running Centos 5.6 and I get 
error messages every time I try to read in a mtz file- unreadable. I check the 
file using mtzdmp and it looks fine (all of the correct columns, etc). 
Has anyone else had a problem reading mtz files with the new version? If so, 
what is the fix? 

Thanks for any help, tom

Re: [ccp4bb] CCP4 6.2.0 reading mtz files

[Tom Peat]

Hello All,

It turns out that the /tmp directory being used by ccp4i had incorrect 
permissions, which gave rise to the problem.  Thanks for the solution! 

Cheers, tom

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tom Peat
Sent: Thursday, 21 July 2011 12:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] CCP4 6.2.0 reading mtz files

I took a quick look via Google and in the FAQ to see if there was anything on 
this topic, but didn't find the clue I was looking for. 
I just installed version 6.2.0 on a linux box running Centos 5.6 and I get 
error messages every time I try to read in a mtz file- unreadable. I check the 
file using mtzdmp and it looks fine (all of the correct columns, etc). 
Has anyone else had a problem reading mtz files with the new version? If so, 
what is the fix? 

Thanks for any help, tom

Re: [ccp4bb] degradation of protein durring freez thaw

[Tom Peat]

Hello again,

I still think it depends on the protein complex, but I agree that the 
consensus/ hearsay/ anecdotal/ old crystallographer's tales revolve around 
complexes not working as well as single proteins for freezing.  I'm not sure 
that has been shown to be the case, although again I would agree that it is 
hard to prove anything like this and one would just have to take a statistical 
approach and say for X% this has happened.

I guess my points are: 1) you have to freeze/thaw the right way, 2) an 
'optimized' buffer system for the complex or even single proteins should be 
used, 3) if you are looking for a single crystal and that is all you need, then 
fresh preps are great.  If you will need more than just one single crystal, 
reproducibility matters, and in the cases I've worked on, freezing is almost 
always more reproducible.

If in the case posted, the freezing was done in an Eppi tube, by putting it in 
the freezer, with a non-optimal buffer and then just pulled out and put into an 
ice bucket- I would expect almost every protein/complex to fall out of 
solution.  If instead the buffer had been optimized, the protein snap frozen 
and snap thawed in a thin walled tube, then maybe one needs to set up the 
protein in crystallization trials as soon as one has purified the complex and 
not freeze or wait around.

Of course, the caveat is that one should never believe generalizations...

Cheers, tom


From: David Briggs [mailto:drdavidcbri...@gmail.com]
Sent: Thursday, 22 April 2010 6:07 PM
To: Peat, Tom (CMHT, Parkville)
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: RE: [ccp4bb] degradation of protein durring freez thaw


Ouch!

I completely agree re: single proteins, but I had always found/heard that 
protein-protein complexes tolerate freezing/thawing less-well that their 
individual components.

Irrespective of generalisations, In the posted case, where apparently, before 
freeze complex is intact and concentrated, after freeze complex is less 
concentrated and degraded, surely the most straight-forward fix is to not 
freeze it?

Dave
--
Delivered via an Android.
On Apr 22, 2010 8:57 AM, tom.p...@csiro.au wrote:
Hello-

Sorry to be disagreeable, but I think it depends on the protein.  We've had 
much better success in getting reproducible crystals when we've snap frozen in 
liquid nitrogen in small aliquots (thin walled PCR tubes work best).  We can 
then screen and optimize using the same protein prep- and again, it is much 
more reproducible than doing a fresh prep each time.  Of course this is only 
for the few hundred proteins I've worked on and it has only been for 98% of the 
cases examined.

Cheers, tom


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David 
Briggs
Sent: Thursday, 22 April 2010 5:27 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] degradation of protein durring freez thaw

  Hi, Obvious answer - don't freeze it. If you cannot set your crystallisation 
screens up straig...

[ccp4bb] Crystallography position at CSIRO, Melbourne

[Tom Peat]

Hello All,

An opportunity is available immediately for a Research Scientist in x-ray
crystallography at the CSIRO Molecular and Health Technologies division. 
Applications are accepted through the CSIRO employment website:

https://recruitment.csiro.au/asp/job_details.asp?RefNo=2009%2F966 

(the job reference number is 2009/966)

International applicants are welcome and encouraged.

Part of the advertisement and a blurb on CSIRO is copied below, but more
information on CSIRO can be obtained on the following web site: 

http://www.csiro.au/ 

And more specifically the CMHT division:

http://www.csiro.au/org/CMHT.html 

Questions can be directed to me (Tom Peat) at the following email address:

tom.p...@csiro.au



We seek an enthusiastic and highly motivated protein crystallographer to
join the Structural Biology research group in Parkville. You will be
responsible for protein structure determination and structure-function
studies. You will work in a team and will liaise with other CSIRO divisions
and external collaborators to carry out their structural biology
requirements. You must have an interest in automation and high throughput
methodology. You will have a PhD or equivalent qualification with
postdoctoral research experience in protein crystallography.

You will be able to operate independently, but will enjoy working in a team
environment. You will develop your own research projects as well as liaising
with collaborators both within and outside CSIRO.  Your interests are
predominately protein structure-function studies, including structure-based
drug design. You will have a strong commitment to safe laboratory practice
and abide by OHSE regulations.

The Commonwealth Scientific and Industrial Research Organisation is
Australia's national science agency and one of the largest and most diverse
research agencies in the world.  At CSIRO Molecular and Health Technologies
our scientists work in partnership to address key challenges in health and
in a range of industries requiring smart materials. Talented scientists are
undertaking biotechnology and advanced materials research drawing on a long
history of chemistry and molecular, cellular and structural biology to
develop and grow their capabilities. We form strong collaborative
relationships with industry, universities, Cooperative Research Centres
(CRCs) and research institutions to deliver leading edge research. Our
vision is to transform industries and improve health and wellbeing by
delivering creative molecular technologies.