Re: [ccp4bb] SAXS models and data

[David Briggs]

Hi Gloria,

There are repositories of SAXS data.

https://www.sasbdb.org/

For example.

Hth,

Dave



Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Tuesday, January 30, 2024 9:29:46 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] SAXS models and data


External Sender: Use caution.

Do we deposit these anywhere?



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Re: [ccp4bb] Resolution Discrepancy in Data Set

[David Briggs]

Hi Liliana,

Two things leap out at me when I look at your data summary.

(1) Your data probably do not go to 1.77Å. The CC1/2 in your outer shell is 
below any of the usual thresholds. There are discussions to be had about what 
the threshold is, but normally CC1/2 values of 0.5 or sometimes 0.3 are used. 
You should also consider I/sigI.

(2) I believe that by default Phenix.refine excludes weaker reflections from 
refinement, which leads to the discrepancy in completeness statistics. As your 
data do not extend as far as what is contained in your mtz file, Phenix 
excludes those essentially "empty" reflections. Judging by the Phenix refine 
output, I would estimate your data goes to somewhere around 1.9Å

The program Pairef can help inform your choice of high-resolution cutoff.

This can be run from CCP4cloud, but is also available for Phenix, I believe.

See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248825/

Hope this helps,

Dave



Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Liliana Margent 

Sent: Wednesday, January 17, 2024 9:19:36 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Resolution Discrepancy in Data Set


External Sender: Use caution.


Hello all,

I hope this message finds you well.

In my current data set, I’ve encountered a discrepancy between the completeness 
in the high-resolution shells in merged statistics vs the refinement 
statistics. For example, when I look at my merged statistics file, output by 
Xia2 dials, the completeness in the high-resolution shells are 97.6%. When I 
take this data and subsequently refine it in PHENIX I get extremely different 
completeness ranges in the high-resolution shells, but I cannot figure out why 
this large difference is occurring. I’m reaching out to you, our esteemed 
community, for any insights or advice you might have. Has anyone else faced a 
similar challenge? If so, how did you navigate through it?  Your experiences 
and suggestions could be invaluable in helping me understand and resolve this 
issue.

Thank you in advance for your time and expertise.

Best regards,
Liliana

see a side-by-side image of the files I mention in the message in 
https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdrive.google.com%2Ffile%2Fd%2F1Y783MzlnqVwXRCtiLeV0Y2EpmkeDL2nd%2Fview%3Fusp%3Dsharing=05%7C02%7C%7Cb15f2dcfd7804db15e8808dc17a23ef1%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C638411232808490947%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=5XcApcqvUkuvjz9oGUsaFpvnfY2p2X1LDE8G4XhFkuo%3D=0



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The Francis Crick Institute Limited is a registered charity in England and 
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Re: [ccp4bb] Alternatives to X

[David Briggs]

Hi Robbie,

you are of course correct - when you join a community, you are bound by their 
rules - so if you join a server that has rules against a particular subject, 
servers pushing those subjects are prone to get defederated.

The solution (AFAIK only available in Mastodon) is to setup your own server. 
You can either pay someone a small fee ( a few EUR/month) to host your server, 
or setup your own server if you are suitably savvy (I believe a small server 
can be run on a Raspberry PI or similar) and then you are lord of your own 
dominion. You get to decide who you federate with and who you see in your local 
various feeds. You can post about whatever takes your fancy.

I realise that this particular phrase has certain Brexity connotations, but 
Mastodon's democratisation/decentralisation of social media really allows one 
to "take back control" 﫠

Best,

Dave


--

Dr David C. Briggs CSci MRSB (he/him)

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

Working hours: Mon-Fri 0900-1700

==

about.me/david_briggs<https://about.me/david_briggs> | 
OrcID<https://orcid.org/-0002-9793-7339> | Google Scholar 
<https://scholar.google.co.uk/citations?user=DRKG5KwJ>


From: Robbie Joosten 
Sent: 06 December 2023 07:56
To: David Briggs ; CCP4BB@JISCMAIL.AC.UK 

Subject: RE: [ccp4bb] Alternatives to X


External Sender: Use caution.


Hi David,

Thank you for the Mastodon links. I like the idea of Mastodon, but many servers 
do blanket bans against other servers, particularly if these are very 
libertarian (e.g. have too much "Freeze Peach"). Your Mastodon 'heritage' seems 
to matter a lot. Nevertheless, it's good to see that engagement is growing. I 
find LinkedIn mostly useful for broadcasting, not really for engagement, so not 
really an X alternative.

Personally, I think it is a shame so many scientists left Twitter/X (or said 
they did/would). Especially if this is for activistic reasons. Yes, it is quite 
unfiltered nowadays, but I actually like to have things in the open whether I 
agree with them or not. It puts more responsibility on the community and the 
community notes help with that. Purely scientific posts don't seem to suffer 
from the new management and your timeline keeps having useful stuff as long if 
you actually engage with (not just follow) users of interest. I do use Twitter 
as a private person, not on behalf of my work. I wonder if that makes a 
difference to my experience.

It is important to note that many X alternatives are not available in the EU to 
avoid regulations (says something about those platforms). That makes Mastodon 
and X the only real options. That is, far behind the CCP4bb.

Cheers,
Robbie



> -Original Message-
> From: CCP4 bulletin board  On Behalf Of David
> Briggs
> Sent: Wednesday, December 6, 2023 08:19
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Alternatives to X
>
> Hi all,
>
> I did a little (and entirely unscientific) test on this with one of our 
> recent papers.
>
> Views : Linkedin came out on top.
>
> Engagement from other scientists : Mastodon.
>
> X didn't really do much, last I checked.
>
> There is a structural biology community on Mastodon and there are several
> servers (a.k.a instances) that are science themed...
>
> at_struct_dot_bio
> at_mstdn_dot_science
> at_biologists_dot_social
> at_cryoEM_dot_social
> at_qoto_dot_org
> at_fediverse_dot_science
>
> Some suppliers are beginning to appear (e.g. Quantifoil) and there is a
> structural biology Mastodon group (struc...@a.gup.pe) that acts a bit like a
> distribution list.
>
> Hth,
>
> Contact me off list if I can help get you started.
>
> Dave
> @xtald...@xtaldave.net
>
> (Apologies if anyone got this twice - the original was pinged back as it 
> tripped
> the spam filter, presumably the list of servers)
>
>
>
>
>
> Dr David C. Briggs CSci MRSB
>
> Principal Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> about.me/david_briggs 
> <https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fabout.me%2Fdavid_briggs=05%7C01%7C%7C04ab752fe0064d7e85a008dbf630dc81%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C638374461962237452%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=nfuQ7Xh1N0mMIdHKTu%2BMh%2Bf6Saoc%2Bjf1cE4Sy8UaBYQ%3D=0<http://about.me/david_briggs>>
>
> 
>
> From: CCP4 bulletin board  on behalf of Marc
> Graille 
> Sent: Wednesday, December 6, 2023 6:33:06 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] Alternatives to X
>
>
>

Re: [ccp4bb] Alternatives to X

[David Briggs]

Speaking of, Dave, did you mean at_fediscience_dot_org for this last instance?
Both!


--

Dr David C. Briggs CSci MRSB (he/him)

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

Working hours: Mon-Fri 0900-1700

==

about.me/david_briggs<https://about.me/david_briggs> | 
OrcID<https://orcid.org/-0002-9793-7339> | Google Scholar 
<https://scholar.google.co.uk/citations?user=DRKG5KwJ>


From: Guillaume Gaullier 
Sent: 06 December 2023 07:57
To: David Briggs 
Cc: CCP4BB@jiscmail.ac.uk 
Subject: Re: [ccp4bb] Alternatives to X


External Sender: Use caution.

Hello,

I can also recommend posting on a Mastodon instance. There is no algorithmic 
promotion of posts or accounts, which means you have to work a bit more to find 
interesting people to follow, but the reward is generally nicer, with more 
genuine interactions (since all the "popularity contest" features are either 
absent or not prominently displayed, like the metrics of answers, reposts, 
stars, etc.).

Speaking of, Dave, did you mean at_fediscience_dot_org for this last instance?

Many people seem to have moved to bluesky, but I would not recommend posting 
there if your goal is for your posts to be visible beyond your peers in your 
field: having an account is mandatory to see any content on bluesky (without an 
account, one only sees the login page), and at the moment new accounts can only 
be registered with an invitation from an already existing account.

Cheers,

Guillaume
@guilla...@fediscience.org<mailto:guilla...@fediscience.org>


On 6 Dec 2023, at 08:18, David Briggs 
mailto:david.bri...@crick.ac.uk>> wrote:

Hi all,

I did a little (and entirely unscientific) test on this with one of our recent 
papers.

Views : Linkedin came out on top.

Engagement from other scientists : Mastodon.

X didn't really do much, last I checked.

There is a structural biology community on Mastodon and there are several 
servers (a.k.a instances) that are science themed...

at_struct_dot_bio
at_mstdn_dot_science
at_biologists_dot_social
at_cryoEM_dot_social
at_qoto_dot_org
at_fediverse_dot_science

Some suppliers are beginning to appear (e.g. Quantifoil) and there is a 
structural biology Mastodon group (struc...@a.gup.pe<mailto:struc...@a.gup.pe>) 
that acts a bit like a distribution list.

Hth,

Contact me off list if I can help get you started.

Dave
@xtald...@xtaldave.net<mailto:xtald...@xtaldave.net>

(Apologies if anyone got this twice - the original was pinged back as it 
tripped the spam filter, presumably the list of servers)


Dr David C. Briggs CSci MRSB
Principal Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs<http://about.me/david_briggs>

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Marc Graille 
mailto:marc.grai...@polytechnique.edu>>
Sent: Wednesday, December 6, 2023 6:33:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Alternatives to X


External Sender: Use caution.

Dear colleagues,

I take advantage of Tim's message about the fact that responsible people have 
resigned from X.
I  really enjoyed Twitter (which I discovered rather late) because it was a 
great tool for announcing news from my laboratory, but also for keeping abreast 
of recent publications or pre-publications related to my research interests.
I notice that many scientists have deserted X in recent months.

Can anyone suggest user-friendly alternatives used by the scientific 
communities to announce recent publications or news in their fields?

Best wishes,

Marc
—
Marc GRAILLE, PhD
DR1-CNRS
Laboratoire de Biologie Structurale de la Cellule  (BIOC; Ex-Laboratoire de 
Biochimie)
UMR7654 du CNRS

Head of the team: “Translation and degradation of eukaryotic mRNAs”

ÉCOLE POLYTECHNIQUE
91128 PALAISEAU CEDEX
FRANCE
: +33 (0)1 69 33 48 90


 : marc.grai...@polytechnique.edu<mailto:marc.grai...@polytechnique.edu> /
Twitter : @GrailleLab<https://twitter.com/GrailleLab>
https://portail.polytechnique.edu/bioc/en/research/coupling-between-translation-and-mrna-degradation-eukaryotes
—



Le 2 déc. 2023 à 10:15, Tim Grüne 
mailto:tim.gru...@univie.ac.at>> a écrit :

Hi Mark,
responsible people are resigning from X.
Cheers,
Tim

Am 01.12.2023 23:24, schrieb Mark J. van Raaij:
just came across this critique of that paper on Twitter:
This exciting paper shows AI design of materials, robotic synthesis.
10s of new compounds in 17 days. But did they? This paper has very
serious problems in materials characterisation. In my view it should
never have got near publication. Hold on tight let's take a look 
[1]
Robert Palgrave (@Robert_Palgrave) on X [1]
twitter.com<http://twitter.c

Re: [ccp4bb] Alternatives to X

[David Briggs]

Hi all,

I did a little (and entirely unscientific) test on this with one of our recent 
papers.

Views : Linkedin came out on top.

Engagement from other scientists : Mastodon.

X didn't really do much, last I checked.

There is a structural biology community on Mastodon and there are several 
servers (a.k.a instances) that are science themed...

at_struct_dot_bio
at_mstdn_dot_science
at_biologists_dot_social
at_cryoEM_dot_social
at_qoto_dot_org
at_fediverse_dot_science

Some suppliers are beginning to appear (e.g. Quantifoil) and there is a 
structural biology Mastodon group (struc...@a.gup.pe) that acts a bit like a 
distribution list.

Hth,

Contact me off list if I can help get you started.

Dave
@xtald...@xtaldave.net

(Apologies if anyone got this twice - the original was pinged back as it 
tripped the spam filter, presumably the list of servers)



Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Marc Graille 

Sent: Wednesday, December 6, 2023 6:33:06 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Alternatives to X


External Sender: Use caution.

Dear colleagues,

I take advantage of Tim's message about the fact that responsible people have 
resigned from X.
I  really enjoyed Twitter (which I discovered rather late) because it was a 
great tool for announcing news from my laboratory, but also for keeping abreast 
of recent publications or pre-publications related to my research interests.
I notice that many scientists have deserted X in recent months.

Can anyone suggest user-friendly alternatives used by the scientific 
communities to announce recent publications or news in their fields?

Best wishes,

Marc
—
Marc GRAILLE, PhD
DR1-CNRS
Laboratoire de Biologie Structurale de la Cellule  (BIOC; Ex-Laboratoire de 
Biochimie)
UMR7654 du CNRS

Head of the team: “Translation and degradation of eukaryotic mRNAs”

ÉCOLE POLYTECHNIQUE
91128 PALAISEAU CEDEX
FRANCE
: +33 (0)1 69 33 48 90

[cid:49F0726D-EF0C-4DF5-8DD6-D36862A03F60@home]
 : marc.grai...@polytechnique.edu /
Twitter : @GrailleLab
https://portail.polytechnique.edu/bioc/en/research/coupling-between-translation-and-mrna-degradation-eukaryotes
—

[cid:1EAA2C1D-C006-430B-A4E9-D6571ACF035D@polytechnique.fr]

Le 2 déc. 2023 à 10:15, Tim Grüne 
mailto:tim.gru...@univie.ac.at>> a écrit :

Hi Mark,
responsible people are resigning from X.
Cheers,
Tim

Am 01.12.2023 23:24, schrieb Mark J. van Raaij:
just came across this critique of that paper on Twitter:
This exciting paper shows AI design of materials, robotic synthesis.
10s of new compounds in 17 days. But did they? This paper has very
serious problems in materials characterisation. In my view it should
never have got near publication. Hold on tight let's take a look 
[1]
Robert Palgrave (@Robert_Palgrave) on X [1]
twitter.com [1]
but I'm not enough of an expert to judge - perhaps some
characterizations were wrong and a lot of the paper does stand.
On 1 Dec 2023, at 20:51, Bryan Lepore 
mailto:bryanlep...@gmail.com>> wrote:
Adding to that literature list a bit outside :
Merchant, A., Batzner, S., Schoenholz, S.S. _et al._
Quote:
"... we show that graph networks trained at scale can reach
unprecedented levels of generalization, improving the efficiency of
materials discovery by an order of magnitude. "
Scaling deep learning for materials discovery.
_Nature_ (2023), November
https://doi.org/10.1038/s41586-023-06735-9
-
To unsubscribe from the CCP4BB list, click the following link:
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-
To unsubscribe from the CCP4BB list, click the following link:
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Links:
--
[1] https://twitter.com/Robert_Palgrave/status/1730358675523424344

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[David Briggs]

Hi Rafael,

For completeness -  there are alternatives to imidazole for eluting proteins 
from Ni-NTA resins:

EDTA - (strips Ni2+, and therefore everything bound to the Ni2+) - 50mM should 
be sufficient, in your favourite buffer/salt system. Obviously not appropriate 
for metalloproteins. You'll need to recharge the column/resin afterwards.

Low pH - Citrate or Acetate buffer with a pH lower than 5.5 (lower still for 
multimers) with an appropriate salt concentration. Obviously test this on a 
small scale first to see if your protein tolerates the drop in pH. You can 
reduce the time of exposure to low pH by eluting the protein straight into some 
1M Tris or HEPES to bring the pH back up to something more neutral.

Hth,

Dave


Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Rafael Marques 

Sent: Tuesday, October 31, 2023 7:21:21 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage


External Sender: Use caution.

Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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with its registered office at 1 Midland Road London NW1 1AT



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[ccp4bb] Job Advert: Drug Discovery Project Research Scientist position, Francis Crick Institute, London, UK.

[David Briggs]

Dear all,

We have a position available at the Francis Crick Institute - please share with 
any soon-to-finish PhD students and postdocs who might be interested:

We seek a talented, highly motivated and independent post-doctoral project 
research scientist to work on a project to design protein kinase C beta 
selective inhibitors for B cell malignancies. Work will be carried out in Neil 
McDonald's laboratory at the Francis Crick Institute, in collaboration with 
clinician-scientist Ingo Ringshausen at the University of Cambridge, and is 
funded by a Cancer Research Horizon Therapeutic Catalyst Award.

The project will be funded for 18 months in the first instance, with the 
potential for extension depending on progress.

The project will build upon prior work within our laboratory to characterise 
and develop new, selective kinase inhibitors. The work will involve both 
optimisation of existing inhibitors, and the parallel discovery of novel 
inhibitors using an XChem Fragment screening approach at Diamond Light Source.

The Francis Crick Institute is situated in central London, and is the largest 
single-site biomedical research institute in Europe, with excellent provision 
for X-ray crystallography and medicinal chemistry to allow rapid 
characterisation and follow-up of any lead compounds.

Prior experience with baculovirus protein expression and X-ray crystallography 
would be advantageous.

Informal enquiries can be made to Professor Neil McDonald 
(neil.mcdon...@crick.ac.uk).
Application Deadline: 1st Sept 2023.

For more details about the role and the application process, please see:

https://crick.wd3.myworkdayjobs.com/External/job/London/Postdoctoral-Project-Research-Scientist---Signalling-and-Structural-Laboratory_R1245-1



--

Dr David C. Briggs CSci MRSB (he/him)

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

Working hours: Mon-Fri 0900-1700

==

about.me/david_briggs | 
OrcID | Google Scholar 


The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] AI and cis-peptides

[David Briggs]

This is very much anecdotal - in my experience, AF2 tends to put the Cys 
residues in the disulfide bridge adjacent to each other, sometimes with roughly 
the correct side chain orientation.

It doesn't build/refine the S-S bond, becuase I don't think it knows how to.

Best,

Dave



Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Guillaume 
Gaullier 
Sent: Thursday, July 13, 2023 5:07:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] AI and cis-peptides


External Sender: Use caution.

Interesting. Has someone similarly looked at whether AlphaFold predicts 
disulfide bridges?

Guillaume


On 13 Jul 2023, at 15:12, Randy John Read 
mailto:rj...@cam.ac.uk>> wrote:

I’m not sure about other methods, but AlphaFold does predict peptides in both 
cis and trans configurations. In a recent paper, Osnat Herzberg and John Moult 
show that it was pretty successful in predicting proline cis-peptides, among 
the novel structures in the CASP15 set of targets 
(https://www.pnas.org/doi/10.1073/pnas.2221745120).

For non-proline cis-peptides, I’m not aware of published work but Tristan Croll 
has shown me examples of correctly-predicted non-proline cis-peptides, 
including cases where some of the related structures in the PDB have an 
incorrect trans configuration. This implies that AlphaFold is not slavishly 
reproducing what it has seen during training.

Best wishes,

Randy Read

On 13 Jul 2023, at 13:56, Oliviero Carugo 
mailto:oliviero.car...@univie.ac.at>> wrote:

Does anybody know if cis-peptides are predicted by the AI tools (AlphaFold2, 
ColabFold, or ESM-2)?



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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] BUSTER on CCP4 Cloud

[David Briggs]

HI Shawn,

the asymmetric unit contents task tells Cloud how many copies of your model 
exist in the ASU, but not necessarily that they are in the correct position 
(think, prior to Molecular replacement - no phase information).

Depending upon where you are in your workflow, you can run a quick MR job, or 
start a new project with the "hop on" mode specifying an already partially 
refined structure.

I am certain you can do this within an existing project, (maybe by importing 
phases?) but I can't recall the right task to do this at the moment (in my 
defence it is still quite early here).

Perhaps someone else can help with that? (please reply to the list because I'd 
like to know as well!)

Best,

Dave



--

Dr David C. Briggs CSci MRSB (he/him)

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

Working hours: Mon-Fri 0900-1700

==

about.me/david_briggs | 
OrcID | Google Scholar 



From: CCP4 bulletin board  on behalf of Shawn R 

Sent: 09 July 2023 05:02
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] BUSTER on CCP4 Cloud


External Sender: Use caution.


Dear All,

I would like to run Buster refinement using CCP4 Cloud. However, the task says 
I am missing a structure revision. This is despite having generated the 
structure revision using the Asymmetric Unit Content task. I can see the output 
of AU Content task is the structure revision, so I am a little confused why it 
does not allow me to run the refinement. Perhaps I am missing some small detail?

Thanks for your thoughts!



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[ccp4bb] Diamond User Committee nominations

[David Briggs]

Dear all,

(Apologies, this is a message primarily intended for UK-based Diamond Light 
Source (and potentially eBIC) users, so if that is not you, you can ignore this 
message)

The Diamond User Committee (DUC) is a cross-discipline group of users who 
provide feedback to the Diamond executive and science group leaders about the 
running of Beamlines and other facilities at Diamond Light source.
You will be expected to attend 2x 1-day-long meetings per year either in-person 
at Diamond, or remotely.

For more information please see: 
https://www.diamond.ac.uk/Users/DUC/Membership.html

The DUC is seeking nominations for three roles that may be of interest to 
Macromolecular Crystallography users:

DUC member: Macromolecular Crystallography (3-year post)
Early Career/Postdocs Biological and Life Science (1-year post, may be renewed)
Student Biological and Life Sciences (1-year post, may be renewed)

The MX DUC member would normally be a tenured scientist or permanently employed 
staff scientist, and a principal investigator, co-investigator, or alternative 
contact on a Diamond BAG.

The ECR/Postdoc and Student posts are 1-year rolling posts and are a new 
initiative by the DUC to get more insight and feedback on training requirements 
for newer Diamond users.


Please use the following link for your nominations – the last day for 
nominations is the 8th of March.  Please use the link twice if you want to 
nominate two persons, or the same person for two different groups:

https://forms.office.com/e/s5GeusbH9k


We will ask you to provide your name, email and affiliation, and the one from 
your nominee. Self-nominations are accepted and existing DUC members are 
eligible for re-nomination. Please ensure that the nominee is in agreement with 
you sharing this information.  A shortlist will be drawn up from nominations 
for each post and elections will take place in April.


Please note: Your personal information will be processed in accordance with 
Diamond’s Privacy Notice available here: 
https://www.diamond.ac.uk/Home/Legal-and-Compliance/Privacy-Notice-.html.
  All nominations will be shared internally at Diamond and externally with the 
current Diamond User Committee (DUC) members. If you wish to nominate another 
person, please make sure you have their permission to share their personal 
information prior to submitting the form.

Note that these positions are for academic users - industrial users have a 
separate committee.

If you have any questions before considering standing, please do not hesitate 
to contact me.

Best wishes, and good luck!

Dave


--

Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] TEV vs HRV3C

[David Briggs]

Hi Gloria,

Both can be made very easily in E.coli.
Both are active at 4°C, but especially 3C, I think.

I have plasmids for both somewhere in the freezer (you might find someone 
closer to you who can send HRV3C, but if you cannot, let me know off list).

I don't see any particular benefit of one over the other, but having both in 
your freezer means you can cleave off tags sequentially as needed by your 
purification strategies.

HTH,

Dave


Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Wednesday, 7 December 2022, 20:26
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] TEV vs HRV3C


External Sender: Use caution.

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G



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with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] EDR (Sophos) and CCP4 compatibility

[David Briggs]

Hi Mark,


Our IT admin has mandated various AntiVirus software on OSX, including Sophos, 
and I have never encountered a problem installing and downloading CCP4, Phenix, 
Coot, Chimera , etc etc

HTH,

Dave



Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Mark J. van 
Raaij 
Sent: Tuesday, 4 October 2022, 10:31
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] EDR (Sophos) and CCP4 compatibility


External Sender: Use caution.


Dear CCP4-ers,

After a hacking scare, in our institution all computers connected to the 
internet will have to have EDR software installed for safety against hackers. 
We will get Sophos here, at least on OSX, to be installed directly by IT 
services, not by ourselves, from an admin account on all our computers 
controlled by them.
Is this compatible with CCP4 installation or should I be resigned to having an 
off-line computer for CCP4?
The other point is that IT services will update the operating systems of those 
computers from now on, and I worry about major OSX or Linux updates breaking 
CCP4 and other necessary programs.
Any comments and experience with EDR software in general and Sophos in 
particular is appreciated, both for OSX and Linux.

Saludos cordiales,

Mark


Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
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Re: [ccp4bb] Unidentified electron density

[David Briggs]

Hi Sayan

Looks interesting. My first thought was a metal ion with attendant waters, but 
it doesn't look quite right for Trigonal co-ordination - maybe trigonal 
bi-pryamidal with a missing/poorly defined ligand above the plane in image 1?

A couple of questions to help refine the search:

  1.  What is the distance from the centre of the nearest positive density blob 
to an Oxygen in the Asp sidechain?
  2.  What are the distances from the middle of the central blob to the centres 
of the surrounding blobs?
  3.  do you have enough data to make an anomalous difference map?
  4.  What was in the crystallisation conditions (thinking specifically about 
cations - any Mg2+, Ca2+?)

Good luck!

Dave

--

Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Sayan Saha 

Sent: 30 June 2022 08:03
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Unidentified electron density


External Sender: Use caution.

Dear All,

I am trying to refine a protein structure. I observed an additional electron 
density (Figures attached) connected to the aspartate residue.

Any suggestion in this regard would be appreciated.

With best regards,
Sayan Saha.



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Re: [ccp4bb] Regarding R factor value (R Free)

[David Briggs]

Hi!

The R factor describes agreement between the refined model and the processed 
diffraction data.

Without the diffraction data, one cannot calculate an R factor just from atomic 
positions.

To me, it seems like your best bet would be to try and retain the R factor 
information in the PDB file header, or recover this information using the PDB 
accession codes. I suspect this can be done relatively easily through the API, 
but others can better advise you on how to do this.

HTH,

Dave











Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Abhilasha Thakur 

Sent: Wednesday, June 1, 2022 7:09:20 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Regarding R factor value (R Free)


External Sender: Use caution.

Hii!!

I have 1500 processed PDB files and I need the R factor value for each pdb 
files. How I get this R factor value, because it's there initially but, after 
processing, some data from PDB is deleted only the atom and coordinate 
information is there no header information in the PDBs. Manually to get R 
factor is not possible.

Please suggest me, how to get this R factor value for the entire PDB ids.



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Re: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide crystallization?

[David Briggs]

Hi Mark,

I also use dis.embl and IUPred in Jalview when trying to come up with construct 
boundaries.

In the specific case I was referring to, we had a secreted human protein with a 
long and atypical secretion tag, which made it difficult to place the 
N-terminus of the mature protein within the sequence.

My initial attempts didn't work at all, and after reviewing the AF2 model at 
the EBI database, I removed a further ~30 residues at the N-terminus, which 
yielded a well expressed, soluble, secreted product.

Speaking more generally, I think that AF2 is probably "just another tool" for 
helping to inform construct boundaries.
I have no empirical evidence that the AF2 pLDDT is more sensitive or accurate 
than other bespoke disorder prediction software.

Because it worked once for me, I'll run it/check the EBI database every time 
from now on, but you're absolutely right that I probably would have done that 
anyway!

Best,

Dave


--

Dr David C. Briggs CSci MRSB

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: Mark J. van Raaij 
Sent: 05 April 2022 14:47
To: CCP4BB@jiscmail.ac.uk 
Cc: David Briggs 
Subject: Re: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide 
crystallization?


External Sender: Use caution.

Hi David,

do you think that alphafold2 dis/order prediction is better than specific 
disorder predictors?

i.e.
http://dis.embl.de<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fdis.embl.de%2F=04%7C01%7C%7C84f2f05bbd5a4598ff5908da170ae108%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637847632751920405%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000=2FjXOEwioG4hdIuef0nPFEM%2B4pV6V%2FB9kYa9phWu4Ds%3D=0>
 , which we've used for construct design
https://prdos.hgc.jp<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fprdos.hgc.jp%2F=04%7C01%7C%7C84f2f05bbd5a4598ff5908da170ae108%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637847632751920405%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000=ckpin7cUBV3sVYnc1Coxe72%2FQNMd1M%2ByXpoRaCbdRgE%3D=0>
 , which I haven't really tried yet
https://iupred2a.elte.hu<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fiupred2a.elte.hu%2F=04%7C01%7C%7C84f2f05bbd5a4598ff5908da170ae108%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637847632751920405%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000=5IVfLHj7g9Wq4BiQdoez1wB0mgsvQ608xAcw3LzY%2BnM%3D=0>
 (which a colleague here likes because it also tries to predict likelihood of 
protein interactions)

Or perhaps it's just because you were going to run alphafold2 anyway?

best wishes,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


On 4 Apr 2022, at 21:30, David Briggs 
mailto:david.bri...@crick.ac.uk>> wrote:

Hi Scott,

I've used AF2 order/disorder prediction (based up pLDDT score) to decided upon 
construct boundaries. We turned a non-expressing construct into a reasonably 
well expressing construct based on the AF2 prediction.

It's part of my construct design process now.

HTH,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs<https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fabout.me%2Fdavid_briggs=04%7C01%7C%7C84f2f05bbd5a4598ff5908da170ae108%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637847632751920405%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000=RqGd8%2Fy%2FuaLk%2B9IjCfiagj%2BZiHQB%2BYwAu269u7C0HFs%3D=0>

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Scott Classen mailto:sclas...@lbl.gov>>
Sent: Monday, April 4, 2022 8:06:38 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide 
crystallization?


External Sender: Use caution.

Hello CCP4,

Has anyone successfully used the available ML/AI protein folding tools to guide 
crystallization construct design? Maybe you had a protein or domain that was 
resistant to crystallization efforts and the folding algorithms  predicted some 
loops or termini that were disordered? Then you trimmed or modified them in 
some way to aid in crystallization? Or if you haven’t done this yourself, are 
you aware of anyone who has?

Thanks,
Scott


~~
Scott Classen, Ph.D.
ALS-ENABLE
TomAlberTron Beamline 8.3.1
SIBYLS Beamline 12.3.1
Advanced Light Source
Lawrence Berkele

Re: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide crystallization?

[David Briggs]

Hi Scott,

I've used AF2 order/disorder prediction (based up pLDDT score) to decided upon 
construct boundaries. We turned a non-expressing construct into a reasonably 
well expressing construct based on the AF2 prediction.

It's part of my construct design process now.

HTH,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs

From: CCP4 bulletin board  on behalf of Scott Classen 

Sent: Monday, April 4, 2022 8:06:38 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide 
crystallization?


External Sender: Use caution.

Hello CCP4,

Has anyone successfully used the available ML/AI protein folding tools to guide 
crystallization construct design? Maybe you had a protein or domain that was 
resistant to crystallization efforts and the folding algorithms  predicted some 
loops or termini that were disordered? Then you trimmed or modified them in 
some way to aid in crystallization? Or if you haven’t done this yourself, are 
you aware of anyone who has?

Thanks,
Scott


~~
Scott Classen, Ph.D.
ALS-ENABLE
TomAlberTron Beamline 8.3.1
SIBYLS Beamline 12.3.1
Advanced Light Source
Lawrence Berkeley National Laboratory
1 Cyclotron Rd
MS6R2100
Berkeley, CA 94720
mobile 510.206.4418
desk 510.495.2697
beamline 510.495.2134
~~




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Re: [ccp4bb] mysterious density

[David Briggs]

MES or HEPES, I reckon.

Both of them can lose definition for the electron density in the 
morpolino/piperazine 6-membered ring due to flexibility between the ring and 
the sulphonic acid group when - leading to the tadpole-like density you 
describe.

HTH,

Dave


--

Dr David C. Briggs CSci

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Group (DUC) MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Xavier 
Brazzolotto 
Sent: 13 March 2022 17:28
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] mysterious density


External Sender: Use caution.


A MES buffer molecule ?

> Le 13 mars 2022 à 18:08, doriano lamba  a écrit :
>
> Glycerol-3-phosphate?
> My 2cents
> DL
> Il 2022-03-13 15:19 Amir Khan ha scritto:
>> Hi,
>> I wonder whether anyone can advise on the ‘tadpole’ like density.
>> The head is currently a phosphate
>> and I’ve placed a water in tail, but it obviously looks connected…
>> would appreciate any help!
>> Green density at 5 sigma (Fo-fc), while blue is 2fo-fc at 1.3 sigma.
>> Crystallization condition is unknown, though it came from sparse
>> matrix commercial (either Wizard, Pact Prem, or JCSG+).
>> Thanks!
>> Amir
>> -
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Re: [ccp4bb] off-topic: glycans

[David Briggs]

Hi Sam,

GlycoMod from Expasy sounds like it might do what you want to do.

https://web.expasy.org/glycomod/

D


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee (MX)

CCP4 WG2

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Sam Tang 

Sent: 29 June 2021 06:12
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off-topic: glycans


External Sender: Use caution.

Dear community

Sorry for an off-topic question here. I wonder if anyone may be aware of any 
glycan modification database where we can predict what is what. For example, if 
I got a mass difference of m/z X on LC-MS, and I would like to have a rough 
idea what it might be, where should I go for?

Thanks!

BRs

Sam



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Re: [ccp4bb] Eukaryotic protein expression

[David Briggs]

Hi Dhiraj,

For transient expression in HEK293 cells in suspension, I generally use 
pcDNA3.1 which also uses a CMV promoter. Yields range from nothing to 50mg/l, 
depending entirely on what it is you're making.

I would suggest that maybe the promoter is not at fault, and perhaps the 
product is. Tweaking construct boundaries and optimizing transfection 
conditions may help.

Good luck,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Sent: Thursday, June 24, 2021 9:42:32 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Eukaryotic protein expression


External Sender: Use caution.

Hi all
I am trying to express my protein in HEK293 cells for crystallization 
purpose but getting poor expression. I am using cmv promoter. Which 
promoter/vector people use to get good expression in eukaryotic expression 
system?

Thank you

Dhiraj



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Re: [ccp4bb] missing covalent bond between asparagine and N-acetylglucosamine using coot carbohydrate module and phenix.refine

[David Briggs]

Hi Jiang Xu,

(Phenixbb added as I suspect this is Phenix not coot that has caused your issue)

Did you check the "add hydrogens" button in Phenix.refine? Sometimes this can 
erroneously add a second H to the ND of glycosylated Asn residues. (I assume 
this is because bond distances in the input PDB file fall outside Phenix's 
definitions of an Asn-NAG link).

If the second H is added, this breaks the refinement of the Asn-NAG bond.

My work around is to run "Ready Set" to add hydrogens as a separate job, and 
then manually inspect and correct the Asn residues as necessary before running 
Phenix.refine and _not_ checking the "add hydrogens" box.

I hope this fixes your problem.

Good luck,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Jiang Xu 

Sent: Tuesday, June 22, 2021 6:47:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] missing covalent bond between asparagine and 
N-acetylglucosamine using coot carbohydrate module and phenix.refine


External Sender: Use caution.

Hello guys,
   I have a problem building and refining an xtal structure. My protein has a 
N-glycosylation site and I want to add N-acetylglucosamine and manose to my 
protein structure. I used Coot's carbohydrate module and let Coot automatically 
build the sugar chain to the electron density. It worked pretty well, but I 
noticed that the bond between the amine group of asparagine and the C1 of 
N-acetylglucosamine is depicted as a dashed line, rather than a solid line in 
Coot. After refinement of the structure using Phenix.refine, I found the bond 
just disappeared, and the amine group still has two hydrogen atoms, which 
should contain one hydrogen atom.  So,  how to solve this problem?
[image.png]
Thank you,
Best,
Jiang Xu
Lin Chen's Research Group
University of Southern California



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Re: [ccp4bb] help needed with link description

[David Briggs]

Hi Gerlind,

Rob Nicholls gave a talk on how to deal with covalent linkages using AceDrg at 
CCP4 study weekend in 2020.

https://youtu.be/p4oTJ0bjD3M

And Paul Emsley has written up a guide about it...

https://pemsley.github.io/coot/blog/2020/06/30/make-a-link.html

Hopefully these will be useful for you.

Good luck,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Gerlind 
Sulzenbacher 
Sent: Thursday, June 3, 2021 7:10:12 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] help needed with link description


External Sender: Use caution.


Dear all,

I am going litteraly mad (or just old).

I would like to make a covalent link between a carbohydrate moiety and
and ASP of my well-cheered protein.

The identifier for the carbohydrate moiety is MAD, chain C, res number 1.

The link is established between C5 of MAD and OD2 of ASP 518, chain A.

In the mad_monlib.cif I added the following:

data_link_list
loop_
_chem_link.id
_chem_link.comp_id_1
_chem_link.mod_id_1
_chem_link.group_comp_1
_chem_link.comp_id_2
_chem_link.mod_id_2
_chem_link.group_comp_2
_chem_link.name
ASP-MAD  ASP  ..MAD  ..
  bond_ASP-OD2_=_MAD-C5
#
data_link_ASP-MAD
#
loop_
_chem_link_bond.link_id
_chem_link_bond.atom_1_comp_id
_chem_link_bond.atom_id_1
_chem_link_bond.atom_2_comp_id
_chem_link_bond.atom_id_2
_chem_link_bond.type
_chem_link_bond.value_dist
_chem_link_bond.value_dist_esd
  ASP-MAD  1 OD2 2 C5.   1.3700.040

In the entry PDB file I added the line:

LINKOD2  ASP A 518C5  MAD C 1

The log file of REFMAC tells me

INFO: link is found (not be used) dist=   1.370 ideal_dist= 1.370
 ch:AAA  res: 518  ASP  at:OD2 .->ch:CCC  res:
1  MAD  at:C5  .

And the expression "not be used" is certainly the case, as the distance
of the covalent bond changes from 1.37 to 1.6 Å.

The data resolution is 1.8 Å.

If somebody could please point out what I am doing wrong, I'd be very
grateful.

With best wishes,

Gerlind


--
Gerlind Sulzenbacher
Architecture et Fonction des Macromolécules Biologiques
UMR7257 CNRS, Aix-Marseille Université
Case 932
163 Avenue de Luminy
13288 Marseille cedex 9
France
Tel +33 491 82 55 66
Fax +33 491 26 67 20
E-mail: gerlind.sulzenbac...@univ-amu.fr



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Re: [ccp4bb] Co crystalization with less soluble ligand.

[David Briggs]

These are my go-to review articles for ideas for getting a ligand into a 
crystal.

https://scripts.iucr.org/cgi-bin/paper?S0907444906047020

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297911/

I have also seen a talk where someone successfully dissolved their 
water-insoluble ligand in Octan-1-ol, and layered that solution over the drop 
after crystals had grown. They incubated for X period of time (I don't recall) 
froze the crystals and found the ligand had diffused into the crystals and was 
in the active site.

Maybe someone else knows of the/a paper describing this?

Hope this helps,

D


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Gourab Basu 
Choudhury 
Sent: 24 April 2021 03:46
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Co crystalization with less soluble ligand.


External Sender: Use caution.

Hello everyone,

I am finding it difficult for getting a ligand density inside the protein as 
the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for 
co crystalization. Kd value of the ligand is near 40um. Any suggestion what to 
do?



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Re: [ccp4bb] Fluka-branded PEG 3000

[David Briggs]

Hi Galen,

Is it possibly the age of the original material, rather than the batch or 
brand? PEG does oxidise over time.

Best,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Correy, Galen 

Sent: Monday, March 22, 2021, 17:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fluka-branded PEG 3000


External Sender: Use caution.

Dear all,

I'm wondering if anyone could spare 50-100 g of Fluka branded PEG 3000 (see 
attached image)?

We have some crystals that diffract better when grown using Fluka PEG compared 
to other brands. Unfortunately, we've exhausted our supply of the Fluka PEG, 
and it's no longer commercially available (the same product code is available 
from Sigma - we've tested it, along with several other brands - but they don't 
seem to have the same magic as the Fluka PEG).

We'll happily purchase a new bottle of Sigma-branded PEG 3000 to replace any 
sent our way.

Many thanks,
Galen



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Re: [ccp4bb] Co-crystallization and thermal shift assay

[David Briggs]

Hi Saif,

Whilst in very general terms, ∆Tm does correlate with binding affinity (there 
will of course always be exceptions to this rule), I don't think there is a 
cutoff beyond which you know that co-crystallisation is feasible. The degree of 
stabilisation will depend very much on the system you are studying.

I've had proteins crystallise with ligands with a very modest ∆Tms (1-2ºC) and 
then failed to get the same protein to crystallise with ligands that give a 
15-20ºC ∆Tm.

I certainly wouldn't let a TSA result dissuade me from trying to co-crystallise 
a protein with a ligand if the hoped-for structure was important for answering 
whatever biological question I'm asking.

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Saif Mohd 

Sent: 25 February 2021 14:55
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Co-crystallization and thermal shift assay

Hello everyone,

1) How much change in Tm (ΔTm) in a thermal shift assay is considered to be 
significant ?

2) A negative  ΔTm infers that the compound is making the protein unstable. In 
such a case, will the co-crystallization be difficult or just impossible or on 
the contrary it shouldn't matter much?


Thanks and best regards,
Saif




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Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

[David Briggs]

I think it is possible, but the reason it hasn't been done and will probably 
never be done is that informed consent is an important part of clinical 
practice.

D

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs

From: CCP4 bulletin board  on behalf of Jacob Keller 

Sent: Wednesday, February 17, 2021 5:33:09 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

It would seem to me that it should be possible to generate versions of the 
Covid virus that would:

A. be extremely contagious and yet
B. be clinically benign, and
C. confer immunity to the original covid virus.

If, then, this virus could be released, with appropriate "kill switch" 
safeguards built in, would this not solve the world's pandemic problems? Is 
there any reason, practically, why this approach would not be feasible?

Maybe we don't really know enough to manipulate A, B, C yet?

Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?

Has this sort of thing been done, or does it have a name?

Jacob
--

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; 
jacobpkel...@gmail.com

Cell: (301)592-7004

+



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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[David Briggs]

A quick note regarding the code that Deepmind released for CASP13 (2018).

It bears the rather important caveat that: "This code can't be used to predict 
structure of an arbitrary protein sequence. It can be used to predict structure 
only on the CASP13 dataset (links below)."

Source: 
https://github.com/deepmind/deepmind-research/tree/master/alphafold_casp13

So whilst we can replicate their previous efforts, we currently can't submit 
our more troublesome sequences to their software, which is (I imagine) 
something that many of us might like to try.

D

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Isabel 
Garcia-Saez 
Sent: Thursday, December 3, 2020, 11:17
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

Dear all,

Just commenting that after the stunning performance of AlphaFold that uses AI 
from Google maybe some of us we could dedicate ourselves to the noble art of 
gardening, baking, doing Chinese Calligraphy, enjoying the clouds pass or 
everything together (just in case I have already prepared my subscription to 
Netflix).

https://www.nature.com/articles/d41586-020-03348-4

Well, I suppose that we still have the structures of complexes (at the moment). 
I am wondering how the labs will have access to this technology in the future 
(would it be for free coming from the company DeepMind - Google?). It seems 
that they have already published some code. Well, exciting times.

Cheers,

Isabel


Isabel Garcia-Saez PhD
Institut de Biologie Structurale
Viral Infection and Cancer Group (VIC)-Cell Division Team
71, Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France
Tel.: 00 33 (0) 457 42 86 15
e-mail: isabel.gar...@ibs.fr
FAX: 00 33 (0) 476 50 18 90
http://www.ibs.fr/




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with its registered office at 1 Midland Road London NW1 1AT



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[ccp4bb] Diamond-II MX beamlines letters of support

[David Briggs]

Dear all,

(Apologies for the slightly off-topic cross-post - hopefully, this will be of 
interest to all MX practitioners, not just Diamond light source users.)

As Diamond User Committee MX representatives, we'd just like to remind you that 
the deadline for letters of support for Diamond-II beamline upgrades is this 
Friday, 20th November.

As you are perhaps aware, the Diamond machine is scheduled for a major upgrade 
to a 4th generation source 2025-2027.
Further information about the upgrade can be found 
here.

All MX beamlines will benefit from a brighter, more focused source – however, 
two MX beamlines have been selected as flagship projects for significant 
upgrades.

I04-1 > K04 Ultra XChem.
The I04-1 fixed wavelength beamline used for the XChem fragment screening will 
be completely rebuilt as K04.
Briefly, K04 will allow higher throughput fragment screening (meaning more 
projects and more fragments can be screened) and it is hoped the improved 
characteristics of the Diamond-II beam will allow XChem methodologies to be 
extended to more difficult targets such as transmembrane proteins.

The K04 proposal outline can be found 
here
 [PDF].

I24 > KMX Kinetic micro crystallography.
The I24 microfocus beamline will be upgraded to enhance the capabilities of I24 
and allowing analysis of smaller crystals, radiation-sensitive crystals, and 
expanding the serial synchrotron crystallography (SSX) functionality of the 
beamline. The abilities to conduct room temperature SSX will also be enhanced 
to allow in-crystal kinetic analysis, with various on-line spectroscopic 
(Raman, UV-Vis, XES) detectors built into the end station.

The KMX proposal outline can be found 
here
 [PDF]

Having read these proposals you will obviously be very excited by the 
possibilities and will want to submit your letters of support for both of them, 
by Friday, 20th November

Letters of support can be submitted via this 
link.

Thanks in advance,

Dave & Arnaud.

--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] File System Problems

[David Briggs]

Hi Sam,

we had a similar issue with CCP4I2 on Linux in the last couple of weeks. 
Installing the very latest updates made the problem go away for us.

I don't know if this Linux issue would also crop up in Windows, but what you 
describe seems similar - try to open a file selection dialogue and the software 
crashes.

D


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Horrell, Sam 
(DLSLtd,RAL,LSCI) 
Sent: 01 October 2020 11:53
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] File System Problems


Hello CCP4bb,



I’m having a serious problem with my CCP4i2 installation on my windows 10 
computer, whenever I try and upload a file via the GUI it fails to open the 
file system and stops responding, then it just hangs until I kill the program. 
I’ve uninstalled and reinstalled CCP4 and rolled back the latest updates but 
nothing has changed. Obviously this makes using CCP4 completely impossible. Any 
suggestions for how I might fix this problem?



Cheers,



Sam





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Re: [ccp4bb] electron density close Histidine side chain

[David Briggs]

Hi Samer,

(1) Given that you say there is a relatively high concentration of Zn2+ ions in 
the crystallisation buffer, I suspect this might be your candidate.

(2) If the Zn2+ ion is correctly represented in the pdb file, your refinement 
program of choice will know what the target co-ordination distances should be, 
and should not give clash issues.

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: samer halabi 
Sent: 21 July 2020 11:08
To: ccp4bb@jiscmail.ac.uk ; David Briggs 

Subject: Re: [ccp4bb] electron density close Histidine side chain

Hello,
Thank you for your kind reply.
If the distances are still less than 2.2Å, would coot and Refmac still consider 
that as a clash. When I try to fit in Imidazole, even if the distance is more 
than 2.8Å, it is still considering it a clash.
Sorry, I should've mentioned how I purified the protein complex.
The protein is secreted in HighFive cells, to purify it  by its 6xHis tag, I 
used Ni sepharose excel beads, eluted with Imidazole and then further purified 
with size exclusion. To get rid of the tags and leucine zipper I used to mimic 
the transmembrane domain, I subjected the protein to V8 edndoproteinase in 0.1M 
Tris pH8.5 (cuts after an exposed Glutamate). Then purified again with Ni 
sepharose excel and size exclusion prior to crystallisation. It crystallised in 
0.2M Zinc acetate, 0.1M Imidazole pH 6.5, 10% PEG 8K. We used glycerol to fish 
the crystals out.
I have worked on three other crystals of the similar molecule, which 
crsytalized in different conditions, and this is the only one I see such blobs.

Thank you again.
Best regards,
Samer


On Monday, July 20, 2020, 06:25:09 PM GMT+1, David Briggs 
 wrote:


Hi Samer,

Did you use a His tag/Ni-NTA during purification? Sometimes Ni2+ ions leach off 
the Ni-NTA -  maybe the two "ears" are  accompanying waters?

Ni-His co-ordination distance is pretty short
(2-2.2Å - table 3 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872550/#!po=0.69<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2872550%2F%23!po%3D0.69=02%7C01%7C%7Cdfc2bafb083e44e4f00e08d82d5e137e%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637309229367599545=u9oHbg6HzNOIeQFMUadyK4OIP1%2BaNFfLAdiPb7OAG8c%3D=0>)
 and might account for your bump when you model in a ligand.

Good luck!

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Nils Marechal 
<4954a024d277-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, July 20, 2020 5:27:37 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] electron density close Histidine side chain

‌Dear Samer,

What king of cryo-protectant did you use ?

Such a bent density, with that size, looks like an ethylene-diol.

Best regards,

Nils Marechal

De : "samer halabi" <30c2162795b2-dmarc-requ...@jiscmail.ac.uk>
A : CCP4BB@JISCMAIL.AC.UK
Envoyé: lundi 20 Juillet 2020 18:17
Objet : [ccp4bb] electron density close Histidine side chain

Hello all,
I have few blobs in an MHC II structure I am working on, especially opposite to 
Histidine as in the accompanying screenshot, that I am confused about.

In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
Whatever ligand I am fitting in I am getting a clash (overlap -1.029), which 
makes me think whether there is a covalent bond forming between Histidine and 
other molecule. Perhaps by oxidation.

I would greatly appreciate if you can advice me about it, whether there is some 
kind of ligand I can try to fit and if this is something that occurs in some 
structures.
Thank you.
Best regards,
Samer



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The Francis Crick In

Re: [ccp4bb] electron density close Histidine side chain

[David Briggs]

Hi Samer,

Did you use a His tag/Ni-NTA during purification? Sometimes Ni2+ ions leach off 
the Ni-NTA -  maybe the two "ears" are  accompanying waters?

Ni-His co-ordination distance is pretty short
(2-2.2Å - table 3 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872550/#!po=0.69) and might 
account for your bump when you model in a ligand.

Good luck!

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Nils Marechal 
<4954a024d277-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, July 20, 2020 5:27:37 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] electron density close Histidine side chain

‌Dear Samer,

What king of cryo-protectant did you use ?

Such a bent density, with that size, looks like an ethylene-diol.

Best regards,

Nils Marechal

De : "samer halabi" <30c2162795b2-dmarc-requ...@jiscmail.ac.uk>
A : CCP4BB@JISCMAIL.AC.UK
Envoyé: lundi 20 Juillet 2020 18:17
Objet : [ccp4bb] electron density close Histidine side chain

Hello all,
I have few blobs in an MHC II structure I am working on, especially opposite to 
Histidine as in the accompanying screenshot, that I am confused about.

In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
Whatever ligand I am fitting in I am getting a clash (overlap -1.029), which 
makes me think whether there is a covalent bond forming between Histidine and 
other molecule. Perhaps by oxidation.

I would greatly appreciate if you can advice me about it, whether there is some 
kind of ligand I can try to fit and if this is something that occurs in some 
structures.
Thank you.
Best regards,
Samer



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Re: [ccp4bb] Commercial source of His-tagged protein

[David Briggs]

Hi Mark,

NEB

https://international.neb.com/products/p8112-tev-protease#Product%20Information

And Sigma

https://www.sigmaaldrich.com/catalog/product/sigma/t4455?lang=en=GB

Both sell His-tagged TEV protease.

Maybe suitable?

HTH,

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
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From: CCP4 bulletin board  on behalf of Mark J van Raaij 

Sent: Thursday, July 2, 2020 10:31:37 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Commercial source of His-tagged protein

Dear All,

Wondering if anyone knows of an economical commercial source of a soluble 
His-tagged protein. In principle any His-tagged protein would suffice, because 
it's for testing coupling to a surface via the His-tag. Asking for an 
international collaborator, who don't have access/expertise of a molecular 
biology lab (except us :-).
We can and will make the His-tagged proteins of interest for them, but if there 
is an economical and reliable commercial source, it might be worth using that 
for initial tests.

Best wishes,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/





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Re: [ccp4bb] Molecular replacement problem

[David Briggs]

Hi Robert,

Have you tried lower symmetry spacegroups? Maybe your crystal is 
'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending to be 
orthorhombic.

Zanuda can do this for you.

https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Robert S 
Phillips 
Sent: 18 June 2020 14:00
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Molecular replacement problem

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net



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Re: [ccp4bb] disinfecting keyboards

[David Briggs]

Have you considered autoclaving the keyboards in between users?

Or maybe autoclave the users?

That'll work, right?

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Wednesday, April 29, 2020 9:19:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] disinfecting keyboards


>I do not know if any of the equipment may suffer damage in the medium or long 
>term due to the incidence of UV light.



Hmm….everything made of cheap plastic….i.e. almost everything made in 
China…that must be the true conspiracy behind the virus!



Cheers, BR



On Wed, Apr 29, 2020 at 2:53 PM Tim Gruene 
mailto:tim.gru...@univie.ac.at>> wrote:

Dear all,

can you make suggestions for how to disinfect computer keyboards, and
instrument panels?

Our facility is going to reboot next week, with shifts so that people
don't meet. The main interface will be the computer keyboards, as well
as the door of our X-ray diffractometer and the mounting of the
crystals.

The keyboard labels may not like alcohols (and the efficiency of
injecting disinfecting through the USB cable is also under discussion,
so I heard).

One way would be to use individual keyboards, and wearing gloves for
replugging, and to use gloves for mounting crystals.

But maybe there are other ways that won't require gloves?

Best regards,
Tim

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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--

Eduardo Rodríguez-Román, PhD

ASM, eLIFE & USERN Ambassador

Biotechnology and Plant Virology Lab

Center for Microbiology and Cell Biology

Instituto Venezolano de Investigaciones Científicas

PO Box 20632, Caracas 1020A, Venezuela.

ORCiD: 
orcid.org/-0001-8717-7527

Phone: +58 (212) 504 1189/1366/1500

Cell phone: +58 (424) 111 0375

E-mail: ejrodrig...@ivic.gob.ve,

or erodriguezro...@gmail.com

Twitter: 
@erodriguezroman

 
[https://docs.google.com/uc?export=download=1_MmmlkLEEx67-CUSQYLGHM9eAuWKgHX0=0B7ogjC4ootvrUi9ITGpVWEd4S01rVnBVTjVvd3p0OUN5d1hRPQ]
 

  
[https://drive.google.com/uc?id=1rHts30EjUWzlunzrJxVquEu_aYsfYmHE=download]
 


[https://docs.google.com/uc?export=download=1eQDqlB4kvjK2qLYX3htqhh54b-l76rcs=0B7ogjC4ootvrNy9vbGtQb0h4YkhUK2J5MmZvczhMK0NBbks0PQ]
 
[https://docs.google.com/uc?export=download=1CekoNvw6Til3qV0zBn5Vohz05SD-nyTR=0B7ogjC4ootvrRFhPTzJiMFBrZ1ZqRUpJYTRDZ0FBRDFkT28wPQ]






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Re: [ccp4bb] CCP4BB vs COVID19

[David Briggs]

For general interest,

The Francis Crick institute in London is offering facilities to public health 
England, and over 300 Crick scientists have volunteered to help with testing.

https://www.crick.ac.uk/news/2020-03-19_francis-crick-institute-offers-assistance-in-emergency-coronavirus-testing

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of David Waterman 

Sent: Saturday, March 21, 2020 11:12:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] CCP4BB vs COVID19

Liz Tunbridge's lab at Oxford are offering PCR machines and expertise to help 
fill the testing shortfall in the UK (see 
https://www.wired.co.uk/article/coronavirus-uk-testing-key-workers).
 This is a worthy initiative, if it is accepted (logistics are the main 
problem). The structural biology community is pretty good with this too. 
Perhaps there are some opportunities to help out here, for those who can still 
get to their wet labs?

-- David


On Fri, 20 Mar 2020 at 22:59, James Holton 
mailto:jmhol...@lbl.gov>> wrote:
You might think that as a structural biologist you won't be able to do
much about COVID-19 anytime soon, but that is not true.  Yes, real-world
therapeutics and vaccines take time, but we have already seen just how
fast we can get started.  There are 21 PDBs already and some even have
bound ligands.  Good job Frank et al. BTW!  And my personal thanks to
all of you out there who are already hard at work on this.

I believe this forum is an ideal place to share information and ideas on
the structural biology of SARS-CoV-2 as we move forward. It's a big
virus, but there are not that many proteins in it.  If all of us
independently do the same bioinformatics and literature searches and end
up trying exactly the same thing in every lab all over the world, then
that would be more than unfortunate.  To that end, I am personally
interested on ORF8 for reasons I will go into below.  Has anyone tried
to solve it yet?  What happened?  Didn't express? Bad diffraction?
What?  Do tell.

Some of us, as you may have heard, are stuck at home, our beamlines and
labs dark while we shelter-in-place.  That doesn't mean our hands are
tied.  We are still allowed to think. The fraction of the human race
that has a snowball's chance in Hades of figuring out this bug is very
very small.  Structure may be your main skill set, but you are still a
biologist.  Do you know how to run a PCR machine?  Do you know how to
pipette?  You might think that anybody can do it, but that is really not
the case. Ever trained a new student on sterile technique?  How many
days did that take?  Now remember that your student was no dummy and
already studying biology.  Everyone reading this will make an excellent
volenteer at the very least.  I'm not saying this to belittle the
average human, only to say that we scientists, moving in the circles we
do, often forget that we have uncommon capabilities.

For example, I also believe we can be useful in assay development. The
void left by the dearth and delay of test results has been filled with
fear, and that is a big problem.  The tests, as defined, are
straightforward, but also extremely regimented like any good laboratory
protocol should be.  The US CDC's instructions for academic labs are here:
https://www.cdc.gov/coronavirus/2019-nCoV/lab/index.html
My question is: how can this test be made faster, using more commonplace
supplies, in high-throughput mode and still valid?  Not just for
clinical but for academic use?  I think more than a few people on this
list could be regarded as experts in making a complex biochemical task
faster, more efficient, high-throughput and nonetheless valid.  Yes,
there are other people who do virus testing for a living, but right now
they are all rather busy.  Maybe if we put our minds to it we can help?

As for why ORF8.  I am basing my interest on the bioinformatics done in
this article: 
https://dx.doi.org/10.1093/nsr/nwaa036.
  Search for
"T8517C" and you will find what I'm 

Re: [ccp4bb] Red trapezoids in Coot

[David Briggs]

Hi Joel,

The red trapezoids are indicative of cis-peptide bonds that are exceptionally 
rare, and usually indicate incorrect tracing of the backbone (unless you have 
good enough data to unambiguously decide that a cis-peptide bond is correct).

I'm afraid I don't know how to get rid of them, other than correcting the 
backbone.

There is a Cis<>Trans button you can add to coot which might correct the issue 
for you (right-click on the icon bar, select "Manage Buttons", and add the 
appropriate button)

HTH

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Joël Bloch 

Sent: 12 March 2020 09:14
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Red trapezoids in Coot

Dear CCP4 community,

Is there any way in Coot 0.8.9.2 to remove/hide the red trapezoids that are 
indicating planar bonds?

I couldn’t find anything on that, neither in the manual nor FAQ.

Thanks a lot for your help!

Best wishes,
Joël



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Re: [ccp4bb] protein expression in human cells

[David Briggs]

For general information, HEKs were derived from a legally aborted foetus the in 
the Netherlands in 1973. Not "killed children".

(As a side note, such use of emotive language is counter-productive and often 
the tool of those who seek remove reproductive rights and autonomy from women. 
I'm certain we wouldn't want to be tarred with that brush.)

That said, use CHOs if you have an issue with HEKs. The only potential problem 
would be if what you wanted to study required authentic human 
post-translational modifications, and even then, I imagine CHOs would still be 
a suitable surrogate in the majority of cases.

Cheers,

Dave


--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs

From: Petr Kolenko 
Sent: Saturday, January 25, 2020 7:10:06 AM
To: David Briggs ; CCP4BB@JISCMAIL.AC.UK 

Subject: RE: [ccp4bb] protein expression in human cells


Dear colleagues,

I wonder if there were a bit less controversial possibility. No matter if that 
was less efficient. Would there be an option of using human cell lines that do 
not origin from killed children? As far as I know, the HEK cells do. Sometimes, 
the science can be pretty cruel.

I am sorry for opening of this topic on a crystallographic forum, but so far, 
nobody has convinced me that I am allowed to work with these cell lines from a 
humanity (and Christian) point of view.

Best regards,

Petr





From: CCP4 bulletin board  On Behalf Of David Briggs
Sent: Saturday, January 25, 2020 7:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein expression in human cells



Hi Gloria,

Another vote here for HEK 293 Expi or Freestyle.

The off-the-shelf transfection reagents are super expensive, but I make my own 
(happy to share protocols with anyone interested) and we normally get a few mgs 
of protein per litre of suspension culture -- certainly enough for 
crystallography.

If budget is an issue, semi-stable transfection of adherent HEK293s is much 
cheaper but the turnaround from vector to protein is much slower (a few weeks 
rather than a few days), and growing up panels of mutants can be quite tedious.

Cheers & good luck

Dave



--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs





From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Rezaul Karim 
<1e364c8f16de-dmarc-requ...@jiscmail.ac.uk<mailto:1e364c8f16de-dmarc-requ...@jiscmail.ac.uk>>
Sent: Saturday, January 25, 2020 1:31:02 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] protein expression in human cells



In our lab, we see Expi293F works very good.



Thanks,
Reza



Md. Rezaul Karim
Graduate student, PhD Program in Integrated Biomedical Sciences

Morsani College of Medicine
University of South Florida
E-mail: reza...@yahoo.com<mailto:reza...@yahoo.com>, 
rez...@health.usf.edu<mailto:rez...@health.usf.edu>

Phone: +1-954-937-8487





On Friday, January 24, 2020, 4:51:11 PM EST, Gloria Borgstahl 
mailto:gborgst...@gmail.com>> wrote:





Hello CCP4-ers,

I was wondering what people have found to be the best human cell line 
expression system for making a large quantity of purified recombinant protein.

Any information and protocols would be greatly appreciated.

Happy 2020, Gloria





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Re: [ccp4bb] protein expression in human cells

[David Briggs]

Hi Gloria,

Another vote here for HEK 293 Expi or Freestyle.

The off-the-shelf transfection reagents are super expensive, but I make my own 
(happy to share protocols with anyone interested) and we normally get a few mgs 
of protein per litre of suspension culture -- certainly enough for 
crystallography.

If budget is an issue, semi-stable transfection of adherent HEK293s is much 
cheaper but the turnaround from vector to protein is much slower (a few weeks 
rather than a few days), and growing up panels of mutants can be quite tedious.

Cheers & good luck

Dave



--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Rezaul Karim 
<1e364c8f16de-dmarc-requ...@jiscmail.ac.uk>
Sent: Saturday, January 25, 2020 1:31:02 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] protein expression in human cells

In our lab, we see Expi293F works very good.

Thanks,
Reza

Md. Rezaul Karim
Graduate student, PhD Program in Integrated Biomedical Sciences
Morsani College of Medicine
University of South Florida
E-mail: reza...@yahoo.com, rez...@health.usf.edu
Phone: +1-954-937-8487


On Friday, January 24, 2020, 4:51:11 PM EST, Gloria Borgstahl 
 wrote:


Hello CCP4-ers,
I was wondering what people have found to be the best human cell line 
expression system for making a large quantity of purified recombinant protein.
Any information and protocols would be greatly appreciated.
Happy 2020, Gloria



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with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] Optimization from needle shaped crystals

[David Briggs]

4. Matrix microseeding. Make a seed stock from these crystals and then re-run 
your primary screens.

https://www.ncbi.nlm.nih.gov/m/pubmed/25195878/

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of chitra latka 

Sent: Sunday, September 8, 2019 12:12:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Optimization from needle shaped crystals

I can share what has worked for my crystals :

1. You can put a grid across your condition with same or altered drop ratios.

2. You can try micro seeding. (This has given me the best results so far).

3. You can try Hampton's additive screen.

On Sun, Sep 8, 2019 at 10:09 AM Prem Prakash 
mailto:prem...@gmail.com>> wrote:
Dear all,
Sorry for a trivial query. I am trying to Co-crystallize my protein with its 
substrate (peptide) using commercial screenings. In one condition of JCSG plus 
(Molecular Dimension) that contains  0.2 M Magnesium chloride hexahydrate,  0.1 
M Tris 8.5 50 % v/v Ethylene glycol, I got needle like crystals (picture 
attached). Does anyone have idea to optimize such needles into better crystals. 
I would appreciate all your suggestions.

Thank you
With kind regards,
Prem Prakash  (Ph.D.)




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--
Regards
Chitra



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Re: [ccp4bb] Another difficult MR case

[David Briggs]

Following on from Ivan's suggestion, SIMBAD might he worth a shot.

https://journals.iucr.org/d/issues/2018/07/00/rr5159/

The other thing you might try is handing the MR phases to the density modifying 
and autobuilding program of your choice, increasing the number of cycles by 
$arbitarylargenumber and then leaving it to run for a few hours/over night.

This has worked for me in the past when resolution was decent, phaser had found 
an obviously correct MR solution, but the domain placed was only ~30-40% of the 
total scattering mass of the ASU, and more conventional refinement was not 
yielding decent maps outside the aforementioned domain.

Good luck!

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Phil Jeffrey 

Sent: Thursday, August 29, 2019 5:24:48 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Another difficult MR case

Are you *sure* there's no translational NCS ?

For example your first molecular replacement solution out of Phenix shows

EULER  293.6   27.7  288.7
FRAC -0.02  0.02  0.02
(that's "first molecule at origin in P1")

and

EULER  294.0   27.9  288.8
FRAC -0.37  0.02  0.02

which is essentially the same orientation, and a translation down one
crystallographic axis (a*)

And this suggests to me that either Xtriage or Phaser is missing
something here.  Does Phaser find translational NCS in its initial data
analysis ?  Unmodeled translational NCS could cause significant problems
with the molecular replacement search.

Phil Jeffrey
Princeton




On 8/29/19 11:28 AM, Napoleão wrote:
> Deal all,
> Sorry for the long post.
> I have a data set obtained from a crystal produced after incubating a
> protease with a protein which is mostly composed by an antiparallel beta
> sheet. I have tried numerous approaches to solve it, and failed.
> Molecular replacement using Phaser, and the protease or the protein as a
> template yields no solution. However, molecular replacement using only
> part of the beta sheet yields LLG=320 TFZ==28.0 (see below).
>
> The apparently good data extends to 1.9 A, as processed by XDS, and the
> space group is P1 (pointless agree). XDS info below:
>
> SPACE_GROUP_NUMBER=1
> UNIT_CELL_CONSTANTS=44.4372.2977.30  97.802  89.939 101.576
>
>   ab  ISa
>   9.647E-01  3.176E-03   18.07
>
>   RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR
> R-FACTOR COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
> LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed
> expected  Corr
>   1.90   24890   19149 23814   80.4%  58.1%
> 63.7%114820.77 82.2%63.8* 30.694 492
>  total  163756  125884146938   85.7%  10.6%
> 10.8%757443.78 15.0%99.0*-30.7615834
>
>
> Xtriage in Phenix 1.16-3549 gives me all green lights (print below),
> suggesting the data presents no twinning, no translational NCS, no ice
> rings and is not anisotropic.
> https://eur03.safelinks.protection.outlook.com/?url=http%3A%2F%2Ffullonline.org%2Fscience%2Fphenix_xtriage_green.pngdata=02%7C01%7C%7Cde40f97d75824ea6519308d72c9d6fe0%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637026927009132543sdata=RMV8abo87ybMyxaqD%2FHgOakRhGvzGgsQXzkuzC4c8HI%3Dreserved=0
>
> Molecular replacement in Phaser yields single solutions like:
>
> Solution annotation (history):
> SOLU SET  RFZ=3.0 TFZ=* PAK=0 LLG=29 RFZ=2.8 TFZ=8.8 PAK=1 LLG=310
> TFZ==27.6
>  LLG=320 TFZ==28.0
> SOLU SPAC P 1
> SOLU 6DIM ENSE ensemble1 EULER  293.6   27.7  288.7 FRAC -0.02
> 0.02  0.02 BFAC
>  -6.03
> SOLU 6DIM ENSE ensemble1 EULER  294.0   27.9  288.8 FRAC -0.37
> 0.02  0.02 BFAC
>  -6.52
> SOLU ENSEMBLE ensemble1 VRMS DELTA -0.1983 RMSD  0.49 #VRMS  0.21
>
> or partial solutions like:
>
> Partial Solution #1 annotation (history):
> SOLU SET  RFZ=3.7 TFZ=* PAK=0 LLG=32 RFZ=2.8 TFZ=13.0 PAK=0 LLG=317
> TFZ==30.2
>  LLG=331 TFZ==30.5 RFZ=2.4 TFZ=7.2 PAK=0 LLG=464 TFZ==18.5 RFZ=2.7
> TFZ=5.7 PAK=1
>  LLG=501 TFZ==6.8 LLG=509 TFZ==6.6
> SOLU SPAC P 1
> SOLU 6DIM ENSE ensemble1 EULER   85.4  153.0  138.5 FRAC -0.01 -0.00
> -0.00 BFAC
>  -12.30
> SOLU 6DIM ENSE ensemble1 EULER   86.2  153.2  139.5 FRAC -0.36 -0.01
> -0.01 BFAC
>  -9.16
> SOLU 6DIM ENSE ensemble1 EULER   83.8  152.3  135.9 FRAC -0.00  0.00
> -0.25 BFAC
>  1.52
> SOLU 6DIM ENSE ensemble1 EULER  191.2  109.1   39.3 FRAC -0.27
> -0.01  0.22 BFAC
>  10.18
> SOLU ENSEMBLE ensemble1 VRMS DELTA -0.0447 RMSD  0.49 #VRMS  0.44
>
>
> However, after 1 refinement round in Phenix_Refine (Final: r_work =
> 0.4881 r_free = 0.5009) I got densities that are part good and part bad,
> and if I delete the bad parts and refine again, the good 

Re: [ccp4bb] SEC and MALS

[David Briggs]

Natesh,

Did you use an experimentally determined dn/dc value?

Is it a glycoprotein, or does it have some other modification?

Default dn/dc values are not appropriate for glycoproteins, and will give rise 
to incorrect Mwt values.

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Natesh 
Ramanathan 
Sent: Tuesday, August 27, 2019 6:57:18 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] SEC and MALS

Dear  Friends,

Can you share your experience with examples of MALS giving lower 
molecular weight (Eg. Monomer) and  SEC giving higher molecular weight (Eg. 
Dimer),  for the same protein sample?

  If you have/know any published paper, can you please point me to that 
reference paper or send me the paper?

Many thanks.
Best regards,
Natesh


--
--
"Live Simply and do Serious Things .. "
- Dorothy Mary Crowfoot Hodgkin OM, FRS

"In Science truth always wins"
- Max Ferdinand Perutz OM FRS
--
Dr. Ramanathan Natesh
Assistant Professor,
School of Biology,
Indian Institute of Science Education and Research Thiruvananthapuram 
(IISER-TVM),
Maruthamala P.O., Vithura,
Thiruvananthapuram,  695551, Kerala, India

nat...@iisertvm.ac.in
http://www.researcherid.com/rid/C-4488-2008
ORCID: 
http://orcid.org/-0002-1145-5962
https://publons.com/author/1520837/ramanathan-natesh#profile
http://faculty.iisertvm.ac.in/natesh

Office Ph. 0091- 471-2778087



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Re: [ccp4bb] Problem in real space - please sign & invite other scientists to sign this letter

[David Briggs]

Dear Daniel,

I could not disagree with you more. As scientists it falls to us to support our 
colleagues (from whatever other discipline) when they provide evidence that has 
profound ramifications for humanity.

Would we hesitate to stand by colleagues who (for example) demonstrate the 
efficacy of vaccination, or that the earth really is an oblate spheroid?

If me being a signatory (not author, important distinction, as noted above) to 
this helps get this message across in even a small way, then it is 2 minutes of 
my time well spent.

As for whether or not scientists from one discipline can make a valuable and 
informed contributions to another, I hope Kevin Cowtan won't mind that I 
suggest you take a look at his work on climate modelling.

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs



From: Edwin Pozharski
Sent: Wednesday, August 21, 06:34
Subject: Re: [ccp4bb] Problem in real space - please sign & invite other 
scientists to sign this letter
To: CCP4BB@JISCMAIL.AC.UK


Dear Daniel,

with all due respect, I do believe that you are making several mistakes in your 
argument.

English is not my native tongue, but I suspect that there is a substantial 
difference between "author" and "signatory".  What people are asked to do here 
is essentially to sign a petition, not to become a co-author in traditional 
sense.  There were 39 signatories to the US Constitution, certainly not all of 
them are considered its authors.

Furthermore, most structural biologists are trained scientists and it is rather 
routine part of our job to evaluate research we are not exactly experts in.  I 
am not a climatologist, but I do take exception to your assertion that I am 
therefore automatically too ignorant to understand basic concepts that pertain 
to global warming and climate change.  A climatologist wouldn't instantly know 
what B-factors are, but is certainly capable of understanding the concept if 
you explain it.

Using physical science and its data to arrive at conclusions regarding 
religion, politics and economic theory (!) is not at all embarrassing.  (And 
letter in question hardly does any "preaching", certainly not about religion)

As for your demand that people stick to structural biology, may I suggest that 
your reaction to exactly one entry in ccp4bb that elicited almost zero follow 
up (until your comment) feels a bit overblown?  If you strongly disagree with 
Dr Ripple and his colleagues, that is fine, but why shouldn't people at ccp4bb 
occasionally share somewhat orthogonal information?  None of us want to see 
inappropriate content, I am just not sure why you feel that this specific post 
is something that needs to be purged.

Just to be clear - your post does create an impression that you might hold the 
opinion that, as they say, "global warming is a hoax".  Please, let's not have 
further discussion online on the specifics, but I think it might be helpful if 
you could confirm or deny this.

Cheers,

Ed.

---
"I disapprove of what you say, but I will defend to the death your right to say 
it"
Evelyn Beatrice Hall, "The Friends of Voltaire"

On Tue, Aug 20, 2019 at 9:23 PM Daniel M. Himmel, Ph. D. 
mailto:danielmhim...@gmail.com>> wrote:
Dear colleagues,

Since when does being a structural biologist make us experts in climatology,
and isn't it a breach of basic ethical practice and professionalism as 
scientists
to sign on as authors to an article for which we have neither contributed
research nor intellectual content of the manuscript?  Are we now going against
the standard to which the editorial policies of leading reputable biological
journals normally hold us as authors?  And doesn't it hurt the credibility
of a serious scientific article, its authors, and the journal in which it 
appears
if biologists with no expertise in earth science/astrophysics appear
without humility as authors to such an article?

Are you not embarrassed to put your name to an article that uses physical
sciences data as a platform for preaching about religion, politics, and economic
theory ("...social and economic justice for all...")?

Does it not upset you when someone unfamiliar with structural biology draws
firm conclusions that heavily depend on the part of a structural model that has 
high
B-factors?  So why are you unconcerned that you may be guilty of an analogous
error when, as structural biologists, you put your name to a controversial 
interpretation
of selected earth science data?  See, for example,
https://blogs.agu.org/geospace/2017/02/24/living-warm-peak-ice-ages/
 about the ways

Re: [ccp4bb] Where is M.Harding's website for metal coordination geometry

[David Briggs]

In addition to MetalPDB there is also Check My Metal.

https://csgid.org/metal_sites

HTH,

Dave




From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Sent: 07 May 2019 10:51
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Where is M.Harding's website for metal coordination 
geometry

Could CCP4 documentation include a link to both Marjories one and the MetalPDB
http://metalweb.cerm.unifi.it


Eleanor

On Tue, 7 May 2019 at 08:42, Louise Jones mailto:l...@iucr.org>> 
wrote:
Dear All

The supporting information for Marjorie's article published in Acta
Cryst. D in 2004 contains a pdf and a zip archive of the website.

The architecture of metal coordination groups in proteins, Acta Cryst.
D60, 849-859, 
https://doi.org/10.1107/S0907444904004081

Best wishes
Louise



On 07/05/2019 00:27, Min, Xiaoshan wrote:
> Hi
>
> I am trying to find out if the website metal coordination groups in
> proteins 
> >
>  is still accessible.   After some
> google search, I still can’t find it except this ccp4 newsletter
> http://www.ccp4.ac.uk/newsletters/newsletter40/15_metal_coord.html
>  .   I
> trust that someone in this list might help me  find an alternative.
>
> Thanks,
>
> Xiaoshan
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] analytical gel filtration columns

[David Briggs]

Hi Mohinder,

Have you tried SEC columns like Sephacryl S-400 or CL-4B?

They have a higher fractionation range than Superose 6

HTH,

Dave

From: CCP4 bulletin board  on behalf of Mohinder Pal 

Sent: 17 April 2019 16:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] analytical gel filtration columns

Dear all,

I would like to gel filter a multi protein complex (1.4MDa) as the final 
purification step. I have tried tandem Superose 6 columns and this complex 
elutes close to void volume as it is a very elongated molecule.


I was wondering if someone could suggest different analytical columns for 
better resolution as I plan to add more components to this existing complex.

Best wishes,

Mohinder Pal

--
"Whatever you’re meant to do, do it now. The conditions are always impossible.”
Doris Lessing
--







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Re: [ccp4bb] pseudo internal symmetry

[David Briggs]

Hi,

This doesn't answer your question directly, but I'd be tempted to reduce the 
symmetry and re-refine in a space group lacking that 2-fold axis.
I'd hope that the scaling in the lower space group would give you better stats 
as well, but maybe the differences in your case are too subtle?

I don't know what PDB rules and regs are though.

HTH,

Dave

From: CCP4 bulletin board  on behalf of Daniele de 
Sanctis 
Sent: 03 April 2019 09:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo internal symmetry

Hi all,

we have a structure with a pseudo internal symmetry along a 2-fold axis that 
sits on a 2-fold crystallographic axis. For refinement purposes we have modeled 
the parts that differ with 50% occupancy, but before depositing the structure 
we wanted to make sure that this is the best way to deal with it and it is in 
agreement with PDB standards.

Did anyone deal with similar cases in the past?

Cheers

Daniele


--

ἀρετή
---
Daniele de Sanctis
Structural Biology Group
ESRF - The European Synchrotron
Grenoble, France
Tel 33 (0)4 76 88 2869

http://www.esrf.eu/id29



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[ccp4bb]

[David Briggs]

Hi,

In Coot 0.8.9.1

Edit -> Preferences -> Others -> Fonts

HTH,

Dave

From: CCP4 bulletin board  on behalf of Sanaz 
Asadollahpour 
Sent: 12 February 2019 08:22
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb]

Dear All,
How can i change the font size of atom labels in Coot?

Regards,
SA



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Re: [ccp4bb] suggestion of crystallization optimization

[David Briggs]

Hi Liuqing,


1) When you say 8Å diffraction - did you test the crystals at room temperature?

2) Change the construct. Trim loops and termini, (re)move tags, etc.


HTH,


Dave


From: CCP4 bulletin board  on behalf of Liuqing Chen 
<519198...@163.com>
Sent: 04 June 2018 11:57:58
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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[ccp4bb] Postdoc Position available - Signalling and Structural Biology Lab, the Francis Crick Institute,

[David Briggs]

Dear all,


A four-year postdoc position is available in the Signalling and Structural 
Biology Laboratory at the Francis Crick Institute, London, UK, focusing on 
cryoEM / X-ray crystallography of Rho-dependent protein kinases assemblies.


Candidates with experience in biochemistry and/or structural biology are 
particularly encouraged to apply.


Closing date for applications is 30th May 2018.


Informal enquiries may be made to Prof Neil McDonald

(neil.mcdon...@crick.ac.uk).


Applications can be made through the Crick HR platform here: 
http://bit.ly/2rTlaoj


Kind regards,


Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT

Re: [ccp4bb] Protein Ligand interaction using SPR technique

[David Briggs]

Hi Praveen,

The BiaCore 3000 is an older model, and is not optimised for protein/small
molecule interactions. The BiaCore T-100 and T-200 have enhanced
sensitivity and are better suited small molecules (assuming you have to use
SPR).

As for chip choice, it depends an awful lot on the behaviour of your
protein. NTA imo is not great as you will always have some loss if protein
during the experiment - covalent immobilisation to CM5 is preferable.

Do you have access to ITC? You might consider this if protein is easy to
make. It's a better experiment than SPR, but is much more thirsty.

HTH,

Dave

On Fri, 31 Mar 2017, 06:04 Praveen Tripathi, <tripathipraveen2...@gmail.com>
wrote:

> Dear all,
> Sorry for off-topic question.
> I want to study protein interaction with few small molecules using SPR
> (Machine- *BIACORE 3000*).
>
> The recombinant protein expressed in bacterial expression system is of 92
> kDa (with His tag), pI= 9.
>
> *Question-*
> 1. What should be the chip of choice- *NTA chip or CM5 chip* of GE
> healthcare?
> 2. Is there any alternative available for chips and machine?
>
> Thanks in advance.
>
> Regards
> --------
> Praveen
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs
<https://about.me/david_briggs?promo=email_sig_source=email_sig_medium=email_sig_campaign=external_links>

Re: [ccp4bb] protein precipitation reg

[David Briggs]

It doesn't cost as much as HEPES, iirc.

On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <kell...@janelia.hhmi.org> wrote:

> A bit off topic, but I’ve always wondered how TRIS got so popular what
> with it’s pKa of 8.3—does anyone know?
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Roger
> Rowlett
> *Sent:* Wednesday, March 29, 2017 11:10 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] protein precipitation reg
>
>
>
> What are you dialyzing against? Your storage solution should typically be
> buffered away from the pI and contain at least a small amount of
> kosmotropic salt, e.g. NaCl. Some proteins will require additional
> stabilizing/solubilizing agents such as glycerol or reducing agents. FYI,
> Tris-Cl, pH 7.5 has very little buffer capacity (about 15% of the total
> concentration in the acid direction). We typically use Tris-Cl pH 8.0,
> which is closer to the Tris pKa and has good buffer capacity for both acid
> and base. For pH 7.5 we would typically use HEPES as the storage buffer.
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>
> On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote:
>
> Dear all,
>
> I am a PhD student doing structural studies on a few proteins from
> Mycobacterium tuberculosis. The gene encoding the proteins I work on are
> cloned into pet22b with c terminal His tag. the proteins are expressing
> well. upon purification I am getting good yield of protein but during
> dialysis, the proteins precipitate. Kindly suggest some solutions to avoid
> aggregation. pI of one protein is 9.7 and that of the other is 5.6
>
> I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM
> beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with
> 20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.
>
>
>
> Thank you
>
> Regards
>
> Akila
>
>
>
> --
>
> Akilandeswari G
>
>
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs
<https://about.me/david_briggs?promo=email_sig_source=email_sig_medium=email_sig_campaign=external_links>

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[David Briggs]

If you "multi-chicken" your 2fo-fc map in coot...

(Extensions > maps > multi-chicken)

... You might be able to pick out where different atoms are by comparing
the peak height with the near-by protein atoms (ADPs & occupancy
notwithstanding). At your resolution you will be able to see that O > N > C
and anything bigger like P or S atoms will appear enormous.

Maybe the nature of the enzyme will help you out here? Does it have a known
substrate or co-factor that might have been pulled through purification?

HTH,

Dave


On Sat, 4 Feb 2017, 21:24 Kevin Jin, <kevin...@gmail.com> wrote:

> According to your first fig. The Ser may carry a dual conformation. If
> then, the occ ration could be ~ 7:3
>
> According to your 1st, 2nd and 3rd figures, the geometry of the density
>  is tetrahedral. Can it be PO4 with the same occ (~70%) ?
>
> If there were more figures available, the geometry of the unknown density
> could be more clear.
>
> Can You try Glucose phosphate withe occ of 70%?
>
> On Sat, Feb 4, 2017 at 7:03 AM, sharifah nur hidayah syed mazlan <
> shn.hida...@gmail.com> wrote:
>
> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>
>
>
> --
> Kevin Jin
>
> Sharing knowledge each other is always very joyful..
>
> Website: http://www.jinkai.org/
>
>
-- 


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[ccp4bb] Coot crashes on starting baton mode.

[David Briggs]

Hi all,

I'm running Coot 0.8.7 (as distributed with the CCP4 package) on OSX
10.11.6.

As soon as I click to enter CA-baton mode, coot crashes (terminal output
pasted below).

All I have done prior to this is load a pdb and an mtz, build skeletons and
pick a start point.

Has anyone seen this before, and if so, how did they overcome the problem?

Thanks in advance,

Dave

Symmetry available for this molecule

INFO:: returning baton atom molecule 12

-- baton_next_ca_options

   4e-12  xyz = (-16.27,-9.644, 24.54)

   4e-13  xyz = (-21.76,-12.59,  26.3)

   4e-13  xyz = (-21.32,-13.01, 25.91)

   4e-13  xyz = (-21.21,-9.022, 20.87)

   4e-13  xyz = (-21.41,-8.895, 20.97)

   4e-13  xyz = (-20.59,-13.48, 23.92)

   4e-13  xyz = (-20.59,-13.51, 24.55)

   4e-13  xyz = (-20.68,-13.39, 25.33)

   4e-13  xyz = ( -20.6,-9.633, 20.66)

   4e-13  xyz = (-20.56,-6.015, 23.94)

   4e-13  xyz = (-19.93,-13.38, 23.31)

   4e-13  xyz = ( -16.4,-8.755,  24.6)

   4e-13  xyz = (-16.27,-9.623, 24.57)

   4e-13  xyz = (-20.62,-11.67, 21.18)

   4e-13  xyz = (-21.17,-12.03, 21.58)

   4e-14  xyz = (-20.65, -6.04, 23.86)

   4e-14  xyz = (-20.78,-6.085, 23.73)

   4e-14  xyz = ( -20.6,-10.28, 20.69)

   4e-14  xyz = (-20.67,-11.09, 20.91)

   1e-15  xyz = (-20.64,-12.43, 21.78)

   9e-16  xyz = (-19.34,-13.12, 22.81)

   4e-16  xyz = (-19.93,-12.74, 22.08)

   0  xyz = (-18.76,-10.22, 27.96)

   0  xyz = (-18.75,-9.646, 27.98)

--

Creating a molecule for Baton Atom Guide Points

!! Warning:: No symmetry available for this molecule

No Symmetry for this model

INFO:: Creating directory coot-backup

INFO:: backup file
coot-backup/Baton_Atom_Guide_Points_Tue_Dec_20_13:53:53_2016_modification_0.pdb.gz

/Applications/ccp4-7.0/bin/coot: line 284: 20534 Segmentation fault:
11  $coot_bin
"$@"

coot-exe: "/Applications/ccp4-7.0/libexec/coot-bin"

coot-version:

/Applications/ccp4-7.0/libexec/coot-bin

platform:

/usr/bin/uname

core: #f
-- 


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Re: [ccp4bb] Nitrate versus Carbonate

[David Briggs]

Assuming it wasn't clear from purification/crystallisation reagents...

Maybe try a high multiplicity anomalous dataset collected in house / at
long wavelength?

P has ~ 75% the f" of S at CuKa.

If you can figure out roughly what anomalous peak height an S atom gives
from a Cys or a Met with similar ADP to your mystery atom, you might be
able to distinguish PO4 & SO4.

You'd need decent data (well & oft recorded anomalous differences), and
usual caveats about occupancy and ADPs apply.

Dave.

On Fri, Nov 11, 2016, 17:50 Gulcin Gulten <gulcingul...@gmail.com> wrote:

> Similarly, how do you differentiate a phosphate ion than sulfate just
> based on electron density if data is not at atomic resolution?
>
> Thanks!
>
>
> On Fri, Nov 11, 2016 at 3:52 AM, Harry Powell <ha...@mrc-lmb.cam.ac.uk>
> wrote:
>
> Hi all
>
> Sticking to the first question, if you don't restrict yourself to _X-ray_
> crystallography but use your local neutron source instead, it should be
> straightforward (subject to all the normal caveats).
>
> On 10 Nov 2016, at 23:02, Tim Gruene wrote:
>
> Dear JPK,
>
> to answer your first question, at atomic resolution you would notice a
> density
> difference between N and C. At a little less resolution you might still
> measure difference in bond length.
>
> Regrds,
> Tim
>
> On Thursday, November 10, 2016 8:41:43 PM CET Keller, Jacob wrote:
>
> Dear Crystallographers,
>
>
> I don't think there is any feasible way crystallographically to distinguish
>
> between nitrate and carbonate or bicarbonate-correct? But that is not my
>
> main question.
>
>
> My main question is: given that nitrate and carbonate are both very
>
> important and also very different physiologically, and therefore they must
>
> be distinguished/recognized by cells, how is this done, since the ions are
>
> so similar in structure? Is there some aspect of these ions that differs
>
> dramatically of which I am not aware? What kind of "handles" could a
>
> protein grab onto to distinguish between nitrate and carbonate/bicarbonate?
>
>
> JPK
>
>
>
> ***
>
> Jacob Pearson Keller, PhD
>
> Research Scientist
>
> HHMI Janelia Research Campus / Looger lab
>
> Phone: (571)209-4000 x3159
>
> Email: kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org
> <kell...@janelia.hhmi.org>>
>
> ***
>
>
>
> --
> --
> Paul Scherrer Institut
> Tim Gruene
> - persoenlich -
> OFLC/102
> CH-5232 Villigen PSI
> phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A
>
>
> Harry
> --
> Dr Harry Powell
> Chairman of International Union of Crystallography Commission on
> Crystallographic Computing
> Chairman of European Crystallographic Association SIG9 (Crystallographic
> Computing)
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> --
> Post-Doctoral Research Associate
> Texas A University
> Dept. of Biochemistry and Biophysics
> Interdisciplinary Life Sciences Building
> 301 Old Main Drive, Lab 2138
> College Station, Texas 77843
>
-- 


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

[David Briggs]

Moral? Donald Trump? Really?

On Wed, Nov 9, 2016, 17:53 John Hardin <jwhcambri...@gmail.com> wrote:

> Greetings,
>
> I fully understand that I will be labeled as ignorant and that I will
> likely be hated because of my views,
> but I am thrilled that Hillary lost this election.
>
> The United States has embarked on a path of immorality,
> and I am excited by the possibility of restoring the moral character and a
> measure of integrity to this once great land (“drain the swamp”).
>
> I hate to see you all go, but perhaps it is best.
> Particularly if you are no longer able see that the pursuit of a morally
> upright society is actually a good and worthy goal.
>
> Best,
> John
>
-- 


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

[David Briggs]

In the UK we have an authoritarian nationalist government seemingly hell
bent on the destruction of our economy, so maybe give it a few years.

Maybe try Germany? Actually - wait until after their election in 2017?
Ditto for France.

On Wed, Nov 9, 2016, 06:33 Ricardo Padua <rpa...@brandeis.edu> wrote:

> Science in Brazil will struggle with the "new" government as well, so I
> wouldn't count on that.
>
> On Wed, Nov 9, 2016 at 12:56 AM, kaiser <kai...@caltech.edu> wrote:
>
> Yeah, given Europe and Canada are obvious, I think Brazil and Japan are
> actually viable alternatives if the first choices are getting too crowded.
> They do have synchrotrons and "internets".
>
>
>
> Sent from my T-Mobile 4G LTE Device
>
>
>  Original message 
> From: "William G. Scott" <wgsc...@ucsc.edu>
> Date: 11/8/16 21:37 (GMT-08:00)
> 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
>
>
>
>
> --
> Ricardo Padua
> Postdoctoral fellow HHMI
> Kern Lab
> Brandeis University
> Waltham, MA
>
> --


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Re: [ccp4bb] OT: Lunch with the FT: Martin Shkreli

[David Briggs]

Time to stock up on crystal screens and loops before he buys Hampton et al
puts 5000% markup on everything.

On Sat, Oct 29, 2016, 20:16 Veronica Fiorentino <
veronicapfiorent...@gmail.com> wrote:

> So the infamous Martin Shkreli is captivated by X-ray crystallography.
>
> https://www.ft.com/content/693ad306-9b6d-11e6-8f9b-70e3cabccfae
> QUOTE
>
> "He speaks of his love of Shakespeare and magical realist literature,
> and describes how he is captivated by science and medicine. “I’ve
> never done any art, but I think if you look at X-ray crystallography,
> it’s beautiful,” he says. "
>
> Google-cache:
>
> http://webcache.googleusercontent.com/search?q=cache:ee0nCvtuV7kJ:https://www.ft.com/content/693ad306-9b6d-11e6-8f9b-70e3cabccfae
>
> Reference: https://en.wikipedia.org/wiki/Martin_Shkreli
>
-- 


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Re: [ccp4bb] Two SGs in one droplet?

[David Briggs]

Hi Sam,

Yes. I've had 2 different crystal forms from the same drop. C2 vs C2221.
Completely different packing, despite that fact that 2 of the cell edges
were within an angstrom or two of each other.
In the above case, I soaked ligand into the crystals, one form had ligand
bound, one didn't, despite the fact that the binding site was available in
both cases.

I think you just need to proceed on a crystal-by-crystal basis and make
zero assumptions during data collection.

HTH,

Dave

On Fri, 28 Oct 2016 at 14:13 Sam Tang <samtys0...@gmail.com> wrote:

> Dear all
>
> Sorry for going a bit off-topic in this thread.
> May I seek your advice as on whether you have experienced that crystals
> being obtained from the same droplet, looking alike under microscope (rod
> shape) and in fact growing possibly from a same nuclei, give two space
> groups after indexing?
>
> I recently obtain crystals for a protein (co-crystallized with a nucleic
> acid ligand) and collected two datasets from synchrotron. Although these
> two crystals are from the same drop, the SG and unit cell dimensions are
> very different:
>
> Xtal1: C121 (156 60 105 90 111 90) (L-test, Pointless shows that there is
> no twinning), ~2.5 Angstrom
> Xtal2: P1 (53 60 79 106 105 98), ~3 Angstorm
>
> Would it be possible that the ligand changes the SG of the crystal so that
> only one of the forms contains the ligand?
>
> Any advice is appreciated and thanks a lot in advance for your input.
>
> Regards
>
> Sam Tang
> Biochemistry Programme, School of Life Sciences, CUHK
>
> --


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Re: [ccp4bb] metal electron density detection

[David Briggs]

Hi Ansuman,

Phenix.refine* will place metal ions if you ask it to:


   - *Ion placement* extends solvent picking to build elemental ions such
   as calcium and zinc. To enable this option, simply enter a list of the
   elements to search for; this will also enable solvent picking as a first
   step. This option is not recommended if you have data worse than 3.0A
   resolution, and works best with anomalous data.

 https://www.phenix-online.org/documentation/reference/refine_gui.html

*other refinement programs are available.

HTH,

Dave


On Tue, 25 Oct 2016 at 10:08 ansuman biswas <
0d48627c03ca-dmarc-requ...@jiscmail.ac.uk> wrote:

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

-- 


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Re: [ccp4bb] Ambiguous metal ion at active site.

[David Briggs]

Dilip,

Depending upon your data and the wavelength you collected it at - can you
generate an anomalous difference map? First off - if there is a significant
anomalous peak, this will exclude Magnesium at it's K-edge is at ~9.5Å

You could also compare the peak heights of your unknown metal ions with
those of identifiable anomalous scatterers, such as well defined CYS or MET
Sulphur atoms (again, depending upon the wavelength you collected at), and
obtain a rough* value for the f value of the anomalous scatterers
responsible for the peaks. You can then use the data here
http://skuld.bmsc.washington.edu/scatter/ to come up with a list of
potential metal ions.

Also, you might check distances from the centre of the peak to the
co-ordinating atoms and compare with known co-ordinating distances for
metal-ions (see http://mespeus.bch.ed.ac.uk/tanna/ for target distances).

You could also look at the co-ordination geometry. Chimera has a fantastic
tool for doing this (just ctrl-double click on the metal ion and select
co-ordination geometry).

HTH,

Dave

*rough, but better than nothing.




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On 9 July 2015 at 10:35, Dilip Kumar dku...@igib.in wrote:

 Dear All

 I have solved a structure of a metal-ion dependent exonuclease enzyme. In
 homologous structures, two or three Manganese ions are present at catalytic
 center. However, I have used 2 mM MgCl2 in protein purification buffer. I
 tried to fit both of these metal ions at catalytic center but in both cases
 it still shows green density (Sigma level ~ 7) in difference map and low
 b-factor (10) for these metal ions. For better understanding I have
 attached the screenshot of metal ions with difference map on. Please
 suggest me the possible reasons or methods to validate the presence of any
 other metal ions at catalytic center.

 Thanks in advance.

 Regards
 Dilip Kumar
 Research Associate
 Chemical and Systems Biology Unit
 CSIR-Institute of Genomics  Integrative Biology
 Delhi-110025

Re: [ccp4bb] How to fit BioSAXS shape to the Structure

[David Briggs]

Yes, SAXS has an enantiomer problem - mirror image DAMMIN/F reconstructions
will give the same fit to the raw scattering data, whereas your protein
structure will only fit one hand.

SUPCOMB can certainly deal with this problem, as detailed in
http://www.embl-hamburg.de/biosaxs/manuals/supcomb.html




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On 26 June 2015 at 12:04, Reza Khayat rkha...@ccny.cuny.edu wrote:

  Hi,

  Follow up question on SAXS. Does SAXS have an enantiomer problem like
 electron microscopy? In other words, does the calculated model possess the
 correct handedness or can both handedness of a model fit the scattering
 profile just as well?

  Best wishes,
 Reza

Reza Khayat, PhD
 Assistant Professor
 City College of New York
 85 St. Nicholas Terrace CDI 12308
 New York, NY 10031
 (212) 650-6070
  www.khayatlab.org

  On Jun 26, 2015, at 6:50 AM, David Briggs drdavidcbri...@gmail.com
 wrote:

  SASTBX has an online tool for achieving this:
 http://sastbx.als.lbl.gov/cgi-bin/superpose.html



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 David Briggs
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 On 26 June 2015 at 11:39, Ashok Nayak ashokgocrac...@gmail.com wrote:

Dear Weifei,

  It can also be done manually in Pymol by changing the mouse mode from 3
 button viewing to 3 button editing and later moving the envelope onto the
 X-ray structure or vice-versa, however the best fit can be achieved in
 SUPCOMB.

  regards
  Ashok Nayak
  CSIR-CDRI, Lucknow
  India

Re: [ccp4bb] How to fit BioSAXS shape to the Structure

[David Briggs]

SASTBX has an online tool for achieving this:
http://sastbx.als.lbl.gov/cgi-bin/superpose.html



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On 26 June 2015 at 11:39, Ashok Nayak ashokgocrac...@gmail.com wrote:

 Dear Weifei,

 It can also be done manually in Pymol by changing the mouse mode from 3
 button viewing to 3 button editing and later moving the envelope onto the
 X-ray structure or vice-versa, however the best fit can be achieved in
 SUPCOMB.

 regards
 Ashok Nayak
 CSIR-CDRI, Lucknow
 India

Re: [ccp4bb] cryo condition

[David Briggs]

If in doubt, try dragging the crystal through a 1:1 mix of Paratone-N and
Mineral oil until most or all the mother liquor from surrounding the
crystal has been removed.

There are more tips here:

http://hamptonresearch.com/tip_detail.aspx?id=99

Good luck!

D

On Mon, 4 May 2015 19:45 Faisal Tarique faisaltari...@gmail.com wrote:

 Hello everyone

 Can anybody suggest me a cryo condition for a crystal obtained in
 MIDAS screen of Molecular Dimension:

 G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

 Crystals are in beautiful cuboid shaped but all sorts of PEG
 combinations and Glycerol formulation failed to prevent it from
 cracking and dissolving.

 Has anybody faced a similar situation as mentioned above and what
 precaution was taken to prevent it from cracking or dissolving.

 Your suggestions will be of immense help

 Thanks in advance
 --
 Regards

 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] Thin plate crystals

[David Briggs]

Hi,

In my experience, additive screens (e.g Hampton's) can change crystal
morphology. You could also re-screen for new conditions either using matrix
micro seeding, or change the protein buffer. Perhaps adding a ligand or a
component from your current crystallisation conditions to your protein
stock?

HTH,

Dave

On Fri, 24 Apr 2015 04:02 Prerana G. tracy...@gmail.com wrote:

 Dear all,

 I am working on a protein (40kDa) which forms very thin plate shaped
 crystals which diffracts at very low resolution. Protein concentration that
 i have used for crystallisation is approx. 8mg/ml. I have attached the
 picture of the protein crystal.


 How can I improve upon the shape of the crystal?

Re: [ccp4bb] Strange Ancient Diffraction Pattern...

[David Briggs]

This looks like a tough cookie.

IMO you'd be crackers to persist with this crystal form. It's certainly not
going to be a piece of cake.

Dr David C Briggs PhD
http://about.me/david_briggs
On 1 Apr 2015 12:07, Keller, Jacob kell...@janelia.hhmi.org wrote:

 Can anyone index this? It's got mostly split spots and a strange diffuse
 scattering background

 JPK

 ***
 Jacob Pearson Keller, PhD
 Looger Lab/HHMI Janelia Research Campus
 19700 Helix Dr, Ashburn, VA 20147
 email: kell...@janelia.hhmi.org
 ***

Re: [ccp4bb] how to reduce protein solubility

[David Briggs]

Hi Francesca,

Try some zinc (1mM + ) in your protein buffer? Zinc tends to make a lot of
things less soluble in my hands.

Dave

On Tue, 17 Feb 2015 04:34 Mattiroli,Francesca 
francesca.mattir...@colostate.edu wrote:

  Hi all,

 I am struggling with a protein complex that is too soluble. I have reached
 about 20 mg/ml but I still observe very little precipitation (clear drops
 in 90-95% of the tested conditions). The proteins are expressed in insect
 cells and going to higher concentration is not easily achievable.
 I have tried different buffer conditions (salt concentration and pH) and I
 am testing temperatures. I am at a loss with what to try next.
 Do you think PTMs (phosphorylation, acetylation) might be causing this?
 Any input on how to decrease solubility?

 Thank you very much in advance,

 Francesca

Re: [ccp4bb] Absence of contact between layers in a crystal

[David Briggs]

Haven'tthat paper and the associated structure been retracted?

http://www.nature.com/news/2009/091222/full/462970a.html

There was a huge scandal when it was discovered that Krishna Murthy had
falsified data, including the structure you refer to.

See http://en.wikipedia.org/wiki/H.M._Krishna_Murthy

A crystallographer with a wikipedia entry for all the wrong reasons...

Dave


On Fri Feb 06 2015 at 11:02:12 AM Kerff Fred fke...@ulg.ac.be wrote:

 Hello,

 Looking at structure 2HR0 (The structure of complement C3b provides
 insights into complement activation and regulation. »,Abdul Ajees, A.,
 Gunasekaran, K.,  Volanakis, J.E.,  Narayana, S.V.,  Kotwal, G.J.,  Krishna
 Murthy, H.M.;  (2006) Nature 444: 221-225), I noticed the absence of
 contacts between layers in the crystal. Is it something that has already
 been observed in other crystals?

 Best regards,

 Fred
 -
 Frédéric Kerff
 Chercheur qualifié F.R.S.-FNRS
 Cristallographie des protéines
 Centre d'Ingénierie des Protéines
 Université de Liège
 17, Allée du 6 Août - Bat B5a
 4000 Liège (Belgium)
 Tel.: +32 (0)4 3663620
 Fax: +32 (0)4 3663772



  Le 6 févr. 2015 à 10:12, Tim Gruene t...@shelx.uni-ac.gwdg.de a écrit :
 
  -BEGIN PGP SIGNED MESSAGE-
  Hash: SHA1
 
  Dear Smith,
 
  The sca file most likely does not contain flags. pointless can read
  the sca file, standardise it to ccp4 standards and freerflag marks
  random reflections. You should use the maximum of 500 unique
  reflections or 5% of the unique reflections, whichever is larger.
 
  Best,
  Tim
 
  On 02/06/2015 09:49 AM, Smith Lee wrote:
  Dear All, I have a sca file. Will you please tell me by which
  software or how I can know whether the sca file contains R-free
  tags? If not, by which software or how I can add the R-free tags?
  And how much of the reflections I add the R-free tags? I am looking
  forward to getting your reply. Smith
 
 
  - --
  - --
  Dr Tim Gruene
  Institut fuer anorganische Chemie
  Tammannstr. 4
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[ccp4bb] X-ray crystallography facility manager position available at Imperial College London

[David Briggs]

Dear all,

A position has come available at Imperial for an X-ray facility manager
position. Responsibilities will include maintenance and training for our
local X-ray diffraction source and robotic crystallisation facilities, as
well as management of BAG time at the Diamond Light Source.

Please go to http:// http://www.imperial.ac.uk/job-applicants/
www.imperial.ac.uk http://www.imperial.ac.uk/job-applicants//
http://www.imperial.ac.uk/job-applicants/job-applicants
http://www.imperial.ac.uk/job-applicants//
http://www.imperial.ac.uk/job-applicants/

and search for job reference NS 2015 003 AB.

Regards,

David

Re: [ccp4bb] secondary structure problem...

[David Briggs]

Mintu,

Can you be a little more specific about what your problem is? Are you
worried about the rfactors (they don't seem too bad) or the lack of
secondary structure?

Regarding the secondary structure - some proteins just don't have much! As
long as the fit to density and geometry are good, I wouldn't worry too much
about this.

Hth,

Dave

On Thu, 15 Jan 2015 07:33 Mintu Chandra mi...@iiserb.ac.in wrote:

 Dear All,
 I am refining one of the structure at 3.1 A resolution with R factor
 and R free of 30 and 34 with setmet  peak data. Some of the region in
 the model look like loops without any secondary structure (helix and
 strands). Can anybody help me out ???

 Mintu.

[ccp4bb] Protein crystallography facility manager position available at Imperial College London

[David Briggs]

Dear all,

A crystallography facility manager position has become available at
Imperial College London.

To apply, p

lease go to the Imperial jobs website http://
http://www.imperial.ac.uk/job-applicants/www.imperial.ac.uk
http://www.imperial.ac.uk/job-applicants//
http://www.imperial.ac.uk/job-applicants/job-applicants
http://www.imperial.ac.uk/job-applicants//
http://www.imperial.ac.uk/job-applicants/

And search for job reference NS 2015 003 AB

Regards,

David

Re: [ccp4bb] asymmetric homotrimer in the asu

[David Briggs]

Hi Hay,

I think SAXS should be more than capable of discriminating between a 12.5
kDa monomer vs ~37.5 kDa trimer.

Lysozyme is a useful standard used in SAXS (as with most structural
biology!), and Lysozyme is only slightly larger than your proteins.

Cheers,

Dave

On Fri Dec 12 2014 at 5:13:26 PM Hay Dvir hd...@tx.technion.ac.il wrote:

 Tanner:
 Thanks, GREAT reference on asymmetric homo oligomers!
 SAXS sounds like a good idea for a bit larger particles. I'm afraid  it
 might be very difficult to get enough resolution to resolve oligomerization
 of a rather small 12.5 kDa protein like ours, but will look into it more
 closely.

 Joes:
 Thanks, we are aware of the serious problem of instability of asymmetric
 homo-oligomers which could essentially polimerize as you nicely explain and
 cite.  Indeed one of the hypothesis we aim to test if we get additional
 evidence about the the trimeric assembly concerns its known function to
 interact with another protein, which could potentially provide the
 complementary quaternary stability. Interface mutational analysis sounds
 like a good approach to take in such cases.

 Thanks again an very best,
 Hay


 On Dec 12, 2014, at 5:39 PM, Tanner, John J. wrote:

  Two thoughts on asymmetric oligomers.

  1.  Here is a recent survey of asymmetric homodimers in the PDB.  I know
 you are looking for trimers, but at least this provides a precedent for
 asymmetric oligomers.

  Swapna LS, Srikeerthana K, Srinivasan N. Extent of structural asymmetry
 in
 homodimeric proteins: prevalence and relevance. PLoS One.
 2012;7(5):e36688. doi:
 10.1371/journal.pone.0036688. Epub 2012 May 22. PubMed PMID: 22629324;
 PubMed
 Central PMCID: PMC3358323.

  2. SAXS is a very effective method for determining whether assemblies
 observed in crystals are stable in solution, since it provides not only the
 oligomeric state, but also the quaternary structure.  The oligomeric state
 can be obtained from the volume of correlation (1) and Porod-Debye analysis
 (2).  The quaternary structure can be deduced by comparing the experimental
 SAXS curve to theoretical curves calculated from oligomer models identified
 by PISA or from manual inspection.   The FoXS server and CRYSOL are good
 tools for this. FoXS also allows ensembles of oligomers (MES) to be used in
 fitting the data (e.g. mixture of monomer + dimer). I believe ATSAS also
 has an ensemble program, but the name escapes me at this time.  We have
 used this approach to show that assemblies that are predicted to be stable
 by PISA are not found in solution (3 and unpublished results).

  1: Rambo RP, Tainer JA. Accurate assessment of mass, models and
 resolution by
 small-angle scattering. Nature. 2013 Apr 25;496(7446):477-81. doi:
 10.1038/nature12070. PubMed PMID: 23619693; PubMed Central PMCID:
 PMC3714217.

  2: Rambo RP, Tainer JA. Characterizing flexible and intrinsically
 unstructured
 biological macromolecules by SAS using the Porod-Debye law. Biopolymers.
 2011
 Aug;95(8):559-71. doi: 10.1002/bip.21638. Epub 2011 Apr 20. PubMed PMID:
 21509745; PubMed Central PMCID: PMC3103662.

  3: Luo M, Singh RK, Tanner JJ. Structural determinants of
 oligomerization of
 δ(1)-pyrroline-5-carboxylate dehydrogenase: identification of a
 hexamerization
 hot spot. J Mol Biol. 2013 Sep 9;425(17):3106-20. doi:
 10.1016/j.jmb.2013.05.027.
 Epub 2013 Jun 7. PubMed PMID: 23747974; PubMed Central PMCID: PMC3743950.

  On Dec 12, 2014, at 4:56 AM, Jose Manuel Duarte wrote:

  Dear Hay

 Your post prompted me to respond, since I think the issue of symmetry is
 extremely important.

 I would like to reinstate here what should be obvious to everyone: a
 stable asymmetric assembly of proteins in solution is essentially
 impossible (or at most very very unlikely), purely because of topological
 reasons.

 This is beautifully explained in a classic paper now 50 years old: Monod,
 Wyman, Changeux (1965) On the Nature of Allosteric Transitions: A
 Plausible Model. The reasoning there is that a homomeric protein in
 solution can only associate in 2 ways: isologous (binding with same surface
 patches in both monomers, necessarily through a 2-fold axis) or
 heterologous (binding through different surface patches in both monomers).
 The isologous case is clearly symmetric (C2). Whilst in the heterologous
 case  the monomers can either assemble infinitely or form a closed
 symmetry. The conclusion that follows is that stable homo-oligomers can
 only be symmetric.

 I especially like this paragraph:

 On the basis of these considerations, it is reasonable to assume that, if
 an oligomeric protein possesses a wide range of stability, it consists of a
 closed structure where all the protomers use the same binding sets; which
 implies, as we have just seen, that the molecule should possess at least
 one axis of symmetry.

 The paper really explains it a lot better than me, it can be found here:
 

Re: [ccp4bb] protein forming dimers

[David Briggs]

Well,

If it is an SDS-PAGE gel, then the act of boiling the protein in detergent
generally denatures the protein and breaks apart non-covalent interactions.

To assess dimerisation, a non-denaturing technique might be more helpful.
Native-PAGE, Size Exlusion Chromatography, Analytical Ultracentrifugation...

SEC with a suitable column for your predicted protein size would probably
be a first thing to try.

If you have SEC with in-line MALLS, even better.

HTH,

Dave



[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs

On 20 October 2014 12:00, arun kumar arungreenlo...@gmail.com wrote:

 Hello everyone,

 I would like to take a advice from you all.  I have protein like 200 KDa
 when i lode in a gel it shows band on 200KDa, Is that this protein gets
 dimer or anything else is happening.
 I would appreciate for some open discussion.

 --

 * Regards,**   Arun *

Re: [ccp4bb] 3 letter code

[David Briggs]

When I google 3 letter code for p nitrophenyl phosphate  the second site
listed is:

http://www.ebi.ac.uk/pdbe-srv/pdbechem/chemicalCompound/complete/4NP

Which looks like it might contain the answer you seek.

HTH,

Dave


[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs

On 2 October 2014 11:50, Faisal Tarique faisaltari...@gmail.com wrote:


 Hello everyone

 I request you to please tell me the 3 letter code for p nitrophenyl
 phosphate..
 --
 Regards

 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] is small dialysis tubes reusable??

[David Briggs]

Hi Manjula,

I imagine that they could be reusable, if you stored them in a buffer with
some sort of preservative like sodium azide (NB Toxic) or maybe a
bacteriostatic agent like EDTA or something.

However, when you use them, a small amount of protein is going to remain in
the cassette after each use, so I would only use each cassette for a
specific protein to prevent cross-contamination. Additionally, I'd worry
about protein from old preps falling apart/denaturing and then
contaminating future fresh preps of protein. I guess it depends at what
step during purification you are using them.

HTH,

Dave



[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs

On 26 September 2014 15:51, Manjula Ramu manjula@gmail.com wrote:

 Hi all,

 I use Slide-A-Lyzer MINI Dialysis Devices from Thermo for dialysis of
 small protein volumes. Are they reusable? if so how can we store them??

 Thanks and Regards,
 Manjula R
 Research Scholar
 Department of Biophysics
 National Institute of Mental Health and Neurosciences
 Bengaluru-29, Karnataka
 INDIA
 E-mail: manjula@gmail.com
 Mobile no:+91-9538553356
 http://www.nimhans.kar.nic.in/
 https://www.nimhans.kar.nic.in/

Re: [ccp4bb] off topic

[David Briggs]

Dear Careina,

Following on from what Herman said, if you have a structure you can use the
propKa server

http://propka.ki.ku.dk/

to predict pKas for amino acids in the local environment as found in your
structure.

Perhaps some of the propKa literature might also be helpful?

HTH,

Dave


[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs


On 13 August 2014 12:40, Careina Edgooms 
02531c126adf-dmarc-requ...@jiscmail.ac.uk wrote:

 Sorry for off topic question, just wondering if anyone has come across a
 study that shows the residue pka of certain amino acids is different in
 vitro compared to in vivo?

 Best
 Careina

Re: [ccp4bb] citation for shelxc and hkl2000

[David Briggs]

HKL2000

http://lmgtfy.com/?q=hkl2000+referencel=1

ShelxC

http://lmgtfy.com/?q=ShelxC+referencel=1

HTH,

Dave


[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs


On 5 August 2014 09:47, Faisal Tarique faisaltari...@gmail.com wrote:

 Dear all

 i request you to please tell me the name of paper required for citing
 shelxC and hkl2000..

 Thanx in advance

 --
 Regards

 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] ITC with heterogeneous protein

[David Briggs]

Hi Sajid,

*Assuming* you have one site per monomer (rather than, say, one site per
dimer), and *assuming* each binding event is completely independent ( I.e
no co-operativity), you might just get away with running the experiment
with the heterogeneous material.

However, you might not be able to confidently make these assumptions, so
imho it would be preferable to separate the monomer and dimer by SEC prior
to ITC. If this is not possible, then pay close attention to the fit when
you run the heterogeneous experiment. Poor fit to a one site model may
indicate that these assumptions are invalid. Can you obtain stoichiometry
information from a different technique? This might be very helpful.

Hth,

Dave

Dr David C Briggs PhD
http://about.me/david_briggs
On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.in wrote:

 Dear All,

 This is an off-topic question. I have protein solution of heterogeneous
 (contains both monomer and dimer). I want to perform ITC with this protein.
 I doubt whether this heterogeneity will interfere the binding study.

 Any advice please.

 Thank you

 Sajid

Re: [ccp4bb] Salt!

[David Briggs]

Hi Catherine,

What buffer  salt do you have your protein in when you set up screens -
maybe this is leading to your salt crystal issues? How long does it take
for the salt crystals to appear?

Have you tried setting up trays at different temperatures? Microbatch
crystallisation under oil?

Failing that - try altering the construct - add / remove a few amino acids
at each end, truncate any predicted loops...

HTH,

Dave


[image: David Briggs on about.me]

David Briggs
about.me/david_briggs
  http://about.me/david_briggs


On 16 July 2014 15:55, Bishop, Catherine E. cati...@ou.edu wrote:

  I have been attempting to obtain a protein crystal of my protein for
 just over 2 years at this point.  We have attempted removing the tag,
 binding the protein to its ligand, removing as much salt as possible
 (crashes at at too low a salt concentration)--this lead us to try reverse
 vapor diffusion--seeding, additive trays, optimization around the
 conditions an ortholog of one of the domains crystallized well in, and a
 plethora of other methods. We do get crystals, but they all diffract as
 salt. Most of these salts are found in wells containing a cation (ie.
 lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride,
 etc.). When crystals are too small to shoot, I do set up optimized trays;
 however, if I get larger crystals, they diffract as salt as well. Most of
 these set ups, at this point, have also been conducted in hands other than
 mine and at other facilities known for their successful crystallization of
 proteins.

 Has anyone else run into this problem and seen light at the other side??
 Any suggestions are appreciated.

Re: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein

[David Briggs]

Hi Lionel,

A few musings/suggestions

If they are bound *inside* the protein, this suggests that the
phospholipids might be very tightly bound.

Do you have an affinity tag on your protein (e.g. His tag)? Perhaps you
might immobilise the protein on a suitable resin and wash with copious
amounts of detergent, then elute as you would normally? One can remove LPS
from a prep this way using Triton X-114, perhaps this might work for you?

I do agree that HIC might be useful as well.

Failing that, and given the fact that these PLs are bound *in* perhaps the
only way to completely remove them from a sample might be to denature the
protein (Urea or Guanidine?), remove the lipids (HIC, Lipidex,
immobilisation/washing as above) and then hope that the protein can be
refolded without the PL present? Perhaps the PLs are an essential
structural component?

Hope this is helpful! Good luck!

Dave

Dr David C Briggs PhD
http://about.me/david_briggs
On 26 Jun 2014 17:16, Lionel lionel.costen...@gmail.com wrote:

 Dear all,

 I would like to remove two phospholipids bound to (actually into) a
 cytoplasmic protein. The protein was expressed in E. coli and the crystal
 structure revealed the presence of two co-purified phospholipids (most
 probably DPPE).

 A web search gave me three methods to remove bound lipids:

 - 1-butanol liquid–liquid extraction
 - Lipidex 1000, VI column at 37°C
 - HIC (aka Hydrophobic Interaction Chromatography) on a Phenyl HP column
 in presence of 1M ammonium sulphate

 All are described in Velkov 2008 (
 http://www.sciencedirect.com/science/article/pii/S1570023208002390).

 I assume these delipidation methods would also work for phospholipid; and
 I am more tempting by the last one, the milder one.

 I would appreciate any comment or practical advice.

 Best regards,
 Lionel

Re: [ccp4bb] Www

[David Briggs]

Solubility issues though?

Dr David C Briggs PhD
http://about.me/david_briggs
On 13 Jun 2014 11:45, Ian Clifton ian.clif...@chem.ox.ac.uk wrote:

 avinash singh avns.si...@gmail.com writes:

  wwdwwwy

 Wow, imagine the fluorescence from that…

 --
 Ian ◎

Re: [ccp4bb] PDB passes 100,000 structure milestone

[David Briggs]

So, a bit like Fold-it but with actual data? :-D

Dr David C Briggs PhD
http://about.me/david_briggs
On 16 May 2014 06:19, Pavel Afonine pafon...@gmail.com wrote:


 What about structures that are obviously wrong based on inspection of the
 density, but no one has bothered to challenge yet?  The TWILIGHT database
 helps some, if that counts, but it doesn't catch everything.


 How about this utopia.. Imagine PDB has two versions: one is the original
 data and model deposited as is, and never ever changed no matter what.
 Another version is a curated one obtained in a quest-like way: anyone can
 take an original entry, improve it and deposit (into the curated version)
 with his/her name tag on it. And of course anyone can take and update that
 improved entry and re-deposit it again with his/her name tag, etc. If
 desired one could keep track of all the revisions, like in svn or so.
 Sounds like a sport with an element of public service that might yield
 crowd-perfected models -:) !

 Pavel

Re: [ccp4bb] metal ion coordination

[David Briggs]

Hi Faisal,

Take a look at Marjorie Harding's website

http://tanna.bch.ed.ac.uk

Loads of information there.

Hth,

Dave

Dr David C Briggs PhD
http://about.me/david_briggs
On 17 Apr 2014 21:16, Faisal Tarique faisaltari...@gmail.com wrote:

 Dear all

 Can anybody please explain what is the classical metal ion coordination
 for Mg2+, Ca+ and Na+ with Oxygen atom and the average distance with these
 metal ions..does the distance vary with the type of metal ion and its
 coordination with oxygen atom..what is the best way to identify the correct
 metal ion in the electron density in the vicinity of negatively charged
 molecule mostly oxygen containing molecule..In one of my paper the reviewer
 has asked me to check whether the octahedrally coordinated Mg+ is  Ca+
 ion..and similarly raised doubt about the identity of the Na+ ion as
 well..his argument was based on metal ion to oxygen distance..I am
 attaching the figure with this mail..i request you to please shed some
 light on this area and help me in clearing some doubts regarding this.

 --
 Regards

 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] Help in getting source of HOLE programme

[David Briggs]

AFAIK, Dr Smart now works at Global phasing.

https://www.globalphasing.com/people/osmart/

HTH,

Dave

Dr David C Briggs PhD
http://about.me/david_briggs
On 12 Mar 2014 19:46, Appu kumar appu.kum...@gmail.com wrote:

 Hello everyone,
 I request all of you to please help in getting
 the source of hole programme which is required to visualize the cavity
 running through the channel in protein structures. This programme is
 written by Dr. Oliver Smart. Despite of exhaustive search on various place
 on web, I am unable to find the source of this programme and therefor
 request to you, if any one has the source or link to get this progammes
 please provide me. I will be highly thank full for the help.

 Thank you
 Appu

Re: [ccp4bb] Large Conformational Change Upon Binding Ligand...

[David Briggs]

Integrin-ligand binding?

See for example:  http://humphrieslab.info/integrins.html

Dr David C Briggs PhD
http://about.me/david_briggs
On 27 Feb 2014 19:43, Keller, Jacob kell...@janelia.hhmi.org wrote:

 Dear Crystallographers,

 Does anyone know of good examples of large, reversible conformational
 changes occurring between ligand-free and -bound states? Could also be a
 non-relevant molecule binding, like sulfate or something inducing dubiously
 -relevant changes. I already know of the calmodulin and periplasmic binding
 protein families, but does anyone know of others out there?

 All the best,

 Jacob Keller

 ***
 Jacob Pearson Keller, PhD
 Looger Lab/HHMI Janelia Farms Research Campus
 19700 Helix Dr, Ashburn, VA 20147
 email: kell...@janelia.hhmi.org
 ***

Re: [ccp4bb] Protein behaves as dimers in 2M NaCl and as high oligomers with 300mM NaCl

[David Briggs]

Hi Tom,

Are your protein or related proteins known to dimerise?

Is the dimer perhaps the natural state, and are the high oligomers
non-specific aggregation?

That would be my first guess, knowing nothing about the protein you are
working on.

If you are absolutely certain that the protein should be a monomer, do you
have any free cys residues you could mutate or reduce with TCEP to split a
disulphide linked dimer?

Just some thoughts,

Dave
On 27 Feb 2014 07:57, Tom Wong wangnan4...@yahoo.co.jp wrote:

 Hello everyone!

 I have run into a problem in a 55kD recombinant human protein
 crystallization (expressed in E.Coil). The purity is pretty good. However,
 it behaves as high oligomers in the buffer with 300mM NaCl and behaves as
 dimers with a little high oligomers in the buffer with 2000mM (2M) NaCl.
 I have already performed several screenings and tried several types of
 buffer, salt or different pHs, etc. Only very small crystals could be
 detected in the dimer drops. High oligomers seem could not be crystallized
 under various of conditions.

 Has anyone ever met the same problem? Could anyone give me some
 suggestions?

 Thanks very much!


 Cheers


 ---
 Tom Wong
 Structural Lab
 ---

Re: [ccp4bb] KD of dimerization, off topic

[David Briggs]

Hi Careina,

I'm not sure you can assume that the ratio of monomer and dimer will stay
constant through the column - as you say, the protein is diluted during the
run, the ratio will change, unless you have a super tight dimer - which
clearly you do not. Also, as the mass and the molar extinction coefficient
will both double in the dimer, the relationship between absorbance and
concentration will be unchanged.

Typically, such these sorts of questions are answered (at least me) by
equilibrium analytical centrifugation.

Hth,

Dave
On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.com wrote:

 Dear CCP4 board

 I have a protein that exists in equilibrium between monomer and dimer and
 I'm trying to calculate KD using size exclusion. The problem is that the
 column dilutes my sample so that if I put 20 uM on to the column, I only
 recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting
 confused as to how to plot my KDs. Do I not regard my initial
 concentrations at all and work only with the final concentrations that come
 off the column?
 I would plot [monomer] squared vs [dimer] and I will assume that the ratio
 of monomer to dimer will stay constant as the protein passes through the
 column. (also I would calculate [dimer] using 2x monomer extinction
 coefficient)

 Does this seem a reasonable way to calculate KDs and reasonable argument?
 Also I am looking for good references for calculating Kds when dealing with
 dimerization
 Thanks and sorry for off topic question
 Careina

[ccp4bb] Isolation of protein-protein complexes.

[David Briggs]

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple groups, and
by multiple techniques - namely ELISA, SPR and DPI.

The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, and as
a first step I attempted to purify the complex by mixing the two proteins
(same protein preps and same buffers as the SPR experiment) and then
running them down a gel filtration column (Superose 6 - predicted size of
the complex is ~500kDa).

Somewhat irritatingly the two proteins separate beautifully on the column
into two distinct peaks. There is no trace of complex formation when the
peaks are analysed by SDS-PAGE.

As far as I am aware, two proteins that interact this strongly should
remain associated during gel filtration, and I was wondering if anyone else
has encountered anything similar in the past, and if they managed to
resolve the problem, how they went about it?

Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs

Re: [ccp4bb] Isolation of protein-protein complexes.

[David Briggs]

Hi all,

Thanks for the incredibly fast and helpful responses, both on and off list.

To provide some further information. I have tried the SEC with several
buffers, including buffers identical to the SPR and DPI experiments.

To-date all SEC and SPR experiments have been conducted at room temperature
- so suggestions to repeat SEC in the cold room are useful and might yield
complex.

I am also fortunate enough that only one protein is (His) tagged, so
co-purification on an IMAC resin is also something that I shall try.

Regarding the SPR, many have highlighted that the most important parameter
vis complex stability is Koff - I don't have those numbers right now (on
the train) but from memory the off rate is slow, and the interaction does
not appear to be the described fast-on/fast-off non-specific interaction.
I always run a blank for all my SPR experiments and no non-specific
interactions with the blank surface were seen

Thanks once again everyone,

Dave
On 21 Jan 2014 15:51, David Briggs drdavidcbri...@gmail.com wrote:

 Dear all,

 sorry for the slightly off topic post,

 I have 2 proteins that have been shown to interact, by multiple groups,
 and by multiple techniques - namely ELISA, SPR and DPI.

 The Kd of the interaction as determined by SPR is on the order of 1 nM.

 I would very much like to crystallise this protein-protein complex, and as
 a first step I attempted to purify the complex by mixing the two proteins
 (same protein preps and same buffers as the SPR experiment) and then
 running them down a gel filtration column (Superose 6 - predicted size of
 the complex is ~500kDa).

 Somewhat irritatingly the two proteins separate beautifully on the column
 into two distinct peaks. There is no trace of complex formation when the
 peaks are analysed by SDS-PAGE.

 As far as I am aware, two proteins that interact this strongly should
 remain associated during gel filtration, and I was wondering if anyone else
 has encountered anything similar in the past, and if they managed to
 resolve the problem, how they went about it?

 Cheers in advance,

 Dave
 
 David C. Briggs PhD
 http://about.me/david_briggs

Re: [ccp4bb] Best compounds for heavy atom soaks

[David Briggs]

Hi Rhys,

It's worth paying close attention to your crystallisation conditions as
well - some heavy atom compounds will not be at all soluble in very
alkaline (they'll form insoluble hydroxides) or phosphate/sulphate
containing mother liquors.

A very low pH may reduce the binding efficiency of some heavy atom
compounds as well - many heavy atom compounds rely on having deprotonated
side chains to interact with - if you are at pH 5 or lower, everything save
your Asps and Glus are likely to be protonated and you'll perhaps have
better luck with Lanthanides (or Uranyl salts).

Good luck!

Dave


David C. Briggs PhD
http://about.me/david_briggs


On 16 January 2014 09:53, RHYS GRINTER r.grinte...@research.gla.ac.ukwrote:

 Hi All,

 A truly herculean response! Thanks everyone, I will process all of the
 information and come up with a strategy.

 Rhys

Re: [ccp4bb] largest protein crystal ever grown?

[David Briggs]

Following on from John's comment, when I did my PhD at Birkbeck in the
early 2000s, one of David Moss's other PhD students (John Bond) grew some
gigantic (1cm edges) crystals of things like HEWL  Myoglobin, which he
then (somewhat perversely) crushed to load into capillaries for powder
diffraction analysis.

IIRC, John fashioned a vapour diffusion setup from a 2l Ice cream tub, and
used a watch glass to support the sitting drop, which was centimetres
across. Truly bucket crystallography.

Regards,

Dave


David C. Briggs PhD
http://about.me/david_briggs


On 24 October 2013 17:08, Jrh jrhelliw...@gmail.com wrote:

 Dear Tobias,
 Take a look at http://dx.doi.org/10.1107/S0108767389012912
 The ribonuclease crystal I used to measure the speed of sound, using laser
 generated ultrasound, was of volume 129 mm3 ie 7.7x6.2x2.7 mm . David Moss
 of Birkbeck College provided it.
 Best wishes,
 John

 Prof John R Helliwell DSc FInstP CPhys FRSC CChem F Soc Biol.
 Chair School of Chemistry, University of Manchester, Athena Swan Team.
 http://www.chemistry.manchester.ac.uk/aboutus/athena/index.html



 On 24 Oct 2013, at 16:33, Tobias Beck tobiasb...@gmail.com wrote:

 Dear all,

 I was just wondering if anyone has some information or references about
 the dimensions of the largest protein crystal ever grown? I am aware that
 for neutron protein crystallography one usually needs crystals with mm
 dimensions. I have found some information on crystallization under
 micro-gravity and how this can enlarge the crystal size. However, I would
 rather be interested in the dimensions for crystals obtained from a regular
 lab setup.

 Thanks, Tobias.

 --
 ___

 Dr. Tobias Beck
 ETH Zurich
 Laboratory of Organic Chemistry
 Wolfgang-Pauli-Str. 10, HCI F 322
 8093 Zurich, Switzerland
 phone:   +41 44 632 68 65
 fax:+41 44 632 14 86
 web:  http://www.protein.ethz.ch/people/tobias
 ___

Re: [ccp4bb] Structural transmission of signal across a membrane

[David Briggs]

Integrins?

Loads of structures and models .
On 24 Sep 2013 16:49, Edward A. Berry ber...@upstate.edu wrote:

 Gloria Borgstahl wrote:

 Hello ccp4ers,
 I am helping a colleague develop a grant and have a vague recollection of
 structures of
 transmembrane protein receptors that signal across the membrane.  Can
 anyone send me
 specific examples?
 Many thanks, G



 I think cytoplasmic and periplasmic domains of the bacterial aspartate
 chemotaxis receptor (tar) were solved and a model of the whole protein was
 constructed.

Re: [ccp4bb] popular piece on X-ray crystallography

[David Briggs]

Following on from that - readers may be interested in Stephen Curry's
post in the Guardian, regarding the Crystallography exhibit at the
London Science Museum.

http://www.guardian.co.uk/science/occams-corner/2013/apr/19/1

regards,

Dave


David C. Briggs PhD
http://about.me/david_briggs


On 19 April 2013 09:44, Peter Artymiuk p.artym...@sheffield.ac.uk wrote:


 Dear all

 In Britain there is a free newspaper that you can pick up on buses called the 
 Metro. My colleague Geoff Ford pointed out this short feature on the history 
 X-ray crystallography in last Monday's Metro newspaper. I think it's rather 
 good.

 http://www.cosmonline.co.uk/blog/2013/04/14/conquering-realm-invisible

 best wishes
 Pete



 Prof Peter Artymiuk
 Krebs Institute
 Department of Molecular Biology  Biotechnology
 University of Sheffield
 Sheffield
 S10 2TN
 ENGLAND

Re: [ccp4bb] PDB crawler

[David Briggs]

Hi Sebastiano,

Phyre Alarm would do something similar to what you suggest.

http://www.sbg.bio.ic.ac.uk/phyre2/html/help.cgi?id=help/phyrealarm

HTH,

Dave

David C. Briggs PhD
http://about.me/david_briggs


On 20 March 2013 12:40, Sebastiano Pasqualato
sebastiano.pasqual...@gmail.com wrote:

 Hi all,
 just wondering if anybody is aware of a service similar to PubCrawler
 (http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and
 unreleased.
 Basically a weekly email that would alert you of freshly deposited
 structures that match some keywords.
 Thanks in advance,
 ciao
 Sebastiano

 --
 Sebastiano Pasqualato, PhD
 Crystallography Unit
 Department of Experimental Oncology
 European Institute of Oncology
 IFOM-IEO Campus
 via Adamello, 16
 20139 - Milano
 Italy

 tel +39 02 9437 5167
 fax +39 02 9437 5990

Re: [ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition

[David Briggs]

Hi Shankar,

I don't claim to be an expert, but I've used ethylene glycol as a
cryoprotectant many times, and 30% (v/v) would probably be sufficient for
proper freezing without any further addition.

Did you try and shoot a crystal at room temperature?

Hth,
Dave
On Jan 2, 2013 5:59 PM, Sankaranarayanan Srinivasan texs...@gmail.com
wrote:

 Dear all,

 A very happy new year to all.

 I would appreciate some expert advice on optimizing a crystallization
 condition in which the initial hits were obtained with ethylene glycol as
 the main precipitant. Here is the summary of things tried.

 We have a protein, size (31Kda) and the starting protein buffer is 0.1M
 Tris pH7.5, 0.1M NaCl, 10% glycerol.
 The initial crystal hit was obtained from the emerald cryo kit condition
 that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals
 were tiny (10-20um). A crystallization matrix to obtain better crystals
 by varying the imidazole pH and ethylene glycol concentrations was tried
 from which the best condition obtained was 0.1M imidazole pH 6.5 , 30%
 (v/v) ethylene glycol. The crystals were slightly bigger 50um.
 On trying the additive screen, bigger crystals (200um) were obtained, but
 putting them under the x-ray beam with direct freezing did not yield any
 diffraction spots.
 Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture
 also yielded similar results.
 Low resolution spots near the beam stop were also not seen. Similarly
 spots indicative of salt was also not seen. It just had hazy ice rings kind
 of stuff. (The beam was definitely on the crystal)
 To check if what we have was salt, a control condition with no protein was
 tried. Also the crystals were run on a gel after thorough washing. Both
 these tests, show that they are definitely protein crystals and not salt.
 Seeding also did not yield any improved crystals.
 I was suggested using di-ethylene glycol, propane diol as alternatives.
 I would greatly appreciate if you can give your opinion on using other
 di-alcohols as precipitants or other ways to improve these crystals.
 I tried searching the PDB to see if someone had actually used ethylene
 glycol as a precipitant, most of them were used as a cryo condition than
 actually as a precipitant.

 Thank you very much in advance.

 Regards
 Shankar Srinivasan

Re: [ccp4bb] Izit dye stained crystal

[David Briggs]

Hi Sarathy,

Are you sure your anomalous scatterers are not the Ca in the
crystallisation buffer? These would also bind to the acidic residues in
your protein, and Ca has a greater f'' (~2.5e compared to less than 1.5e
for S) at the wavelength you used.

Just another possibility - unless you already solved the structures and see
density for the izit in your density, of course!

Cheers,

Dave


David C. Briggs PhD
http://about.me/david_briggs


On 30 November 2012 21:19, Sarathy Karunan Partha sarathyus...@gmail.comwrote:

 Dear all,



 We did some Izit dye staining to test our crystal (salt or protein) and we
 observed that the crystal didn’t take up the dye well. But, showed nice
 protein diffraction (home source KCr 2.2909 A) and we collected a dataset
 (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The
 data collection statistics looks great and most interestingly we saw some
 anomalous signal for this data (see attached XSCALE.LP).



 This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5.
 Izit dye is basically methylene blue which contains a sulfur atom
 (phenothiazine ring) and also has some basic dimethylamio groups. Our
 protein has many acidic residues that could enhance binding of this basic
 dye.We think the anomalous signal could be from this dye and the heavy atom
 search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms.


 Did anyone come across similar situation with using this dye and also
 welcome any suggestions about using this data for S-SAD phasing.


 Thanks,
 Sarathy

Re: [ccp4bb] ResearchGate?

[David Briggs]

Hello Adrian,

I use Research Gate and there are a few occasions where I have found
it useful, particularly the questions feature.

HTH,

Dave

David C. Briggs PhD
http://about.me/david_briggs


On 30 October 2012 16:13, Adrian Goldman adrian.gold...@helsinki.fi wrote:
 Hi,

 At the risk of starting another series of rants, and somewhat off-topic, is
 anyone actively using ResearchGate?  It is bombarding me with email
 messages, but I am uncertain as to whether people are really using it or
 whether it is just scientific spam.

 Adrian Goldman


 Adrian Goldman

 Institute of Biotechnology, University of Helsinki, Finland

Re: [ccp4bb] Mercury phenylglyoxal

[David Briggs]

Hi Vijay,

Not an answer to your question, but I tried and failed to source any
about 10 years ago. I had a chemist friend try to make some following
the a protocol we found on the web - which didn't work.

The consensus on the ccp4bb back then was that it was a bit of
mythical beast, and I never progressed beyond this.
http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00958.html

We made some Iodo-phenylglyoxal though... never got it to work (as a
derivative) in action.

Cheers,

Dave


David C. Briggs PhD
http://about.me/david_briggs


On 11 July 2012 13:37, Vijay Reddy red...@scripps.edu wrote:
 Hello all,

 Could someone please advise me on where to purchase Mercury Phenylglyoxal
 from?

 Many thanks,
 Vijay

 
 Vijay S. Reddy, Ph.D.
 Associate Professor
 Department of Molecular Biology, TPC6
 The Scripps Research Institute,
 10550 North Torrey Pines Road,
 La Jolla, CA 92037

 E-mail: red...@scripps.edu
 WWW:http://www.scripps.edu/~reddyv
 VIPERdb:http://viperdb.scripps.edu

 Phone:(858) 784-8191
 FAX:(858) 784-8896
 

Re: [ccp4bb] Do my SAXS data agree with the crystal structure?

[David Briggs]

Dear Xun,

Regarding your monomer vs dimer, theoretical vs observed crysol plots
- yes - they are significantly different.

If you focus at the very lowest q part of the curve - the deviation
there in your monomer plots indicate that there is a significant size
difference between your PX monomer and your SAXS data - the PX dimer
is a much better fit at low q.

This should be enough to demonstrate to a reviewer that the dimer you
see in PX is also present in solution.

Other experiments that could support this are SEC-MALLS or perhaps AUC.

HTH,

Dave

David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB



On 17 June 2012 06:11, Xun Lu xlun...@gmail.com wrote:
 Drs.Caldwell, Briggs, and Gupta,

    Thank you very much for the advices.   I regret that I didn't show any
 figure in the earlier post.  Here I've attached a figure showing the data
 quality and some fittings.
    Data look OK, right? This question may sound silly, but I just want to
 make sure.
    As I said in the earlier post, I tried Crysol.  I used the crystal
 structure (dimer+DNA) as the model, and the fitting was OK, right?  In fact,
 I also tried monomer+DNA as the model (I simply deleted one monomer from the
 PDB file).  This kind of comparison may be meaningless, but I was just
 curious.  I am wondering how people judge whether the fit is good or not.


     Another question, I tried to generate an envelope from SAXS data using
 Gasbor and Dammin (people say Dammin is better at protein-DNA complex,
 although it still uses the same bead for both DNA and protein?).  The
 generated envelope was nothing like my crystal structure.  As people have
 pointed out, protein and DNA scatter differently.  SANS is the way to go.
  So I should give up on modeling SAXS data?  I've almost given up, because
 anyways I have the crystal structure, and SAXS is only a small part of this
 paper.



 Thanks,

 Xun



 On Sat, Jun 16, 2012 at 6:36 PM, Kushol Gupta kushol.gu...@gmail.com
 wrote:

 Two cents -



 A good deal of caution must be exercised when working with composite
 particles such as a protein-DNA complex in SAXS because of the contrast
 problem.  Simply, protein and DNA scatter differently in x-rays, with a bias
 towards the DNA component.  As a result, experimental Rgs could be slightly
 deflated versus what their true values would be at infinite contrast.  Mass
 estimation by I(0) analysis with a protein standard of known mass and
 concentration is not really valid because the contrast terms are different.
 Because the particle is heterogeneous in composition and distribution, shape
 reconstruction from SAXS alone, which assumes homogeneity, can also be
 misleading (although in practice it is still reasonably instructive).  It is
 for these reasons that SANS and the contrast variation approach can be
 extremely useful.



 With those caveats, the strategy you describe - comparison of experimental
 and theoretical profiles from an experimental structure using CRYSOL or FoxS
 is definitely the best way to go in the case of a protein-DNA complex with
 SAXS alone.  Showing comparisons of the experimental with the calculated
 should make the point.  Test other possible models inferred from lattice
 packing to further your point (if applicable).



 Regarding populations of monomer and dimer -



 · it is generally good to constrain your interpretation of
 scattering data with other orthogonal solution measures which demonstrates
 the homogeneity of your complex in comparable experimental conditions, such
 as sedimentation velocity or gel filtration.



 · Have some determination of affinity of the complex in the same
 solution conditions (including temperature!).  This will allow you to argue
 that your sample concentrations are well in excess of any monomer-dimer
 association behavior (eg, mixtures!).  Scattering of mixtures can undermine
 your ability to accurately assess the structural properties of your complex.



 · Collect a concentration series and extrapolate to infinite
 dilution, if possible, to ensure elimination of the S(q) term from your
 data.  Interparticle interactions can be an issue with complexes containing
 DNA if the buffers aren’t quite right. (I’ve seen this a lot)



 Lastly, remember that the scattering profile represents the solution
 average of the particle, not just a single snapshot.  Some discrepancies
 like those you note should be expected.



 Hope that helps,



 Kushol



 Kushol Gupta, Ph.D.

 Research Associate - Van Duyne Laboratory

 HHMI / Perelman School of Medicine

 University of Pennsylvania

 kgu...@mail.med.upenn.edu

 215-573-7260 / 267-259-0082





 -Original Message-
 From: CCP4 bulletin board 

Re: [ccp4bb] Do my SAXS data agree with the crystal structure?

[David Briggs]

Hi Xun,

it is difficult to judge without seeing the P(r) plots, but seeing as
you have a dimer in your crystal structure and a dimer in your SAXS,
AND your Chi2 value seems reasonable for a good match between PX and
SAXS, I'd say you've got what you need.

A matching P(r) plot would be nice, but the crysol plot comparing your
SAXS scattering curve and the theoretical scattering curve from your
crystal structure should be enough for a figure.

HTH,

Dave


David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB



On 16 June 2012 19:29, Xun Lu xlun...@gmail.com wrote:
 Dear all,


       I have solved a protein-DNA structure, and I also did SAXS to get some 
 ideas of the solution structure.  The SAXS data were good, no aggregation at 
 all three tested concentrations.  I tried to use Crysol to see if my crystal 
 structure fits the SAXS. The fitting to the scattering profile seems good to 
 me and the Chi2 is 1~1.4.   Then I wanted to see how the P(r) looked like 
 (wanted to make a figure for my paper:).  I calculated the theoretical 
 scattering profile of the crystal structure from an online server (FOXS).  I 
 then run GNOM to make P(r).  To my surprise, this theoretical P(r) looks a 
 little different from the P(r) of SAXS data.  There's a very small bump that 
 was peaked at 70A (Dmax is 108A, which seems reasonable from the crystal 
 structure).   The major peak was at 25A.  As some people said, P(r) is indeed 
 quite sensitive to subtle differences.

        The protein is a dimer in the crystal, although it can also bind DNA 
 as a monomer (much  more loosely).  The estimated MW from SAXS indicates it's 
 a dimer in solution as well.   It seems that I got the information I wanted 
 from the SAXS experiment, but maybe not.  Due to the low resolution of SAXS, 
 maybe I can only say that the majority is a dimer??  Would it be possible to 
 see the monomer if there's only 10% of them in the solution?  How to 
 interpret the discrepancy between the P(r) from crystal and the P(r) from 
 SAXS?


 Any comments are welcome!




 Xun


 Sent from my iPad

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

[David Briggs]

Trollus, Trollum, Trolli, Trollo, Trolli, Trollos, Trollorum, Trollis.



David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB




On 1 April 2012 21:27, Kendall Nettles knett...@scripps.edu wrote:
 What is the single Latin word for troll?

 Kendall

 On Apr 1, 2012, at 3:06 PM, Kevin Jin kevin...@gmail.com wrote:

 “I hope and believe that this is not the case.  Even basically-trained
 crystallographers should be able to calculate and    interpret difference
 maps of the kind described by Bernhard.  And with the EDS and PDB_REDO
 server, one does not even need to know how to make generate a difference
 map...”

 You are right!

 Actually, I am not an experienced protein crystallographer. I have learnt a
 lot from CCP4BB. I may have paid too much attention to bonding angle and
 bond length, like in small molecule. This may be an example to share with
 you.

 When I worked on those nitroreductase complexed with FMN in 2009 (?), I
 always observed that the flavin ring presented a strange geometry after
 refinement. Indeed, I had used the definition of FMN from CCP4 library all
 the time.

 In some cases, the methyl group at position of either 7a or 8a was bent off
 the aromatic ring, if the whole the rest of flavin was restrained in a flat
 plane.  According to my limited knowledge from organic chemistry, carbon of
 7 and 8 on the flavin ring is sp2 hybridized in a coplanar manner. How could
 those methyl groups be bent as sp3 hybridization? Any chemistry behind?

 With increased resolution (1.6 ~ 1.8 Ang), I observed that the electron
 density map was a bent along the N5-N10 axis. The bend angle was around ~16
 degree.   Again, I questioned myself why it was bent? Should this be
 correct?

 According to my limited knowledge in chemistry, N10 should be sp3
 configuration even if FMN is in its oxidization form, in which the flavin
 ring should be bent. A quick “google” immediately gave me a link to a very
 nice paper published by David W. Rodgers in 2002.

 http://www.jbc.org/content/277/13/11513.full.pdf+html

 According to this paper, Yes!  “In the oxidized enzyme, the flavin ring
 system adopts a strongly bent (16°) conformation, and the bend increases
 (25°) in the reduced form of the enzyme,…”

 When I reported this in the group meeting, I was laughed and told that this
 is just a model bias. It was over interpreted.  Nobody has such sharp vision
 on electron density map.  If this was correct, why nobody could find this
 and report to CCP4 within last 7 years?

 Eventually, a senior team member emailed to CCP4 about this issue. Since
 then, the definition of FMN was updated, according to my suggestion.

 I was asked “how did you find it?”……. “why you believed you are so right?”
  I really don’t how to answer.

 Je pense donc je suis

 Kevin


 On Sun, Apr 1, 2012 at 8:09 AM, Paul Emsley paul.ems...@bioch.ox.ac.uk
 wrote:
 On 31/03/12 23:08, Kevin Jin wrote:


 I really wish PDB could have some people to review those important
 structures, like paper reviewer.


 So do the wwPDB, I would imagine.

 But they can't just magic funding and positions into existence...

 If the coordinate is downloaded for modeling and docking, people may not
 check the density and model by themself. However this is not the worst
 case,
 since the original data was fabricated.


 1. All of data was correct and real,


 Hmmm...

  It will be very difficult for people to check the density and coordinated
 if he/she is not a well-trained crystallographer.


 I hope and believe that this is not the case.  Even basically-trained
 crystallographers should be able to calculate and interpret difference
 maps
 of the kind described by Bernhard.  And with the EDS and PDB_REDO server,
 one does not even need to know how to make generate a difference map...

 Paul.





 --
 Kevin Jin

 Sharing knowledge each other is always very joyful..

 Website: http://www.jinkai.org/

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[David Briggs]

Hi Matt,

This doesn't really answer your question directly, but sidesteps
around the issue -
I wrote a little something on this exact subject not so long ago -
http://xtaldave.wordpress.com/2012/02/23/on-protein-crystallisation/
(You can ignore the first 5 paragraphs of intro - it was written with
a lay audience in mind)

Basically, I setup fine gradient screens in deep well blocks by
titrating stock solutions across a row - which I can then put straight
into use with our Phoenix crystallisation robot. In most situations I
would hope to get decent diffracting crystals from a standard 3-well
intelliplate setup this way.

HTH

Dave

David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB




On 26 March 2012 18:57, Matthew Lalonde mattc...@gmail.com wrote:
 Dear All,

 I have searched the archives and would like more information about
 reproducing robot tray hits using 24-well hand trays. I reproducibly get
 crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
 precipitate in 1-2 ul sitting drops in 24-well hand plates.

 What parameters should I vary to reproduce crystals in hand plates?
 References that discuss this procedure exhaustively would also be
 appreciated.

 Thanks,
 Matt

[ccp4bb] Envelope Phasing.

[David Briggs]

Hi CCP4bb,

I would like to ask about envelope phasing - specifically with SAXS data.

There are papers (1) and tutorials (2) describing how this might be
done, but I have also found comments on the ccp4bb, such as this one
(http://www.proteincrystallography.org/ccp4bb/message11690.html) which
are somewhat less optimistic.

I get the impression from my reading around that SAXS envelope phasing
is somewhat difficult to do unless you have some NCS you can use to
help the phase extension process. Does anybody have any
opinions/evidence/examples/anecdotes/tips about how SAXS envelope
phasing can be done successfully?

Cheers,

Dave

(1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081
(2) - eg - 
http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model


David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB