[ccp4bb] Fully funded BBSRC PhD opportunity at the University of Liverpool

[Daniel Rigden]

Pleas make any suitable candidates aware of this project

*https://www.findaphd.com/phds/project/working-at-the-intersection-of-protein-bioinformatics-and-structural-biology-exploiting-protein-structural-ensembles-to-facilitate-structure-solution-from-x-ray-crystallography-and-cryo-em/?p117359*

This exciting project involves developing novel tools, using advanced 
structural bioinformatics methods, to facilitate protein structure 
determination by X-ray crystallography and cryo-EM. With networking 
opportunities via the leading software consortia CCP4 and CCPEM, and 
exposure to experimental methods through time spent in the lab of Owen 
Davies in Newcastle, this is a chance to work at the cutting edge of 
applied bioinformatics.


thanks

Daniel

--
Prof Daniel Rigden
Institute of Integrative Biology
Room 101, Biosciences Building
University of Liverpool
Crown St., Liverpool, L69 7ZB

(+44) 151 795 4467
pcwww.liverpool.ac.uk/~drigden/




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Re: [ccp4bb] coiled coil molecular replacement

[Daniel Rigden]

Hi Tommi

Yes, AMPLE does well with coiled-coils. The QUARK route is the easiest 
to try. In your position I would simply trim a bit of sequence off 
either end. Maybe you can see homologs that are a bit shorter at one 
terminus? In any case, that's unlikely to affect the modelling and we've 
seen QUARK make good models of coiled-coil proteins.


For Rosetta modelling we simply recommend you use the Robetta server 
http://robetta.bakerlab.org/fragmentsubmit.jsp for fragment libraries. 
Unless you're processing large numbers of sequences it's not worth 
getting to grips with local fragment library generation. At the moment 
CCP4online takes a file of ready-made models, not carrying out the 
actual modelling for you. Doing local Rosetta modelling via AMPLE is as 
described here 
https://ample.readthedocs.io/en/latest/examples/rst/abinitio.html#example-abinitio


Recently, in collaboration with Owen Davies in Newcastle, we've made 
some big improvements to AMPLE's abilities with coiled-coils, involving 
more bespoke modelling protocols, both of a single chain and, where the 
information is known, of a parallel oligomer. I gather Owen has been in 
touch with you about these. The code is available but is not in the 
current CCP4 distribution. We're also planning to improve the AMPLE 
documentation in the next few months and we'll include an example of use 
of this new coiled-coil mode at that time.


Best wishes

Dan

On 07/10/2019 22:33, Kajander, Tommi A wrote:

Hello,

We have a bit tricky case of coiled coil protein with good data (2.05Å) for 
dimeric coiled coil (dimer in AU)  - looks like AMPLE might be a way
to solve such cases, if you know other good programs please suggest (Better yet 
if there is a clear how-to manual)

Some technical tips on usage for generation of fragments for AMPLE would be of 
help, not completely on top of that… (running the QUARK server, the real 
sequence is bit over 200 aa so not sure what is the best approach here? 
Rosetta? any how-to for that.. well i am running Robetta fragments too).

with AMPLE can I do this with the online server or better run locally (need 
Rosetta installed I take it?)

Suppose I could try Rosetta-MR also, but to my recollection that requires some 
kind of a phaser hit first to be improved, and i dont think I am there.

Thanks for any comments,

Best,
Tommi






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Institute of Integrative Biology
Room 101, Biosciences Building
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Crown St., Liverpool, L69 7ZB

(+44) 151 795 4467
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Re: [ccp4bb] MR for coiled coil structure

[Daniel Rigden]

Dear Shengyang


We'll be happy to help you sort this out. I suggest we discuss it 
between the two of us and then post a summary to the bulletin board.



Could you please tell me, privately not to the board, which gui you are 
using, which version of CCP4 and which options you are trying to select?



Best wishes


Dan


On 10/07/2019 09:00, Shengyang Jin wrote:

Dear all,

We recently acquired a data set (2.0 A, P222) for a coiled coil 
protein (according to Itasser, QUARK, Robetta, and Phaser).
Matthews coefficient indicates 1 copy of protein per ASU. Sequence of 
the protein is quite novel with no apparent homolog in PDB.
We tried to to MR with various models (ab initio or homology based) 
but with little success.


We then tried to use AMPLE, but in ccp4 it always returned this error:
__main__.py: error: unrecognized arguments: -use_arpwarp True
(if we untick arpwarp and choose buccaneer instead, it returns 
-use_arpwarp False)


Could anyone help?

Thank you very much.


Shengyang Jin
Nanyang Technological University
Singapore



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Institute of Integrative Biology
Room 101, Biosciences Building
University of Liverpool
Crown St., Liverpool, L69 7ZB

(+44) 151 795 4467
pcwww.liverpool.ac.uk/~drigden/




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[ccp4bb] Postdoctoral position in structural bioinformatics/structural biology

[Daniel Rigden]

Applications are invited for a postdoctoral fellow position in the 
laboratory of Dan Rigden at the University of Liverpool. The position 
involves translating protein bioinformatics methods to applications in 
structural biology.


As part of the CCP4 software consortium, we are looking for a PDRA to 
develop methods that exploit evolutionary covariance-derived information 
for the benefit of structural biologists. The work, some in 
collaboration with CCP4 partners, will use covariance information to 
help parse domains, to guide and validate map interpretation and to 
develop novel methods to address the phase problem in X-ray crystallography.


You should have a PhD degree (or equivalent experience) in Informatics, 
Bioinformatics or in a Biological Science with experience of 
bioinformatics. You should have excellent programming skills and 
experience of research in computational or structural biology. The post 
is available from October 2019 for four years.


Full details of the post and the application process are available here

https://recruit.liverpool.ac.uk/pls/corehrrecruit/erq_jobspec_version_4.display_form?p_company=1_internal_external=E_display_in_irish=N_process_type=_applicant_no=_form_profile_detail=_display_apply_ind=Y_refresh_search=Y_recruitment_id=011968

Informal approaches to drig...@liv.ac.uk are welcome.

--
Prof Daniel Rigden
Institute of Integrative Biology
Room 101, Biosciences Building
University of Liverpool
Crown St., Liverpool, L69 7ZB

(+44) 151 795 4467
pcwww.liverpool.ac.uk/~drigden/




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Re: [ccp4bb] Protein-Protein Interaction prediction

[Daniel Rigden]

Dear Sayli

Here are some options from Table 10.1 of this book chapter

https://link.springer.com/chapter/10.1007/978-94-024-1069-3_10

cons-PPISP


Uses sequence profiles and solvent accessibility for residues and their 
neighbours




http://pipe.scs.fsu.edu/ppisp.html



Zhou and Shan (2001 
)


meta-PPISP



Metaserver using cons-PPISP, PINUP and Promate predictions



http://pipe.scs.fsu.edu/meta-ppisp.html



Qin and Zhou (2007 
)


CPORT



Consensus prediction from six different methods



http://haddock.science.uu.nl/services/CPORT



de Vries and Bonvin (2011 
)


PRISE



Atomic composition, residue type and solvent exposure of a central 
surface residue plus its neighbours




http://prise.cs.iastate.edu/index.py



Jordan et al. (2012 
)


VORFFIP



Considers a variety of structural, energetic, evolutionary and 
crystallographic parameters




http://www.bioinsilico.org/VORFFIP



Segura et al. (2011 
)





i



I don't know if they are all still functional: unhappily there tends to 
be quite a high turnover of such servers


Best wishes

Daniel

On 02/03/18 07:47, Sayli Dalal wrote:

Dear all,

Can anyone help me with a web server  or method for prediction of 
interfaces for protein-protein interaction?


Thanks and best regards,

Sayli Dalal
Post-Doctoral Fellow
Dept. of Biophysics
NIMHANS





--
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] 3D Structure Search

[Daniel Rigden]

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.,
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Re: [ccp4bb] cavities in protein structures

[Daniel Rigden]

Hi Claudia

Two options I've used recently, liked and displayed without fuss in 
PyMOL are ProFunc 
(https://www.ebi.ac.uk/thornton-srv/databases/profunc/) and Ghecom 
(http://strcomp.protein.osaka-u.ac.jp/ghecom/). Ghecom gives you PDB 
files directly. ProFunc gives you rasmol scripts but they open fine with 
PyMOL. Other options are listed in this book 
chapterhttps://link.springer.com/content/pdf/10.1007/978-94-024-1069-3_10.pdf 
which I can email if you don't have access.


Best wishes

Daniel


On 30/01/18 11:51, Claudia Binda wrote:

Hi everyone,

I need suggestions to calculate and represent cavities of protein 
structures. For years I have been using Voidoo that produces maps in 
ezd format which could be converted in map format (ccp4) using the 
online server http://xray.bmc.uu.se/cgi-bin/gerard/mapman_server.pl. 
However, this does not work anymore. Is there another way to do it? 
What is the best tool to calculate cavities and draw them by Pymol or 
ccp4mg?


Thank you
Claudia







--
Claudia Binda
University of Pavia
Dept. Biology and Biotechnology
via Ferrata 1, 27100 Pavia - Italy
Phone: +39-0382-985535 
Fax: +39-0382-528496 
E-mail: claudia.bi...@unipv.it 
Web: http://www.unipv.it/biocry


--
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] NMR or Homology Model as a MR model

[Daniel Rigden]

Hi Nishant

AMPLE can work with ensembles from NMR structures 
(http://ample.readthedocs.io/en/latest/examples/rst/nmr_ensemble.html#example-nmr-ensemble; 
see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817692/) or sets of 
distant homologues 
(http://ample.readthedocs.io/en/latest/examples/rst/homologs.html#example-dist-homologs), 
trialling a large number of edited variant ensembles (from mildly 
truncated to brutally) in an automated fashion. With AMPLE you select 
Phaser and/or Molrep for the actual MR step and, resolution permitting, 
can use Shelxe's statistics to give you a particularly clear indication 
of success/failure.


Good luck

Dan

On 29/08/17 13:31, Randy Read wrote:

Hi,

Molecular replacement gets progressively more difficult as the 
sequence identity drops, though the correlation between sequence 
identity and model quality is not perfect.  With the best models 
having sequence identities around 24%, success is by no means 
guaranteed, so you might have to try lots of strategies or resort to 
experimental phasing.  NMR models do tend to be worse for molecular 
replacement, so that will probably lower the chances of success further.


In addition to what others have said already, it's worth mentioning 
that, in a number of very difficult structure determinations (best 
models around 20% sequence identity), the key thing that made the 
difference between failure and success was pruning off the parts of 
the ensemble that do not agree among the members of the ensemble, 
leaving just the conserved core.  This can be done, for instance, in 
the Ensembler program with the "trim=True" flag.  For this to work 
best, it's helpful if you happen to have a number of potential models 
that are also relatively distantly related to each other, to really 
highlight what is conserved in the fold.


Best wishes,

Randy Read

On 29 Aug 2017, at 12:42, Andreas Forster > wrote:


Dear Nishant,

Rosetta is a good suggestion.  You can also use an ensemble of 
several related (superposed) structures as your search model.  This 
will improve your chances of success.


All best.


Andreas



On Tue, Aug 29, 2017 at 12:50 PM, Nishant Varshney > wrote:


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




--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills Road  E-mail: rj...@cam.ac.uk 
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk 





--
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.

[ccp4bb]

[Daniel Rigden]

Dear Shijun

As the message suggests, AMPLE has not been able to find the executable 
of THESEUS, the program it uses for maximum likelihood superposition of 
structures. This error message is nothing to do with Rosetta. THESEUS is 
distributed with more recent versions of CCP4. To continue with your 
current version you can install THESEUS (if you haven't already) from 
http://theseus3d.org/ . You can then either put it in your $PATH or use 
the flag


  -theseus_exe /path/to/theseus

Best wishes

Daniel

On 31/07/17 12:55, 张士军 wrote:


Hi all

  It always fail when I run ccp4-AMPLE, and log file says


Cannot find executable theseus in PATH. Please give the path to theseus
***
* Information from CCP4Interface script
***
The program run with command: 
/usr/local/programs/ccp4/ccp4-6.4.0/bin/ccp4-python -u 
/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample -mtz 
/home/xingqiang/ZSJ/kif5b/430-566/A12P422output.mtz -fasta 
/home/xingqiang/ZSJ/kif5b/430.seq -nmodels 500 -name MVD0 -run_dir 
/home/xingqiang/ZSJ/kif5b/430-566 -nproc 4 -make_models True 
-rosetta_dir /usr/local/programs/rosetta -frags_3mers 
/home/xingqiang/ZSJ/kif5b/aat000_03_05.200_v1_3 -frags_9mers 
/home/xingqiang/ZSJ/kif5b/aat000_09_05.200_v1_3 -make_frags False -F F 
-SIGF SIGF -FREE FreeR_flag -early_terminate True -use_shelxe True 
-use_arpwarp False

has failed with error message
Traceback (most recent call last):
File "/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample", line 505, in
amopt.d['theseus_exe'] = ample_util.check_for_exe('theseus', 
amopt.d['theseus_exe'])
File 
"/usr/local/programs/ccp4/ccp4-6.4.0/share/ample/python/ample_util.py", 
line 93, in check_for_exe

raise RuntimeError,msg
RuntimeError: Cannot find executable theseus in PATH. Please give the 
path to theseus

***
#CCP4I TERMINATION STATUS 0 "Traceback (most recent call last): File 
"/usr/local/programs/ccp4/ccp4-6.4.0/bin/ample", line 505, in 
amopt.d['theseus_exe'] = ample_util.check_for_exe('theseus', 
amopt.d['theseus_exe']) File 
"/usr/local/programs/ccp4/ccp4-6.4.0/share/ample/python/ample_util.py", 
line 93, in check_for_exe raise RuntimeError,msg RuntimeError: Cannot 
find executable theseus in PATH. Please give the path to theseus"

#CCP4I TERMINATION TIME 31 Jul 2017 19:49:31
#CCP4I MESSAGE Task failed
 Anyone know what's wrong with it? Does it mean I don't install 
Rosseta successfully? Thanks


best regards

shijun



--
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.,
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Re: [ccp4bb] Hydrophobic hotspots

[Daniel Rigden]

Dear Hugh

If you know Aggrescan then I assume you're away of its 3D counterpart?

http://biocomp.chem.uw.edu.pl/A3D/

It even allows you to consider whether structures reached by 
coarse-grained MD are more aggregation-prone than your starting structure.


Good luck

Daniel


On 26/07/17 18:05, Hugh Morgan wrote:

Hi all, can anyone recommend a program for identifying hydrophobic 
(aggregation) hotspots using either the amino acid sequence and/or structural 
data.
Thanks in advance for your help
Hugh

Ps. Have tried aggrescan but would like to try a few others and compare, 
ideally a more structural based program.


--
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] ample

[Daniel Rigden]

Dear Pat

Yes, AMPLE does it's own, purely structure-based, alignment of the 
homologs provided using GESAMT. You provide a directory of structures to 
work with and, crucially, give AMPLE the


-homologs True

flag so that it knows the inputs have different sequences (unlike in 
modelling mode), and need to be dealt with accordingly.


There's more explanation and example files here

http://ample.readthedocs.io/en/latest/examples/rst/homologs.html#example-dist-homologs

In this mode AMPLE first finds the common core shared by all inputs and 
then produces graded further truncations of that with different side 
chain treatments. This sampling is often necessary in the hardest cases. 
What this means, however, is if the input set are ultra-diverse, then 
the GESAMT shared common core can be quite small. It can therefore also 
be worth trying a smaller set of not quite so diverse structures which 
share a somewhat larger core structure.


Best wishes

Daniel

On 20/07/17 23:13, Patrick Loll wrote:

I’m intrigued by the prospect of using AMPLE to test multiple distant homologs 
in a MR problem. I’ve used HHPRED to identify about 20 high-probability 
homologs of known structure, each of which has about 20-25% identity with the 
unknown protein. However, it’s not clear to me from the documentation whether 
the program will use the alignments from HHPRED, and, if so, how I should 
provide that information.

Or does AMPLE perform its own alignment? I.e., do I simply point the program to 
a directory containing 20 different PDB files and stand back?

Thanks for any insights.

Cheers,

Pat
---
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


--
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] AW: [ccp4bb] high Rfree

[Daniel Rigden]

Hi there

AMPLE works very well for coiled-coil structures, irrespective of 
parallel/anti-parallel etc


http://journals.iucr.org/m/issues/2015/02/00/lz5005/

Resolution permitting, SHELXE tracing and phase modification, built into 
the AMPLE pipeline, gives statistics that differentiate very clearly 
between success and failure.


I also understand that ARCIMBOLDO works very well for coiled-coils

Good luck

Daniel


On 21/07/17 08:37, herman.schreu...@sanofi.com wrote:


Hi ???,

Coiled coil structures can be very tricky with MR, so your solution 
may not be correct. You could try to split your search model and run 
MR with the separate coils. If you have high enough resolution (> ~2.3 
Å?) and your MR solution is basically correct, you may be able to 
solve your problem by rebuilding and refinement, but this may be a lot 
of work. Without any details (resolution of the data, current 
Rwork/Rfree etc.) it is difficult to give any more specific advice.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *???

*Gesendet:* Freitag, 21. Juli 2017 03:17
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short 
fragment recently, then I want to solve a longer fragment structure 
with phenix-MR using this short fragment structure as a model.The 
result is not good because of the Rwork and Rfree is high.So 
 
I think the longer fragment will be parallel coiled-coil which is 
different with the shorter one. I am wondering whether there are any 
other methods to handle this phenomenon besides heavy atom phasing? 
Thanks a lot!!!




--
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] Se-Met and Se-Cys double labelling

[Daniel Rigden]

Dear Vito

AMPLE is another option to easily and automatically find conserved cores 
among a set of distant homologues. You point it to a directory 
containing your homologues and use the


-homologs True

flag. You can use the command line or CCP4i. It will then use GESAMT to 
find the multiple structural superposition and then generate a series of 
ensemble search models containing 100, 95, 90...% of the superimposable 
set of residues shared among the structures. The processing uses 
information about structural variance to trim progressively down to 
smaller but more structurally homogeneous cores. Very often this further 
processing, more dramatic than would be attempted manually, is necessary 
for success.


If you only have a single homologous structure we have had some success 
using it as the basis to generate ensembles using distance geometry 
(CONCOORD) and treating the result in the same way as a set of ab initio 
models.


These approaches work best where resolution allows for automated Shelxe 
main chain tracing and phase modification since the resulting statistics 
very clearly indicate success or failure.


These methods will be submitted for publication in Study Weekend papers. 
In the meantime you can hear some explanation here

https://www.youtube.com/watch?v=3B1-Qr00zXk=2s
from about 19:37

There's also a multiple homolog tutorial here
https://amplemr.wordpress.com/programs/ample/ample-tutorials/ample-tutorial-3/

Do feel free to contact us if you need a hand trying this approach.

Best wishes

Daniel Rigden


On 22/06/17 16:37, Randy Read wrote:
Yes, I agree that MR is worth a shot, though depending on the 
resolution your life may be vastly easier with experimental phases! 
 We've had several cases where the following kind of procedure works: 
find related structures with a very sensitive homology search (we like 
HHPRED, though other options probably work), use the corresponding 
sequence alignment to prune the models (with sculptor or chainsaw or 
other tools) and make a trimmed ensemble with ensembler.  The last 
step can be very important, in trimming off any bits of the collection 
of models that do not constitute a conserved core.  Then provide the 
ensemble to Phaser as a molecular replacement model.  The trimmed 
ensemble is usually the best model, in our experience, but one of the 
individual models may be better in some cases, so that's also worth 
testing.


Also, the MR-Rosetta pipeline has had a substantial number of 
successes when the highest sequence identity was in the range of 15-25%.


Best wishes,

Randy Read

On 21 Jun 2017, at 17:08, Mark J van Raaij <mjvanra...@cnb.csic.es 
<mailto:mjvanra...@cnb.csic.es>> wrote:


If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride 
binding to free Cys. You may have to experiment with different 
soaking times and protocols.
Finally, don't give up on MR too early, what matters is the 
structural similarity, not directly the sequence identity. We've had 
success once with 19% identity.
Native protein may be much easier to produce than the SeMet and 
SeMet/SeCys versions (and may differ a lot between proteins).


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
http://wwwuser.cnb.csic.es/~mjvanraaij 
<http://wwwuser.cnb.csic.es/%7Emjvanraaij>


On 21 Jun 2017, at 17:46, Vito Calderone <calder...@cerm.unifi.it 
<mailto:calder...@cerm.unifi.it>> wrote:


I am working on a protein having 360 residues. In its sequence there 
are 3

Met and 5 free Cys.
I will need MAD to solve the structure since based on the sequence the
closest homologue has 20% identity匢 suppose MR would be very 
unlikely to

work卻o I would like to express a selenium derivative to exploit MAD.
Looking in the literature 1 Se-Met every 120 residues seems not to 
comply
the threshold to get a good anomalous signal. For this reason I 
would like
to exploit both Met and Cys so I would have 8 seleniums per 360 
residues.

Could somenone suggest a reference to a protocol to express the double
mutant protein in NON auxotrophic strains of E. coli which you have
experienced working efficiently?
Thanks




--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills Road  E-mail: rj...@cam.ac.uk <mailto:rj...@cam.ac.uk>
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk 
<http://www-structmed.cimr.cam.ac.uk>




--
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.,
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Re: [ccp4bb] secondary structure assignment to PDB file

[Daniel Rigden]

A variety of alternatives to DSSP are easily available here

http://2struc.cryst.bbk.ac.uk/

As has been pointed out, methods other than the well-known DSSP may be 
particularly appropriate at lower-resolution or with model structures 
where perfect geometry, H-bond formation etc are not to be expected.


Dan


On 29/01/17 17:11, Pavel Afonine wrote:
Tools like DSSP and such rely on model geometry to annotate SS 
elements, so GIGO applies. For example, something that by eye looks 
like an obvious helix but has enough distortions is unlikely to be 
annotated correctly.

Pavel

On Sun, Jan 29, 2017 at 3:50 AM, Antonio Ariza 
> wrote:


I always use DSSP, which I believe has been the gold standard for
secondary structure assignment from pdb files for many years.
PYMOL also has a DSSP plugin that can be used to override its own
secondary structure assignment, which is nowhere near as good as DSSP.

Best

Tony

--
*
Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE*
**
*Links to my public profiles:*
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LinkedIn 
GoogleScholar

ORCID 

*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK
] on behalf of chemocev marker
[jirivit...@gmail.com ]
*Sent:* 29 January 2017 10:41
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* [ccp4bb] secondary structure assignment to PDB file

Hi
Is there any tool that can assign secondary structure to the PDB
file. The problem is if I used different modelling tools, there
are regions in the protein which does not remain consistent and
looks different in different application.

best

J. Vitali




--
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] problem with AMPLE

[Daniel Rigden]

Dear Vikram

AMPLE tries to create ensembles by clustering the ab initio models from, 
in this case, Rosetta. It has failed here because it has not found any 
pair of models that are similar enough by its criteria to superpose into 
a cluster. This is a sign that your 500 models are very diverse and, in 
turn, that the modelling has not produced reliable results: large 
clusters of similar models are the basic indication that ab initio 
modelling may have produced something promising.


The explanation for the poor results lies in the size of your target. 
377 residues is far above the threshold below which ab initio modelling 
is expected to work well. It is currently considered to be worth trying 
up to around 150-200 residues for globular proteins, somewhat larger for 
membrane proteins. That limit is raised by the use of extra information 
from predicted contacts 
(http://journals.iucr.org/m/issues/2016/04/00/lz5010/) but 377 residues 
is much larger than we have had success with using ab initio models.


It looks to me as if you may have distant homologues available. Making 
an ensemble of these and seeking a well-conserved core to use as a 
search model ensemble can be a productive approach, solving structures 
that would not fall out easily with any single distant homologue. AMPLE 
can also apply a cluster and truncate logic to distant homologues in 
such cases - see 
https://amplemr.wordpress.com/programs/ample/ample-tutorials/ample-tutorial-3 
- automatically producing a range of differently sized shared core 
ensembles and trialling them.


Feel free to contact us offline or through ccp4bb if you need more help.

Regards

Daniel


On 04/11/16 05:28, Vikram Dalal wrote:

Hi everyone,

I have run the ample for a 377 amino acids protein and generated 500 
models. But I did not get any  ensemble model and got error message 
that could not load any ensembles after running create_ensembles. I am 
here attaching the ample.log and debug.log file.

Any one suggest me why this happened?

Thanks and regards


*
*








--
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] Help in running AMPLE

[Daniel Rigden]

Hi Vikram

They are regular text files. You don't need to view/edit. Just right 
click on the link and 'Save as' somewhere locally.


Daniel


On 21/10/16 11:12, Vikram Dalal wrote:

Hi Daniel,

Thank you for suggestion. I want to ask that in which format i have to 
saved both these files. I am not getting any option to directly 
download these result. So, I have to paste these results in notepad 
files. I am not able to get that in which format i have to save it then.





*
*

Thanks & Regards,


VIKRAM DALAL
Research Scholar
Macromolecular Crystallographic Unit
Department of Biotechnology
Indian Institute of Technology
Roorkee (INDIA)







On Fri, Oct 21, 2016 at 3:27 PM, Daniel Rigden 
<drig...@liverpool.ac.uk <mailto:drig...@liverpool.ac.uk>> wrote:


Boxbe <https://www.boxbe.com/overview> Daniel Rigden
(drig...@liverpool.ac.uk <mailto:drig...@liverpool.ac.uk>) is not
on your Guest List

<https://www.boxbe.com/approved-list?tc_serial=27255294745_rand=1911667988_source=stf_medium=email_campaign=ANNO_MWTP_content=001=kFDoMfChV%2F2Qn7zYPGqSNRzHkBdkiVkNRSI7BNGTCOY%3D=1BoyEjpGEcScg13fggRG0BY28lIKTFoWsgWx1K67RiXJxnkUc6te4znbTa8S%2FX1D>
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Dear Vikram

Your file aat000_03_05.200_v1_3

<http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_03_05.200_v1_3>
contains fragments of 3 residues length, the other one contains
9-residue fragments. Download these to your own computer and then,
in the CCP4i interface to AMPLE, use the Browse buttons in the
Fragment Files section to locate them as 3mers and 9mers
respectively. Â  Once you've told AMPLE where your Rosetta
installation is, and filled in the other Input Files as normal,
you're good to go.

Best wishes

Daniel

On 21/10/16 10:50, Vikram Dalal wrote:

Hi eveyone,

I want to run the ample in ccp4i. I have generated the files for
my sequence using robetta fragment library server. But, I do not
know how to use these fragments in in ccp4i (3 mers and 9 mers).
I got these results after submission of my sequences in robetta.

Your results are available at:

Result Details
http://rosettaserver.bakerlab.org/fragmentqueue.jsp?id=45590
<http://rosettaserver.bakerlab.org/fragmentqueue.jsp?id=45590>

Download Fragment Files

http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_03_05.200_v1_3

<http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_03_05.200_v1_3>

http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_09_05.200_v1_3

<http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_09_05.200_v1_3>
*
*



Thanks & Regards,


VIKRAM DALAL
Research Scholar
Macromolecular Crystallographic Unit
Department of Biotechnology
Indian Institute of TechnologyÂ
Roorkee (INDIA)








-- 
Dr Daniel John Rigden Tel:(+44) 151 795 4467

Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpoolhttp://pcwww.liverpool.ac.uk/~drigden/
<http://pcwww.liverpool.ac.uk/%7Edrigden/>
Crown St.,
Liverpool L69 7ZB, U.K.


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

Re: [ccp4bb] Help in running AMPLE

[Daniel Rigden]

Dear Vikram

Your file aat000_03_05.200_v1_3 
 
contains fragments of 3 residues length, the other one contains 
9-residue fragments. Download these to your own computer and then, in 
the CCP4i interface to AMPLE, use the Browse buttons in the Fragment 
Files section to locate them as 3mers and 9mers respectively.   Once 
you've told AMPLE where your Rosetta installation is, and filled in the 
other Input Files as normal, you're good to go.


Best wishes

Daniel

On 21/10/16 10:50, Vikram Dalal wrote:

Hi eveyone,

I want to run the ample in ccp4i. I have generated the files for my 
sequence using robetta fragment library server. But, I do not know how 
to use these fragments in in ccp4i (3 mers and 9 mers). I got these 
results after submission of my sequences in robetta.


Your results are available at:

Result Details
http://rosettaserver.bakerlab.org/fragmentqueue.jsp?id=45590 



Download Fragment Files
http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_03_05.200_v1_3 

http://rosettaserver.bakerlab.org/downloads/fragments/45590/aat000_09_05.200_v1_3 


*
*



Thanks & Regards,


VIKRAM DALAL
Research Scholar
Macromolecular Crystallographic Unit
Department of Biotechnology
Indian Institute of Technology
Roorkee (INDIA)








--
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] molecular replacement with poor model

[Daniel Rigden]

Dear Ursula

AMPLE is also worth trying, especially but not exclusively for smaller 
and predominantly helical targets.  It was originally conceived for 
novel folds but you may well find ab initio modelling methods get closer 
to the real structure of distant homologs than the nearest available 
templates.  AMPLE will produce a range of search models of different 
sizes, through clustering and truncation of a model set, and this 
sampling can be helpful.  You can install Rosetta for the modelling or, 
thanks to Yang Zhang's efforts, take a tar file direct from his Quark 
server (http://zhanglab.ccmb.med.umich.edu/QUARK/) and give it to AMPLE. 
Higher resolution helps generally, but we have had success out to 2.9A.


Good luck

Dan


On 13/12/14 10:44, Claudia Millán Nebot wrote:

Dear Ursula,

If you have a resolution around 2.0 A you can try some of the following:

- Expand the partial solution with shelxe autotracing feature.

- Do a search with ARCIMBOLDO_LITE using the partial solution. You can 
fin the program at http://chango.ibmb.csic.es/arcimboldo_lite. Then, 
instead of searching for ideal alpha helices, you can input your 
solution and search for 2 copies.


Hope it helps. Best,

Claudia



Claudia Millán (cmn...@ibmb.csic.es mailto:cmn...@ibmb.csic.es)

Crystallographic Methods Group

http://chango.ibmb.csic.es

Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

Barcelona, Spain

LinkedIn: es.linkedin.com/in/claudiamillan/ 
http://es.linkedin.com/in/claudiamillan/http://es.linkedin.com/pub/claudia-mill%C3%A1n/60/a76/821/


ResearchGate: 
https://www.researchgate.net/profile/Claudia_Millan?ev=hdr_xprf




2014-12-12 22:38 GMT+01:00 Ursula Schulze-Gahmen 
uschulze-gah...@lbl.gov mailto:uschulze-gah...@lbl.gov:


I am trying molecular replacement with a very poor model. The
model consists mainly of 1 long helix and two slightly bent
antiparallel helices. After dividing it into 2 fragments, I was
able to find a solution for one of the fragments ( at least I
think so after looking at maps, packing, refinement etc). But even
if I place the first solution as fixed ensemble in phaser, I
cannot find a solution for the second fragment ( 18% sequence
identity). From the structure of the model and the packing it
seems clear where the fragment should go roughly.

Are there any other programs other than phaser that might be able
to solve this problem? I tried already epmr and mr_rosetta without
success.
I also tried to just superimpose the complete model onto the
partial solution. This results in quite nice packing, but doesn't
refine. Is there a rigid program refinement program with very
large convergence?

Ursula

-- 
Ursula Schulze-Gahmen, Ph.D.

Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220 tel:94720-3220
(510) 643 9491



--
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.liv.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

[ccp4bb] Postdoctoral position at the University of Liverpool

[Daniel Rigden]

Post-doc position in structural biology at the School of Biological
Sciences, University of Liverpool, UK


An enthusiastic and dedicated individual is sought for a 2 year
BBSRC-funded project that will explore the potential of ab initio
protein models for X-ray crystal structure solution by Molecular
Replacement. Recent high-profile advances in ab initio modeling have
generally employed large scale computing resources. Nevertheless, we
have shown (Rigden et al. (2008), Acta Cryst. D64, 1288) that
appropriate processing and combination into ensembles of main-chain only
models, generated on PCs, can produce search models that successfully
solve structures. This project will test automated procedures for
generating ranked search models from the results of ab initio modeling,
principally by locally installed ROSETTA. The method will also be
extended to the use of ab initio models for individual domains in
multi-domain proteins. The resulting processes will be incorporated into
the MR pipeline MrBUMP for eventual distribution as part of the CCP4
crystallography software package.


You should have a PhD in Bioinformatics or a relevant discipline;
experience of protein structure modeling and/or protein crystallography
is highly desirable. In-depth knowledge of an appropriate scripting
language eg Python and the ability to deal with large amounts of data
are essential. You will generate protein models for a series of chosen
test cases, cluster and process them, then systematically analyse the
success of MR using PHASER or other software. You will participate in
manuscript preparation.

Full details and instructions on how to apply are here

http://www.liv.ac.uk/working/job_vacancies/research/R-572008.htm

Informal enquiries may be made to me.

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

Re: [ccp4bb] 3D modeling program

[Daniel Rigden]

I agree with previous posts that the reality of inferring evolutionary
relationships is often messy, but there is no excuse for being unclear
on the concepts and, in particular, for use of the % homology construct,
still far too common in supposedly good journals.

BTW, %identity is clear but not always unambiguous...

May AC. Percent sequence identity; the need to be explicit.
Structure. 2004 May;12(5):737-8.  PMID: 15130466

Dan



On Sat, 2008-12-06 at 21:33 +0100, Anastassis Perrakis wrote:
 I think we are getting a bit too philosophical on a matter which is  
 mainly terminology .
 
 1. To quantify how similar two proteins are, one should best refer to  
 'percent identity'. Thats clear, correct and unambiguous.
 2. One can also refer to similarity. In that case it should be  
 clarified what is considered to be similar, mainly which comparison  
 matrix was used to quantify the similarity.
 3. Homology means common evolutionary origin. One understanding is  
 that homology refers to the genome of 'LUCA', the hypothetical last  
 universal common ancestor. I am not an evolutionary biologist, but I  
 would clearly disagree that homology is a leftover pre-Darwinian term.  
 The very notion of homology is only meaningful in the context of  
 evolution.
 
 Thus, to me:
 
 1. These proteins are 56% identical is clear.
 2. These proteins are 62% similar is unclear.
 3. These proteins are 62% similar using the Dayhoff-50 matrix is Ok.
 4. These proteins are homologous is clear, but can be subjective as  
 to what homology is.
 5. These proteins are 32% homologous is simply wrong.
 
 Sorry for the non-crystallographic late evening blabber.
 
 A.
 
 On 6 Dec 2008, at 21:09, Dima Klenchin wrote:
 
  Having a generic dictionary definition is nice and dandy. However,  
  in the present context, the term 'homology' has a much more  
  specific meaning: it pertains to the having (or not) of a common  
  ancestor. Thus, it is a binary concept. (*)
 
  But how do we establish phylogeny? - Based on simple similarity!  
  (Structural/morphological in early days and largely on sequence  
  identity today). It's clearly a circular logic: Lets not use  
  generic definition; instead, lets use a specialized definition; and  
  lets not notice that the specialized definition wholly depends on a  
  system that is built using the generic definition to begin with.
 
  Plus, presumably all living things trace their ancestry to the  
  primordial soup - so the presence or a lack of ancestry is just a  
  matter of how deeply one is willing to look. In other words, it's  
  nice and dandy to have theoretical binary concept but in practice it  
  is just as fuzzy as anything else.
 
  IMHO, the phylogenetic concept of homology in biology does not buy  
  you much of anything useful. It seems to be just a leftover from pre- 
  Darwinian days - redefined since but still lacking solid foundation.
 
  Dima
-- 
Dr Daniel John Rigden Tel:(+44) 151 795 4467
School of Biological Sciences FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool
Crown St.,
Liverpool L69 7ZB, U.K.