Re: [ccp4bb] 3D Structure Search

[Briggs, David C]

Also,


TopSearch https://topsearch.services.came.sbg.ac.at/

CAME - TopSearch :: Structure Search
topsearch.services.came.sbg.ac.at
Sequence Analysis Highlight identical residues Jmol Mode HTML5 Java. Cite 
TopSearch ©

Dave


==

Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Daniel Rigden 

Sent: 16 February 2018 08:29:29
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3D Structure Search

Another topology-independent method is CLICK, available as a server here

http://cospi.iiserpune.ac.in/click/

You might also want to try FATCAT which considers potential flexibility
of separate subdomains in its structural alignment

http://fatcat.burnham.org/fatcat-cgi/cgi/fatcat.pl?-func=search

Dan


On 15/02/18 23:25, Ethan A Merritt wrote:
> On Thursday, February 15, 2018 3:16:50 PM PST Nicola Evans wrote:
>> I have recently solved a novel structure which previously did not have any 
>> structural homologues (as related by sequence identity). I was wondering if 
>> anyone could recommend a 3D structural search engine? I used the Dali server 
>> which has been recommended to me in the past (and with past success) but the 
>> top few hits this time aren't similar structurally (although I haven't 
>> exhausted the list yet). I just want to confirm if the folds are truly novel 
>> or not.
> Dali is sensitive to the order of secondary structure elements.
> This is relevant if you are looking for evolutionary relatedness, but
> not if you just want to ask "does it look like this".
>
> For the latter question, I suggest the VAST server at NCBI.
> https://www.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml
>
>Ethan
>

--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool   http://pcwww.liverpool.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

Re: [ccp4bb] question regarding sequence numbering

[Briggs, David C]

Hi Tony,

When I've had similar issues, I've numbered them sequentially (i.e.  7,8,9) and 
remarked in the PDB header that they are vector-derived sequence.

I believe that is what the PDB ask you to do in situations like this (maybe 
they can comment?).

If they are not numbered sequentially, then often graphics and refinement 
software won't treat them as linked.

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs



From: Antonio Ariza
Sent: Tuesday 19 September, 13:15
Subject: [ccp4bb] question regarding sequence numbering
To: ccp4bb@jiscmail.ac.uk


Hi all,

Here's a problem I haven't come across before. I'm working on a structure whose 
expression plasmid was designed to remove the first 9 amino acids from the 
protein of interest and to which an N-terminal tag was added. After cleaving 
the tag I am left with 3 amino acids (GPM) followed by the original sequence. 
Obviously the residues of interest should follow the numbering of the original 
sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?

Cheers,

Tony

--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
Structural insights into the function of 
 
ZRANB3 in replication stress 
response

Re: [ccp4bb] NMR or Homology Model as a MR model

[Briggs, David C]

Dear Nishant,

You can use anything you want as a molecular replacement search model including 
NMR models/ensembles or homology models. Whether or not it will be successful 
is another matter!

For borderline cases, some have reported success using Phaser in combination 
with the Rosetta modelling suite.

E.g.
https://www.phenix-online.org/documentation/reference/mr_rosetta.html

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs



From: Nishant Varshney
Sent: Tuesday, 29 August, 11:51
Subject: [ccp4bb] NMR or Homology Model as a MR model
To: ccp4bb@jiscmail.ac.uk


Dear Crystallographers,

I am working to solve an human protein structure which has a domain sequence 
identity of 24% with domain of another protein.  As Phaser as well as Molrep 
failed to give any definite solution (TFZ=3.7 from MR), I want to ask, if 
solution structure of another protein having domain sequence similarity of 25% 
or a homology model can be used as a template for MR?
Many thanks and Regards
Nishant

--
Dr. Nishant Kumar Varshney,
Research Associate,
C/O Dr. Sameena Khan,
Drug Discovery Research Center,
Translational Health Science and Technology Institute (THSTI)
NCR Biotech Science Cluster,
3rd Milestone, Faridabad – Gurgaon Expressway,
Faridabad – 121001 (HARYANA), India
Ph: +91- 0129-2876477
Mob: 8390564690

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Briggs, David C]

Hi Chris,

What is the theoretical pI of your protein? If it is around pH 7.5, you might 
try gel filtering your protein into a different buffer/pH combination. Try 
changing by at least 1 pH unit in either direction.

If the pI isn't a problem, then you might try try solubility screening as 
outlined...

http://scripts.iucr.org/cgi-bin/paper?dz5020

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Chris Fage 

Sent: Thursday, July 13, 2017 11:40:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein rapidly precipitates when off ice

Dear CCP4BB Community,

This week, I purified a nicely overexpressing protein by Ni-NTA followed by gel 
filtration. In a 4 C centrifuge, I concentrated my gel filtration fractions to 
~1 mL, transferred the spin filter to ice, and then collected 2 uL for 
measurement on the Nanodrop. Sadly, the protein precipitated heavily in the 
pipet tip before I could dispense it onto the Nanodrop pedestal, directly 
adjacent to my ice box. This effect seems to be abated at 4 C, as the protein 
remained stable in cold room-chilled pipet tips. However, the protein also 
precipitated heavily when overnight at 4 C in 1 mL gel filtration buffer (150 
mM NaCl, 10 mM HEPES pH 7.5), but not overnight at 4 C in 10 mL Ni-NTA buffer 
(500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) prior to gel filtration. Has 
anyone experienced and resolved a similar issue before? Do any useful additives 
come to mind?

Things I have tried with the gel filtration sample:
-Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g. 500 
mM).
-Exchanging buffer to add 10% glycerol.
-Simply diluting the protein in gel filtration buffer to rule out concentration 
dependence.

In each case, the protein precipitates to a milky solution within about a 
minute of removal from ice (I am working with 20-50 uL volumes in PCR tubes).

Many thanks for any suggestions!

Best,
Chris

Re: [ccp4bb] weird diffraction pattern

[Briggs, David C]

Hi,

I'd need to see more processing stats to figure out your data issues. Streaky 
spots in certain directions can be indicative of anisotropy and/or lattice 
disorders.

However, if your Rfree is above 0.5, it is likely that your molecular 
replacement solution is poor/bad/wrong.

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of 唐晨骏 
<0910010...@cau.edu.cn>
Sent: Thursday, July 13, 2017 8:56:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] weird diffraction pattern

hello everyone,
I would like to seek your opinion on my crystal hits. I am working on a helicase

of which the native structure is solved and the all solution statistics are

fine. I am trying to crystallize and solve the structure of the protein/ssDNA

complex. I recently got some hits from commercial screens using sitting drop

vapor diffusion. After crystallization optimization, these crystals diffract

weakly but to 3.2 Angstroms for the longer exposure time. However, when the

crystals rotate between 120 degrees to 180 degrees, the spots become streaky

(attached), no matter the crystals are hexagonal or flaky. I have tried to

determine the structure by molecular replacement method, but the Rwork/Rfree

values are huge (above 0.5) and can’t be reduced further. I suspect the

obtained crystals quality and resulting processed statistics is the reason for

the observed high Rwork/Rfree values. Are there any suggestions?

All comments will be appreciated!

Best,
Chenjun Tang

Re: [ccp4bb] long loop

[Briggs, David C]

I know that this might be considered heresy on a crystallography mailing list, 
but do you have any friendly NMRists at your institution?


D


Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of dongxiaofei 
<18811070...@163.com>
Sent: 04 July 2017 16:22:39
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] long loop

Dear ALL,
I want to make a protein crystal,but there is a long loop between domains of 
protein , which contains two small domains owning about 40 amino acids 
respectively and a loop about 70 amino acids.
Loop is so long and flexible ,but I don't want to delete some fragments,because 
it may be  important for protein's function of a histone demethylase.
Besides, the  surface charge of  protein is whole negative .
I have tried a long time but it is hard to me to get crystal.

Would be very grateful for any advice!
Thanks
Dong Xiao

Re: [ccp4bb] Correcting 3-letter codes based on protonation states in a PDB file

[Briggs, David C]

I believe the ProPka or Pdb2pqr webservers can do this.

ProPka.org

http://nbcr-222.ucsd.edu/pdb2pqr_2.0.0/

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Sampson, Jared 

Sent: Wednesday, June 28, 2017 11:34:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Correcting 3-letter codes based on protonation states in a 
PDB file

Dear all -

I'm working with a PDB file with explicit hydrogens where many of the 
histidines are in protonated form due to crystallization at low pH.  
Unfortunately, although the additional protons are present in the model for the 
positively charged histidines, the residues in question are indicated in both 
the SEQRES and the ATOM records as 3-letter code `HIS` regardless of 
protonation state (i.e. instead of `HIP` for positively charged, and `HID` or 
`HIE` for the neutral tautomers).

Are there existing tools available to determine the proper 3-letter residue 
code for titratable amino acid residues based on which hydrogens are present, 
and output a corrected PDB file?

Thank you in advance for your suggestions.

Cheers,

Jared Sampson
Ph.D. Candidate
Columbia University

Re: [ccp4bb] mystery disappearance of CCP4 website(s)

[Briggs, David C]

http://www.ccp4.ac.uk/ works for me, (from Sunny London).


Maybe it was just a glitch in the matrix?




Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Peter Keller 

Sent: 25 May 2017 14:29:00
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] mystery disappearance of CCP4 website(s)

http://downforeveryoneorjustme.com/www.ccp4.ac.uk and
http://www.isitdownrightnow.com/ccp4.ac.uk.html both report that the
CCP4 web site is down, so I don't think that there is any question that
it has really gone.

Regards,
Peter.

On 25/05/17 14:25, David Schuller wrote:
> I can't reach it.
>
> A newspaper site I frequent, www.startribune.com, 
> was down earlier this
> morning, but is back up already.
>
>
> On 05/25/17 09:12, Andrew Sharff wrote:
>>
>> You are not alone. They are unreachable from here too!
>>
>>
>> Andrew
>>
>>
>> On 25/05/17 14:09, R. Michael Garavito wrote:
>>> I have been trying to reach some of the CCP4 websites
>>> (www.ccp4.ac.uk/ ) from the US and they all
>>> seem to be unreachable, even from the http://www.ccp.ac.uk/ site.
>>>  Any ideas? Or did I miss some announcement.  Most of the other CCPs
>>> seem to be up.
>>>
>>> Michael
>>>
>>> //
>>> /R. Michael Garavito, Ph.D./
>>> /Professor of Biochemistry & Molecular Biology/
>>> /603 Wilson Rd., Rm. 513///
>>> /Michigan State University/
>>> /East Lansing, MI 48824-1319/
>>> /Office:(517) 355-9724Lab:(517) 353-9125/
>>> /FAX:(517) 353-9334Email:  rmgarav...@gmail.com
>>> /
>>> //
>>>
>>>
>>
>> --
>> --
>> *Dr. Andrew J. Sharff D.Phil**
>> *Research Scientist / Software Developer
>> Global Phasing Ltd
>> Sheraton House
>> Castle Park
>> Cambridge CB3 0AX
>> UK
>> Tel: +(0)1223 353033
>> Fax: +(0)1223 366889
>
>
> --
> ===
> All Things Serve the Beam
> ===
> David J. Schuller
> modern man in a post-modern world
> MacCHESS, Cornell University
> schul...@cornell.edu
>

--
Peter Keller Tel.: +44 (0)1223 353033
Global Phasing Ltd., Fax.: +44 (0)1223 366889
Sheraton House,
Castle Park,
Cambridge CB3 0AX
United Kingdom

Re: [ccp4bb] Cys modification - deciding between CSS and SMC

[Briggs, David C]

Hi Mohamed,

Did you try CSO or CSD?

I've seen oxidation of Cys residues in the active site of Cysteine-dependent 
Phosphatases before.

In addition, oxidation is more easily explainable than CSS and SMC, especially 
if reducing agents were omitted from final purification steps.

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs



From: Mohamed Noor
Sent: Sunday, 21 May, 13:00
Subject: [ccp4bb] Cys modification - deciding between CSS and SMC
To: ccp4bb@jiscmail.ac.uk


I am working on a model at a resolution of 2.1 A. The active site Cys in both 
copies have a positive density towards the S end of the residue and these blobs 
are there in FEM and Polder maps. When I replace these residues with either CSS 
or SMC, I get the following statistics. What is the best way of deciding which 
one is correct? CSS: R/Rfree - 0.2016/0.2299 Local CC (chain A/B) - 0.950/0.937 
Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8 
Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3 No 
residual positive density left. SMC: R/Rfree - 0.2087/0.2299 Local CC (chain 
A/B) - 0.903/0.886 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 
43.9/47.9/46.5/42.1/51.8/64.4/66.5 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 
43.4/45.0/47.3/45.4/51.8/59.4/66.1 Crescent shaped positive density left 
towards the CS atom end. Although CSS seems more plausible, the high B factors 
of SD make me wonder if they are correct. Thanks. Mohamed

Re: [ccp4bb] BSA as additive

[Briggs, David C]

(Sorry... over-enthusiastic sending)


As for an actual reference - I'm not sure.


It's a bit like lab folk-lore.


Dave


Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Ha Sin 

Sent: 08 May 2017 13:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] BSA as additive

[Briggs, David C]

Hello,


I've seen BSA used to "block" membranes of centrifugal concentrators when 
proteins were sticking to them.


It appeared to work (proteins could be concentrated with less loss), but I'm 
not sure how much BSA ends up in your sample.


I can imagine the answer is "a bit" and therefore for some techniques this 
might not be appropriate.


Dave


Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Ha Sin 

Sent: 08 May 2017 13:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin