Re: [ccp4bb] ccp4 development resources

[Phil Evans]

Dear Lothar

[I’ve copied this to the CCP4 team in case they have suggestions]

I don’t think there is any specific guidance for writing a “CCP4 program” - the 
programs distributed in the suite have been written in a diversity of styles 
and languages over many years - but I can offer a few thoughts

As a former Fortran programmer, I wouldn’t recommend Fortran now. There some 
really useful libraries to handle common crystallographic calculations, written 
in C++, and accessible also from Python, but not easily from Fortran. These 
include the Clipper library in CCP4 (from Kevin Cowtan); and cctbx, which is 
used by Phenix, but is also distributed by CCP4. These libraries also provide 
access to the file formats for reflection lists and coordinates, see below. 
Personally, I write in C++, but the use of Python at the high level along with 
C++ libraries to do the compute-intensive parts is also popular, and this 
approach is used in Phenix and DIALS

Reflection lists should be read and written using the binary MTZ file format, 
using the libraries. The “normal” merged MTZ files (each hkl occurring only 
once) can most easily be accessed using the Clipper or cctbx libraries, though 
the lower level CCP4 libraries can also be used. Unmerged reflection lists 
(multiple observations fro each hkl), in the data reduction steps, are a bit 
more complicated. Traditionally MTZ files may contain multiple types of data 
for each hkl, identified by column lables,e.g. different observed intensities, 
or Fs, phases, figures of merit, etc, but the recent ccp4 GUI ccp4i2 stores 
“miniMTZ” files containing only one type of data, e.g. observed I/sigma(I), 
phase/figure-of-merit, map coefficients etc, and this may be more used in future

Coordinate files also should be read and written using libraries, as this 
allows automatic switching between old-style PDB format and mmCIF. Clipper 
provides easy APIs for simple use, built on top of the more elaborate but 
flexible MMDB2 library

I find the CCP4 build system inconvenient for development work, though is good 
for distribution:  I just use an editor and a Makefile, old school, but other 
people probably have other more modern ways of working

Looking at existing (preferably reasonably recent) programs is useful to see 
various ways of accessing the general framework of reflection lists and 
coordinate lists which are central to most crystallographic calculations

Good luck
Phil


> On 13 Jul 2017, at 15:54, esse...@helix.nih.gov wrote:
> 
> Dear Prof. Evans,
> 
>  I am writing to you to solicit your expertise as a ccp4 developer. The
> principal investigator of our group has interest in developing ccp4-based
> programs and has done so with some success. However, I was wondering if
> there is a comprehensive set of instructions how to write ccp4 programs well
> and which tools to use incl. perhaps IDEs like Eclipse or so.  I know about
> the developer page on ccp4's website but it seems to be heavy on interfaces
> i.e. I have not found a clear comprehensive set of tools for ccp4 developers
> that includes how to write makefiles etc. I think the PI is mostly
> interested in small stand-alone fortran program that do not require much
> graphics. Can you give me a hint ? It is possible that I cannot see the
> forest for all the trees - in which case I apologize.
> 
> Thanks and kind regards,
> 
>  Dr. Lothar Esser
> 
>  National Institutes of Health
>  National Cancer Institute.
> 

[ccp4bb] A challenging Molecular replacement

[CDaddy]

I am a structural biologist who is teaching X-ray crystallography. Recently I 
noticed that BrlR structure (5XQL) was solved using molecular replacement with 
a search model of very low similarity. I am very interested in this structure 
because I think this a very good example to show students how to solve phase 
problem using molecular replacement, especially when the model and the target 
protein share a low sequence identity. However, when I downloaded the data from 
PDB, I found that I cannot solve the phase problem using Phaser as mentioned by 
the authors. During this procedure BmrR (PDB:1R8E) was used as the search 
model. I tried to consult the authors for help but receive no response by now. 
Since the description of this issue in the literature is very brief, could 
anyone please spend a little time on this molecular replacement and give me 
some advices on this issue? I like to learn some valuable tricks. Your 
assistance will be highly appreciated.


All the best,


Richard.

Re: [ccp4bb] A challenging Molecular replacement

[Tanner, John J.]

Richard,

I can’t help you with 5XQL.

However, I can point out a recent structure from my group that might be useful 
for teaching. The structure was solved by MR with a search model that had 33% 
sequence identity and represented only 46% of the target structure. The Methods 
section of the paper has a detailed description of the phasing procedure. We 
used BALBES, then did autobuilding in phenix from the BALBES/REFMAC map.

https://www.ncbi.nlm.nih.gov/pubmed/28420730

John J. Tanner
Professor of Biochemistry and Chemistry
Chair, Biochemistry Department Graduate Admissions Committee
Department of Biochemistry
University of Missouri-Columbia
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Fax: 573-882-5635
Email: tanne...@missouri.edu
http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A

On Jul 17, 2017, at 11:01 AM, CDaddy 
<2295867...@qq.com> wrote:

I am a structural biologist who is teaching X-ray crystallography. Recently I 
noticed that BrlR structure (5XQL) was solved using molecular replacement with 
a search model of very low similarity. I am very interested in this structure 
because I think this a very good example to show students how to solve phase 
problem using molecular replacement, especially when the model and the target 
protein share a low sequence identity. However, when I downloaded the data from 
PDB, I found that I cannot solve the phase problem using Phaser as mentioned by 
the authors. During this procedure BmrR (PDB:1R8E) was used as the search 
model. I tried to consult the authors for help but receive no response by now. 
Since the description of this issue in the literature is very brief, could 
anyone please spend a little time on this molecular replacement and give me 
some advices on this issue? I like to learn some valuable tricks. Your 
assistance will be highly appreciated.

All the best,

Richard.

Re: [ccp4bb] A challenging Molecular replacement

[Ethan Merritt]

On Tuesday, 18 July 2017 00:01:59 CDaddy wrote:
> I am a structural biologist who is teaching X-ray crystallography. Recently I 
> noticed that BrlR structure (5XQL) was solved using molecular replacement 
> with a search model of very low similarity. I am very interested in this 
> structure because I think this a very good example to show students how to 
> solve phase problem using molecular replacement, especially when the model 
> and the target protein share a low sequence identity. However, when I 
> downloaded the data from PDB, I found that I cannot solve the phase problem 
> using Phaser as mentioned by the authors. During this procedure BmrR 
> (PDB:1R8E) was used as the search model. I tried to consult the authors for 
> help but receive no response by now. Since the description of this issue in 
> the literature is very brief, could anyone please spend a little time on this 
> molecular replacement and give me some advices on this issue? I like to learn 
> some valuable tricks. Your assistance will be highly appreciated.


I have no familiarity with either structure, but even a cursory glance at the
cartoon depiction of 5XQL in the PDB suggests that you would want to chop
it into at least 3 pieces in order to use it for molecular replacement.
Did you try placing the N- and C- terminal domains separately after
chopping out the long connecting helix?

Ethan

-- 
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

[ccp4bb] suggestions are welcome

[高艺娜]

Hi all ,

It has been reported the Negative stain EM of a protein A-B complex, but 
according to my gel filtration results (I purified A and B respectively for 
incubation) , I found that A could not bind to B, of course I tried different 
buffer condition with various pH value, even the binding condition only had 50 
mm Kcl. Do you have any suggestion or methods that I can try to get the protein 
A-B complex?

Any suggestion is welcome，

Thank you all ,

Best,

Re: [ccp4bb] suggestions are welcome

[El Sahili Abbas (Dr)]

Hi,

This might indicate that the binding affinity of your complex is not high 
enough for it to resist the gel filtration dilution. Maybe running a native 
PAGE gel would allow you to visualise a complex.

Best
A

> On 18 Jul 2017, at 10:25, 高艺娜  wrote:
>
> Hi all ,
>
> It has been reported the Negative stain EM of a protein A-B complex, but 
> according to my gel filtration results (I purified A and B respectively for 
> incubation) , I found that A could not bind to B, of course I tried different 
> buffer condition with various pH value, even the binding condition only had 
> 50 mm Kcl. Do you have any suggestion or methods that I can try to get the 
> protein A-B complex?
>
> Any suggestion is welcome，
>
> Thank you all ,
>
> Best,


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Re: [ccp4bb] A challenging Molecular replacement

[chenzhonghao...@163.com]

Dear Dr.Richard,

  Your paper gave a good example for us.
Is it possible that you send me all the related files? It will be very helpful 
for students to learn the detail of solving difficult protein structures.

Thanks in advance!

best,

Zhonghao Chen
China agricultural university.



chenzhonghao...@163.com
 
From: Tanner, John J.
Date: 2017-07-18 00:34
To: CCP4BB
Subject: Re: [ccp4bb] A challenging Molecular replacement
Richard, 

I can’t help you with 5XQL.  

However, I can point out a recent structure from my group that might be useful 
for teaching. The structure was solved by MR with a search model that had 33% 
sequence identity and represented only 46% of the target structure. The Methods 
section of the paper has a detailed description of the phasing procedure. We 
used BALBES, then did autobuilding in phenix from the BALBES/REFMAC map. 

https://www.ncbi.nlm.nih.gov/pubmed/28420730 

John J. Tanner
Professor of Biochemistry and Chemistry
Chair, Biochemistry Department Graduate Admissions Committee 
Department of Biochemistry
University of Missouri-Columbia
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Fax: 573-882-5635
Email: tanne...@missouri.edu
http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A

On Jul 17, 2017, at 11:01 AM, CDaddy <2295867...@qq.com> wrote:

I am a structural biologist who is teaching X-ray crystallography. Recently I 
noticed that BrlR structure (5XQL) was solved using molecular replacement with 
a search model of very low similarity. I am very interested in this structure 
because I think this a very good example to show students how to solve phase 
problem using molecular replacement, especially when the model and the target 
protein share a low sequence identity. However, when I downloaded the data from 
PDB, I found that I cannot solve the phase problem using Phaser as mentioned by 
the authors. During this procedure BmrR (PDB:1R8E) was used as the search 
model. I tried to consult the authors for help but receive no response by now. 
Since the description of this issue in the literature is very brief, could 
anyone please spend a little time on this molecular replacement and give me 
some advices on this issue? I like to learn some valuable tricks. Your 
assistance will be highly appreciated.

All the best,

Richard.