Re: [ccp4bb] recombinant protein-based fluorophores

[Smith Liu]

eGFP, for example, can be fused to the C-
terminal of your target protein. Fluorescent proteins were usually highly 
soluble, especially if you select to express in insect cells, which has the 
tendency to express proteins in soluble states.

Smith



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On 12/07/2021 07:21, samer halabi wrote:
Dear All,
Sorry for disturbing you all with my current inquiry. However, I am in the 
process of designing and expressing soluble proteins and I thought to ask for 
your valuable advice.


1-What would be your first choice (protein-based) fluorophore to link it to the 
soluble protein at its C-terminus?
2-Would this fluorophore be suitable for carrying later experiments in vivo and 
in vitro withstanding such conditions and still having detectable fluorescence 
signal (medium to high)?
3-Would this fluorophore of choice stand denaturing and renaturing conditions, 
as refolding the protein chimera (soluble protein + the linked fluorophore) 
from bacterial inclusion bodies that were dissolved in 8M Urea or 6M 
Guanidine-HCl?
4-Have you had success with producing APC or R-PE in such manner (secreting 
them from insect cells or refolding them from bacterial inclusion bodies)?


Thank you in advance.
Best regards,
Samer





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Re: [ccp4bb] AW: Re: [ccp4bb] AW: Re: [ccp4bb] coordinate transformation

[Smith Liu]

sorry, how i move the mtz into the transformed pdb for the question in my 
previous email?




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在2017年12月18日 23:37，Smith Liu 写道：
thanks. i may mean something other. for example, if i rotate the pdb by 30 
degree (or 29.5 degree), or i shift the pdb along x-axis by something for 
example 0.123*a, then how i move the mtz map correspondingly for the fitting of 
mtz into the transformed map?




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Smith Liu
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在2017年12月18日 21:58，herman.schreu...@sanofi.com 写道：

Dear Smith,

The map extends through the whole crystal. What happens is that the map is 
calculated around the atom you clicked on during centering. So by centering on 
your transformed pdb, you will sample the same map at a different position. 
Just load your transformed pdb and untransformed mtz and try.

If the transformed pdb does not fit the map, something went wrong during the 
transformation of your pdb. If you have applied an origin shift (is not equal 
to applying a crystallographic symmetry operation), you have to recalculate the 
mtz, e.g. by running another round of refinement.

I hope this is clear so.

Herman

 

Von: Smith Liu [mailto:smith_liu...@163.com]
Gesendet: Montag, 18. Dezember 2017 14:52
An: Schreuder, Herman /DE
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Re: [ccp4bb] coordinate transformation

 

you mean the mtz map will transform simutaneously?




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Smith Liu

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邮箱：smith_liu...@163.com

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签名由 网易邮箱大师 定制






在2017年12月18日 21:26，herman.schreu...@sanofi.com写道：

If you use coot with on the fly map calculation (e.g. you load an mtz and not a 
map file), you do not need to transform the map. Otherwise I would recommend to 
run one more round of refinement and produce a new map your usual way. This 
will also get rid of any rounding errors due to the transformation.

 

Best,

Herman

 

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu
Gesendet: Montag, 18. Dezember 2017 14:16
An:CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] coordinate transformation

 

Dear All,

 

If I have a set of PDB with the corresponding density map, after I transform 
the PDB based on the suggestion of everybody, is any way to transform the map 
so that the map will be fit with the transformed PDB?

 

Smith






At 2017-12-18 18:39:34, "Eleanor Dodson" 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

I showed you pdbset ..

Find the centre of mass for your assembly.

Move it where you will

pdbset xyzin mow.pdb

end

Find  CoM 0.7 1.3 -0.2

Hmm - a little thought - centre at 1 -1 0   say

pdbset yzin now.pdb xyzout changed.pdb

symgen x , y-2, z 

end

New CoM  0.7 -0.7  -0.2

Eleanor

 

 

 

On 18 December 2017 at 00:19, Edward A. Berry <ber...@upstate.edu> wrote:

Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? or 
0.1,0.1,0.1 if I still want it near the origin but biased toward the inside of 
the positive-going cell?
eab



On 12/14/2017 07:23 PM, James Holton wrote:

What I usually do for this is make a copy of the PDB file and change all the 
atom x-y-z positions to "1.000".  Then I use something like reforigin or my 
"origins.com" script to shift the original coordinates via allowed symmetry 
operations, origin shifts, or perhaps indexing ambiguities until it is as close 
as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 
0,0,0 because there are generally at least two symmetry-equivalent places that 
are equidistant from the origin. Declaring the reference to be a bit off-center 
breaks that ambiguity, and also biases the result toward having all-positive 
x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:


Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...


Thanks,

Tommi

 

 

 

 

Re: [ccp4bb] AW: Re: [ccp4bb] AW: Re: [ccp4bb] coordinate transformation

[Smith Liu]

thanks. i may mean something other. for example, if i rotate the pdb by 30 
degree (or 29.5 degree), or i shift the pdb along x-axis by something for 
example 0.123*a, then how i move the mtz map correspondingly for the fitting of 
mtz into the transformed map?




| |
Smith Liu
|
|
邮箱：smith_liu...@163.com
|

签名由 网易邮箱大师 定制




在2017年12月18日 21:58，herman.schreu...@sanofi.com 写道：

Dear Smith,

The map extends through the whole crystal. What happens is that the map is 
calculated around the atom you clicked on during centering. So by centering on 
your transformed pdb, you will sample the same map at a different position. 
Just load your transformed pdb and untransformed mtz and try.

If the transformed pdb does not fit the map, something went wrong during the 
transformation of your pdb. If you have applied an origin shift (is not equal 
to applying a crystallographic symmetry operation), you have to recalculate the 
mtz, e.g. by running another round of refinement.

I hope this is clear so.

Herman

 

Von: Smith Liu [mailto:smith_liu...@163.com]
Gesendet: Montag, 18. Dezember 2017 14:52
An: Schreuder, Herman /DE
Betreff: [EXTERNAL] Re: [ccp4bb] AW: Re: [ccp4bb] coordinate transformation

 

you mean the mtz map will transform simutaneously?




|

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Smith Liu

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邮箱：smith_liu...@163.com

|

签名由 网易邮箱大师 定制






在2017年12月18日 21:26，herman.schreu...@sanofi.com写道：

If you use coot with on the fly map calculation (e.g. you load an mtz and not a 
map file), you do not need to transform the map. Otherwise I would recommend to 
run one more round of refinement and produce a new map your usual way. This 
will also get rid of any rounding errors due to the transformation.

 

Best,

Herman

 

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu
Gesendet: Montag, 18. Dezember 2017 14:16
An:CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] coordinate transformation

 

Dear All,

 

If I have a set of PDB with the corresponding density map, after I transform 
the PDB based on the suggestion of everybody, is any way to transform the map 
so that the map will be fit with the transformed PDB?

 

Smith






At 2017-12-18 18:39:34, "Eleanor Dodson" 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

I showed you pdbset ..

Find the centre of mass for your assembly.

Move it where you will

pdbset xyzin mow.pdb

end

Find  CoM 0.7 1.3 -0.2

Hmm - a little thought - centre at 1 -1 0   say

pdbset yzin now.pdb xyzout changed.pdb

symgen x , y-2, z 

end

New CoM  0.7 -0.7  -0.2

Eleanor

 

 

 

On 18 December 2017 at 00:19, Edward A. Berry <ber...@upstate.edu> wrote:

Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? or 
0.1,0.1,0.1 if I still want it near the origin but biased toward the inside of 
the positive-going cell?
eab



On 12/14/2017 07:23 PM, James Holton wrote:

What I usually do for this is make a copy of the PDB file and change all the 
atom x-y-z positions to "1.000".  Then I use something like reforigin or my 
"origins.com" script to shift the original coordinates via allowed symmetry 
operations, origin shifts, or perhaps indexing ambiguities until it is as close 
as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 
0,0,0 because there are generally at least two symmetry-equivalent places that 
are equidistant from the origin. Declaring the reference to be a bit off-center 
breaks that ambiguity, and also biases the result toward having all-positive 
x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:


Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...


Thanks,

Tommi

 

 

 

 

Re: [ccp4bb] coordinate transformation

[Smith Liu]

Dear All,


If I have a set of PDB with the corresponding density map, after I transform 
the PDB based on the suggestion of everybody, is any way to transform the map 
so that the map will be fit with the transformed PDB?


Smith







At 2017-12-18 18:39:34, "Eleanor Dodson" 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

I showed you pdbset ..

Find the centre of mass for your assembly.

Move it where you will


pdbset xyzin mow.pdb

end

Find  CoM 0.7 1.3 -0.2


Hmm - a little thought - centre at 1 -1 0   say


pdbset yzin now.pdb xyzout changed.pdb


symgen x , y-2, z 


end


New CoM  0.7 -0.7  -0.2


Eleanor









On 18 December 2017 at 00:19, Edward A. Berry  wrote:
Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? or 
0.1,0.1,0.1 if I still want it near the origin but biased toward the inside of 
the positive-going cell?
eab


On 12/14/2017 07:23 PM, James Holton wrote:
What I usually do for this is make a copy of the PDB file and change all the 
atom x-y-z positions to "1.000".  Then I use something like reforigin or my 
"origins.com" script to shift the original coordinates via allowed symmetry 
operations, origin shifts, or perhaps indexing ambiguities until it is as close 
as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 
0,0,0 because there are generally at least two symmetry-equivalent places that 
are equidistant from the origin. Declaring the reference to be a bit off-center 
breaks that ambiguity, and also biases the result toward having all-positive 
x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:

Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...


Thanks,

Tommi

Re: [ccp4bb] secondary structure prediction

[Smith Liu]

yes




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在2017年12月07日 09:11，zheng zhou 写道：
Thanks for many advices. I was not clear in the previous email. I know
the close homologous protein (20% identity, total 500aa), but the
fragment hits (30~40aa, identity 40~50%) are from other proteins in
PDB. I am trying to see whether the fragments from non-homologous
proteins may help the secondary structure prediction.

Best,
Z

On Wed, Dec 6, 2017 at 10:14 PM, zheng zhou <zhengzho...@gmail.com> wrote:
> Dear CCP4 community,
>
> Sorry for the off-topic question. I am trying to design constructs for
> structure studies. It only has a homolog structure in PDB with
> sequence identity ~20%. When I blast against PDB sequence, there are
> quite a few motif hits (30~40aa, identity 40~50%). Any prediction
> tools utilize this information?
>
> Thanks for your advice in advance.
>
> Best,
>
> Zheng

Re: [ccp4bb] secondary structure prediction

[Smith Liu]

Rosetta




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Smith Liu
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在2017年12月06日 22:14，zheng zhou 写道：
Dear CCP4 community,

Sorry for the off-topic question. I am trying to design constructs for
structure studies. It only has a homolog structure in PDB with
sequence identity ~20%. When I blast against PDB sequence, there are
quite a few motif hits (30~40aa, identity 40~50%). Any prediction
tools utilize this information?

Thanks for your advice in advance.

Best,

Zheng

Re: [ccp4bb] script for shape complementarity

[Smith Liu]

how about modify your selected part of protein into two molecules






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签名由 网易邮箱大师 定制




在2017年12月03日 16:21，joy yang 写道：
Dear All, 



can anyone shed a clue on how to define the suface area used in ccp4 sc (shape 
complementarity) script? The script that I could find on internet is as 
following:


sc XYZIN a22.pdb SURFIN1 A.grasp_surf SURFIN2 B.grasp_surf \
   SURFOUT1 A.sc_surf SURFOUT2 B.sc_surf <

Re: [ccp4bb] RMSD calculation for large assemblies

[Smith Liu]

how about mean square deviation of the rmsd of each c alpha？





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签名由 网易邮箱大师 定制




在2017年12月02日 22:48，Reza Khayat 写道：

Hi,





I'm analyzing the RMSD between 60 subunits of a virus. Can someone identify a 
program that can generate a spread for the RMSD between equivalent C-alpha 
atoms? For example, the C-alpha atom for amino acid 39 may have RMSD values 
from 0.1 to 1.5. Coot does a nice job of automatically detecting and 
calculating RMSD, but I'd like to have the spread for each atom in the final 
graph that Coot generates. Thank you.




Best wishes,
Reza 





Reza Khayat, PhD
Assistant Professor 
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Vacuum pump in cold room

[Smith Liu]

use the brand of  vacuum pump without oil


发自网易邮箱大师


在2017年10月29日 01:17，Denis Rousseau 写道：
Does anyone have experience with a vacuum pump in cold room? We have been told 
they may not start because of the viscosity of the oil?


Thanks


Denis Rousseau

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Schulz, 
Eike-Christian [eike.sch...@mpsd.mpg.de]
Sent: Friday, October 27, 2017 4:31 PM
To:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] using CC1/2 to define resolution limit in Xscale



Dear all,

 

I would like to compare > 15 datasets and would like to use a common CC1/2 
value as an objective criterion to determine the resolution cut-off.

 

All data were integrated in XDS.

 

Is there a convenient way to apply this in XSCALE or in any of its alternatives?

 

With best regards,

 

Eike

 

 

 

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

[Smith Liu]

suppose protease is the issue, then avoid overnight dialysis


发自网易邮箱大师


在2017年10月26日 22:18，Anamika Singh 写道：
Dear All,


I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM 
Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the 
precipitation problem during dialysis but with the addition of 50mM L-Arg 
somehow managed to overcome the precipitation issue. But this time I have seen 
after overnight dialysis there are degradation products on SDS page. I have 
used protease inhibitor (PMSF) in my sonication buffer. 


Please suggest me to overcome this problem. 



Thanks
--

Anamika

Re: [ccp4bb] Off topic: denaturing urea gels

[Smith Liu]

your enzyme cannot give definite fragment. thus smear


发自网易邮箱大师


在2017年09月30日 19:28，Mohammad Khan 写道：
Dear all,


I am working with an exonuclease and I run the digested DNA on a 
8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 15 
well comb. I run my gels at 70 V for as long as 4 hours till my undigested DNA 
reaches half the gel distance. I use 20-30 nt long susbtrates.


I am mostly not able to get distinct bands of the digested products but rather 
get a smear. Is there any way to make sure that I get distinct digested 
products rather than a smear?


I am looking forward for suggestions from all!


Thank you.


Ciao!

Re: [ccp4bb] Biotin binding protein

[Smith Liu]

if necessary, phage or yeast display to screen



发自网易邮箱大师


在2017年09月24日 07:05，Reza Khayat 写道：
Hi,

Sorry for the non crystallography question. Is anyone aware of monomeric 
proteins (<20kDa) that can bind to biotin? We need this for a couple of 
different projects where biotin covalently modifies a ligand of interest. We'd 
like to complex the biotin to a protein Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Difficult purification with imac columns

[Smith Liu]

change buffer，or use different resin matrix
from another compny


发自网易邮箱大师


在2017年09月15日 19:53，Narayanan Ramasubbu 写道：
Hi. We are working on a periplasmic protein that breaks naked glycans in 
peptidoglycans. There is truncated structure available but our target is the 
full length protein. The difficulty us that it strongly binds to the resin with 
or without his.tag. Changing the resin to acrylamide did not help.
Has anyone come across similar problem and how was it resolved.
The pdb structure is the catalytic domain and mussing a region that, in my 
opinion, binds to the resin.  
Thank you in advance
Sent from my iPhone

Re: [ccp4bb] Fast searching of articles related to your PubMed indexed paper

[Smith Liu]

can you updated the server so that the most recent articles can be found?



发自网易邮箱大师


在2017年07月25日 11:49，Yaoqi Zhou 写道：
Fast searching of articles related to your PubMed indexed paper  

Are you spending too much time searching the literature to prepare your grant 
proposals, manuscripts and keeping track of research happening in your field? 
We have built a literature-based search engine 
(http://pubmed.ict.griffith.edu.au/) which is powered by a combination of 
state-of-the-art methods to locate relevant articles to your published 
PubMed-indexed papers within a few seconds.

While providing you the ability to locate relevant articles, we are seeking 
your feedback regarding the performance of these algorithms. By annotating 
recommended articles as relevant, somewhat relevant, or irrelevant, you are 
participating in a community-wide effort of establishing a gold-standard 
benchmark for relevant literature search.

The accuracy of this benchmark is ensured by your domain knowledge and 
experience as your judgements will be based in the context of your own article. 
With your expertise, the entire process will take only a few minutes to 
complete (1 paper, 6 recommendations). Upon completion, this community-wide 
dataset will be available to freely download and redistribute, encouraging 
development of next-generation literature searching and retrieval 
methodologies, something we all desperately need.

Due to current data availability, this relevant-paper search is limited to 
papers indexed by PubMed between January 1, 2007 to December 31, 2016 (the end 
of last year). Click http://pubmed.ict.griffith.edu.au/ to start. As this is a 
product in development, any constructive comments and suggestions will be truly 
appreciated.

If you like this idea of community-wide benchmark for literature 
recommendation, please forward this email to your colleagues in the scientific 
community.

Initial feedbacks to our server. “The process was certainly fast!”, “Although 
the articles found in the search were not necessarily exactly relevant, many 
are articles that I don't think I would have come across easily with a 
traditional search”, “Very nice work. I think there can be a longer list” 
(limited to 30 currently).

Thank you!

Yaoqi

Professor Yaoqi Zhou | Research Leader
Institute for Glycomics
Griffith University | Gold Coast campus | QLD 4222 | Institute for Glycomics 
(G24) Room 2.10
T +61 7 5552 8228 | F +61 7 5552 9040 | email yaoqi.z...@griffith.edu.au 

http://sparks-lab.org (Group webpage)

http://griffith.edu.au/institute-glycomics (Institute Webpage)

On 25 Jul 2017, at 9:01 AM, CCP4BB automatic digest system 
 wrote:


There are 14 messages totaling 9878 lines in this issue.

Topics of the day:

 1. Buccaneer places residues in different asymmetric units (3)
 2. Primer design (7)
 3. PhD position at University of Oslo
 4. Postdoctoral position at Boston Children’s Hospital
 5. About weighting factor settings in new ccp4i2 (2)

--

Date:Mon, 24 Jul 2017 16:56:51 +0800
From:Lingxiao Zeng 
Subject: Buccaneer places residues in different asymmetric units

Dear All,

I tried to use buccaneer to build a model. The starting model is a partial 
model, after model building the Rwork and Rfree are reasonable but buccaneer 
places residues in different asymmetric units and the model looks really weird.

Is there any way to build the model into the same ASU or put different parts 
together after model building? Thanks!



Best,

Alice

--
Lingxiao Zeng
PhD candidate
School of Biomedical Sciences
The University of Hong Kong

--

Date:Mon, 24 Jul 2017 10:35:17 +0100
From:Jon Agirre 
Subject: Re: Buccaneer places residues in different asymmetric units

Dear Alice,

there's an option in both ccp4i and ccp4i2 interfaces that lets you tell
Buccaneer that you want to build the new model in the same place as the
partial model supplied - see my screenshots for reference. It might not be
on by default and perhaps it should be.

Please be aware that most newer developments and improvements will be put
on the ccp4i2 interface to Buccaneer - it would be helpful if you could
have a go and let us know what you think!

Hope this helps,

Jon

On 24 July 2017 at 09:56, Lingxiao Zeng  wrote:

Dear All,


I tried to use buccaneer to build a model. The starting model is a partial
model, after model building the Rwork and Rfree are reasonable but
buccaneer places residues in different asymmetric units and the model looks
really weird.


Is there any way to build the model into the same ASU or put different
parts together after model building? Thanks!




Best,

Alice

--
Lingxiao Zeng
PhD candidate
School of Biomedical Sciences
The University of Hong Kong




--
Dr Jon Agirre
York Structural Biology Laboratory / Department of 

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Smith Liu]

May i ask, whether the fluoresnce anisotropy method was reliable enough to 
determine the stoichiometry of a protein complex?


发自网易邮箱大师


在2017年07月22日 03:44，Phoebe A. Rice 写道：
You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?




++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton 
[eilise.mil...@gmail.com]
Sent: Friday, July 21, 2017 9:44 AM
To:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement


Completely agree, you need a higher DNA concentration. We have had luck with 10 
nM DNA.


Also, bubbles have a HUGE impact on how the fluorescent signal is measured. 
Make sure you spin your plates down (assuming you are using them) to remove any 
bubbles. We just had an undergrad read his anisotropy assay and the data looked 
horrible. He then realized he had not spun his plate down, after doing so the 
data was much more consistent.


Are you doing technical replicates? We do at least triplicates per plate.


All the best,

Morgan





Morgan E. Milton, PhD




On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi  
wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.

[ccp4bb] 回复：[ccp4bb] suggestions are welcome

[Smith Liu]

please characterize your a and b to see they really what they are



发自网易邮箱大师


在2017年07月18日 10:25，高艺娜 写道：
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] long loop

[Smith Liu]

Fab fragment binding towards long loop



在2017年07月04日 23:22，dongxiaofei 写道：
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] Separating Monomers and Dimers

[Smith Liu]

it was not stable for frozen storage. if necessary，using protein fresh without 
frozen

发自网易邮箱大师


在2017年06月27日 20:22，jai mohan 写道：
Dear all,
I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 
27kDa. After purification I ran a SDS page, the band at 27kDa confirms the 
monomer. The protein was stored at -20C, a week later again I ran a gel, this 
time I saw another new band between 50-60kDa, it confirms the protein solution 
contains both monomers and dimers. I would like to know, what is the best way 
to separate the monomers and dimers? One of my colleague advice me to go for 
sucrose gradient centrifugation and size exclusion chromatography.
However, I seek all your valuable suggestions and advice.


With best regards
Dr. S.M.Jaimohan

[ccp4bb] Fw:[ccp4bb] on Cell & Symmetry in coot

[Smith Liu]

Dear All,


I mean if the radius set in the Coot "Cell Symmetry" was too small, not enough 
monomers (less than 6) can be displayed to show the "continuous helix with a 
six-fold screw axis". If the radius was too large, as for the  "continuous 
helix with a six-fold screw axis" can be regarded as a "rod", too large radius 
will lead to show several rods in one window. But with the Coot window, it 
cannot distinguish which monomer was from which rod. Thus I cannot identify 6 
monomers forming the single rod, i.e., a  "continuous helix with a six-fold 
screw axis".


Can anyone explain in this situation how can I identify the 6 monomers in the 
Coot "Cell and SYmmetry" windows forming a single "continuous helix with a 
six-fold screw axis"?


Smith
 






 Forwarding messages 
From: "Smith Lee" <0459ef8548d5-dmarc-requ...@jiscmail.ac.uk>
Date: 2016-12-09 18:12:20
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] on Cell & Symmetry in coot

  Dear All,


There is a pdb, once opended in coot, it was a monomer (space group   P 65 2 2 
). But in the correspondence paper, it writes, "the subunits form a continuous 
helix with a six-fold screw axis".


I have tried to view with Coot the "six-fold screw axis" formed by 6 monomers. 
But in the "Cell & Symmetry" in Coot, if the radius is small, 6 monomers cannot 
be shown. If I increase the radius, more than 6 monomers would occur in the 
window, and it can hardly distinguish the 6 monomers forming the "six-fold 
screw axis".


In this situation, will you please let me know how to use Coot to identify the 
6 monomers forming the "six-fold screw axis"? In addition, suppose  6 monomers 
forming the "six-fold screw axis" have been identified in Coot, in order to 
save the pdb of each monomer, I need to click each monomer in mouse, then by 
"Save symmetry coordinates" to save the pdb of each monomer, right?


I am looking forward to getting your reply.


Smith

[ccp4bb] on R-free label

[Smith Liu]

Dear All,


Will you please tell me how to know whether my mtz file has the R-free label or 
not?


SMith

Re: [ccp4bb] Rfree below Rwork

[Smith Liu]

If both the PDB and mtz for the pdb have been assigned to P1 space group for 
some reason, can this lead to Rwork higher than Rfree during refinement?



If after converting my PDB and mtz to P1 space group, and I have forgotten what 
is the original space group for my PDB and mtz before conversion to P1 space 
group, is any method which can recover the original space group for my PDBand 
mtz, so that in the following refine Rwork would be lower than Rfree?


Smith








At 2015-07-01 01:55:22, Eleanor Dodson eleanor.dod...@york.ac.uk wrote:

I suppose if I was the referee for this structure and your FreeR is so close to 
the Rfactor I would ask you to ensure you had the right space group - is the 6 
fold NCS actually 2 fold NCS with a crystallographic 3 fold..
Cases occur where R32 is indexed as C2.. 


Certainly if the Rfree set is assigned randomly to reflections which are 
symmetry equivalents then you see this phenomena of Rfree = Rfactor


Eleanor


On 30 June 2015 at 18:26, Gerard Bricogne g...@globalphasing.com wrote:
Dear Wolfram,

 I have a perhaps optimistic view of the effect of high-order NCS
on Rfree, in the sense that I don't view it as a problem. People
have agonised to extreme degrees over the difficulty of choosing a
free set of reflections that would produce the expected gap between
Rwork and Rfree, and some of the conclusions were that you would need
to hide almost half of your data in some cases!

 I think it is best to remember that the idea of cross-validation
by Rfree is to prevent overfitting, i.e. ending up with a model that
fits the amplitudes too well compared to how well it determines the
phases. In the case of high-order NCS (in your case, the U/V ratio
that the old papers on NCS identified as the key quantity to measure
the phasing power of NCS would be less than 0.1!) the phases and the
amplitudes are so tightly coupled that it is simply impossible to fit
the amplitudes without delivering phases of an equally good quality.
In other words there is no overfitting problem (provided you do have
good and complete data) and the difference between Rfree and Rwork is
simply within the bounds of the statistical spread of Rfree depending
on the free set chosen.

 You are lucky to have 6-fold NCS, so don't let any reviewer
convince you that it is a curse, and make you suffer for it :-) .


 With best wishes,

  Gerard.

--

On Tue, Jun 30, 2015 at 12:58:44PM -0400, wtempel wrote:
 Hello,
 my question concerns refinement of a structure with 6-fold NCS (local
 automatic restraints in REFMAC) against 2.8 A data. The size of my free set
 is 1172 selected in thin resolution shells (SFTOOLS) and corresponding to
 4.3 % of reflections.
 A refmac run of 10 cycles of TLS and 10 cycles of CGMAT starts out at
 Rfree/Rcryst 0.271/0.272. After the 10th TLS cycle I have 0.227/0.224. Yes,
 Rfree  Rcryst. At the end of CGMAT I have 0.2072/0.2071.
 I understand that NCS stresses the independence assumption of the free set.
 Am I correct in believing that Rfree *may* be smaller than Rcryst even in
 the absence of a major mistake? My hope is that the combined wisdom of
 ccp4bb followers can point out my possible mistake,  suggest tests that I
 may perform to avoid them and, possibly, arguments in defense of a
 crystallographic model with Rfree  Rcryst.
 Many thanks,
 Wolfram Tempel


--

 ===
 * *
 * Gerard Bricogne g...@globalphasing.com  *
 * *
 * Global Phasing Ltd. *
 * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
 * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
 * *
 ===

[ccp4bb] on mosaicity estimation by iMosflm

[Smith Liu]

Dear All,

When I do mosaicity estimation by iMOSFLM, it shows, The mosaicity estimation 
has not worked for some reason. Message from Mosflm is - Unable to estimate 
mosaicity automatically from this image-determine a value visually. You should 
enter an estimated value to replace 0.05.

First I have input a lot of images, I think  Unable to estimate mosaicity 
automatically from this image-determine a value visually should be  Unable to 
estimate mosaicity automatically from theseimages-determine a value visually. 
Secondly, will you please tell me how can I determine a value visually? And in 
which step I can get a reliable mosaicity estimation?

I am looking forward to getting a reply from you.

Smithy.

[ccp4bb] on secondary structure restraint

[Smith Liu]

Dear All,


Even with the Coot secondary structure (for example helix) restraint selected 
and by this way we keep the secondary structure, I find the protein secondary 
structure formed in this way was not so typpical, for example, not all CO ( i) 
and NH (i+4) forms H-bonds, and there are H-bonds formed by CO ( i) and NH 
(i+3). I checks some RCSB PDBs, and find this kind of untypical H-bonds for the 
Helix backbone were not rare.


Do we accept this kind of untypical H-bonds containing PDB as normal, or do we 
think we need further refine the structure based on the map?


Smith

[ccp4bb] on resolution and explaination of the intersubunit bonds

[Smith Liu]

Dear All,
 
In order to acceptably explain the salt bridges, hydrophobic interactions and 
H-bonds among subunits in the crystal structure of a protein complex, is there 
a threshold resolution of the crystal, for example, if the crystal is poorer 
than 4A or 5A, the crystal structure solved cannot be used to acceptably 
explain the intersubunit interactions at the non-covalen bond level?
 
Smith
 
 

[ccp4bb] on b-factor

[Smith Liu]

Dear All,
 
In the PDB file, the b-factors were only determined by the quality of the map, 
is this view right or not?
 
Smith

[ccp4bb] HIS related crystallography issue

[Smith Liu]

Dear All,
 
Suppose the protein crystal resolution is about 2-3A, then in the map it should 
be rather difficult to distinguish the C and N in the sidechain of HIS. In this 
way we may regard the sidechain of HIS is flippable. But suppose in one flipped 
conformation of the HIS, the free N in the sidechain of HIS can form H-bond 
with the neighbour H of the OH of the Thr, in the other flipped conformation of 
the HIS, the free N in the sidechain of HIS cannot form H-bond with the 
neighbour H of the OH of the Thr (caused by distance issue).
 
Suppose whether the H-bond forms between the free N in the sidechain of HIS and 
the neighbour H of the OH of the Thr was very important biologically, in this 
situation how can we distinguish whether the H-bond forms?
 
Smith

[ccp4bb] HIS related crystallography issue

[Smith Liu]

Dear All,
 
Suppose my protein has 4 same subunits (not exactly 4 subunits, in order to 
explain things clear, it contains not less than 2 identical subunitss), each 
subunit has the potential H-bond involving N of HIS. I have run the 
optimization by both PDB_REDO and the Phenix refine with the phenix flips 
checked.
 
I have checkecd the PDB_REDO refine results and the Phenix refine results, the 
answer is, suppose in PDB_REDO it tells us subunit A has that specific H-bond 
formed, Phenix refine rells us that subunit B has that specific H-bond formed.
 
In fact, supposing it has 4 subunits, the symmetry determines that the 4 
subunits should be identical, and they should all contains the specific 
H-bonds, or all do not contains the specific H-bonds.
 
Any more suggestions welcome.
 
Smith


At 2015-05-20 22:55:13, Robbie Joosten robbie_joos...@hotmail.com wrote:

Hi Smith,

Just to contrast Pavel's phenix plug. PDB_REDO does HQN-flips automatically 
based on WHAT _CHECK results and refines your model in Refmac.

Cheers,
Robbie

Sent with my Windows Phone
Van: Robbie Joosten
Verzonden: ‎20-‎5-‎2015 14:30
Aan: Smith Liu; CCP4BB@JISCMAIL.AC.UK
Onderwerp: RE: [ccp4bb] HIS related crystallography issue


You can typically assign the histidine orientation based on analysis of the 
hydrogen bond network. In ambiguous cases you might have to look a few residues 
deep. WHAT_CHECK does this for you by a global optimization of the hydrogen 
bond network.

Cheers,
Robbie

Sent with my Windows Phone
Van: Smith Liu
Verzonden: ‎20-‎5-‎2015 14:12
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] HIS related crystallography issue


Dear All,
 
Suppose the protein crystal resolution is about 2-3A, then in the map it should 
be rather difficult to distinguish the C and N in the sidechain of HIS. In this 
way we may regard the sidechain of HIS is flippable. But suppose in one flipped 
conformation of the HIS, the free N in the sidechain of HIS can form H-bond 
with the neighbour H of the OH of the Thr, in the other flipped conformation of 
the HIS, the free N in the sidechain of HIS cannot form H-bond with the 
neighbour H of the OH of the Thr (caused by distance issue).
 
Suppose whether the H-bond forms between the free N in the sidechain of HIS and 
the neighbour H of the OH of the Thr was very important biologically, in this 
situation how can we distinguish whether the H-bond forms?
 
Smith

[ccp4bb] how to actively assign H to N of His

[Smith Liu]

Dear All,
 
After CCP4 or phenix refinement, I find the H position of N in specific His 
residues needs to be regularized based on the function of that His.
 
Will you please advise on how to actively assign H to one or two of the 2 N in 
the His residue in the PDB file? Or do we have a server, which can help us to 
assign H to the N of the His in the PDB based on the function of the His, or 
based on our intention which N we need to have H in the His?
 
In addition, after H regularuzed in the His in the PDB, do you think whether 
further CCP4 or phenix refinement is needed?
 
Smith

[ccp4bb] on the contour level in Coot

[Smith Liu]

Dear All,
 
For the contour level in the Properties in the Dssplay of the Display Manager 
in Coot, the contour level should be exacly the sigma value we see in the 
published crystallography paper, am I right? But I have paid attention to a 
paper which has a sigma level of 6-7 for its densty. Will you please explain 
why the sigma can be so high and how can we make it so high?
 
Smith

[ccp4bb] on space group

[Smith Liu]

Dear All,
 
Alhough there are on-line explainations on the space group, I found it was 
difficult to fully understand. Here we take P 1 21 1 as an exmaple, will you 
please explain to me with easy language what each number indicates？Or do we 
have a on-line server which can demonstrate the meaning of each number? How can 
we get out crystal arrangement with repeating the unit cell in the format of 
space group?
 
Smith
 
 

[ccp4bb] on the contour level in Coot

[Smith Liu]

If for both a mtz density and mrc map I set the contour level as 0.15, does the 
015 has the comparable significance for the mtz density and mrc map?
 
Smith

Re: [ccp4bb] self-rotation in the absence of NCS

[Smith Liu]

I recently solved a 4-identical chain  protein structure. First I got it from 
initial model with NCS, good enough. Then from the initial model I process it 
without NCS, quality better than solved with NCS. 


Smith





At 2015-04-30 02:07:31, Eleanor Dodson eleanor.dod...@york.ac.uk wrote:

Hmm - I think these peaks MUST be related to some internal symmetry in the 1200 
aa solved structure. Is there some arrangement of helices or other features 
which are replicated in another part of the structure? 


A phi value of ~ 19 degrees can't be explained by a different related space 
group I don't  think


Eleanor


On 29 April 2015 at 15:56, Ian Tickle ianj...@gmail.com wrote:


Peer, you didn't say which program you are using for this?  Polarrfn or Molrep? 
 Do you get the same results with both programs?  Also did you try sharpening 
the Fs with Ecalc and/or using all your data?  In my experience sharpening 
works better with self- than with cross-rotation functions because the 
differences between NCS-related monomers are usually much less those than 
between non-isomorphous ones.


Cheers


-- Ian



On 29 April 2015 at 15:00, Peer Mittl mi...@bioc.uzh.ch wrote:
Zbyszek Otwinowski and  Fred Vellieux suggested to run the self-rotation on 
Fcalcs. This suggestion solved the problem, since there are similar peaks on 
the kappa=180° planes as well. However, I wasn't able to get rid of those peaks 
by playing around with resolution and integration radius. I must say that I am 
surprized, because - as Eleanor pointed out - I also expected to find peaks on 
the kappa=180° planes only in case of P6522 symmetry. Anyway, this experience 
reminds me to run some simple tests beforehand.
-Peer



On 29.04.2015 15:31, Eleanor Dodson wrote:
Well - PG P6/mmm (possible SG P6522)   will have peaks at kappa = 180 omega = 
90 phi = 0 30 60 etc..

But if there is only one molecule / asymm unit there cant be an extra 2-fold.

How big are the relative domains? Your interesting domain couldnt just be 
cleaved off could it?
Eleanor






On 29 April 2015 at 12:59, Peer Mittl mi...@bioc.uzh.ch 
mailto:mi...@bioc.uzh.ch wrote:

We are working with a multi-domain protein crystallized in SG P6_5
with one molecule per asymmetric unit. The structure was refined
at 2.00 A resolution with reasonable R-factors but unfortunately
the domain we are most interested in seems to be disordered.
Interestingly, the self-rotation function shows peaks on the
kappa=180° plane (omega=90°, phi=19° (and every 30°)), with more
than 50% origin peak height. Therefore, we are wondering if
perhaps the space group assignment might be sub-optimal. Any
explanations how these self-rotation peaks could occur and how we
could extract meaningful information to resolve the disordered
domain are welcome.

Best regards,
Peer

P.S. Some additional information: pointless suggests SG P6_5, the
data doesn't seem to be twinned (L-test), the refined part of the
structure has no internal symmetry and refinement in P1 doesn't
reveal the lost domain.

[ccp4bb] on the electron microscopy function of refmac5

[Smith Liu]

Dear All,
 
The CCP4 document says refmac can process electron microscopy map for 
refinement. But I cannot localize that function in the refmac5 of the CCP4.
 
Will you please advise how to have the refmac process the electron microscopy 
map?
 
Smith

Re: [ccp4bb] on NCS restraint

[Smith Liu]

Dear Jurgen,
 
My understanding is that NCS restraint can significantly enhance the speed of 
calculation, but considering the subunits even with the eactly same sequence 
may not be identical, to have NCS restraint may be not necessary or may be not 
good for the refinement, am I right?
 
Smith







At 2015-04-17 09:09:05, Jurgen Bosch jbos...@jhu.edu wrote:
yes.
Have two sets of NCS operators one that describe the four subunits and one 
describing the two subunits. If during the refinement of your structure you 
should find out that the subunits are not identical to each other you can relax 
the NCS weights.


Jürgen 

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


On Apr 16, 2015, at 9:02 PM, Smith Lee 
0459ef8548d5-dmarc-requ...@jiscmail.ac.uk wrote:



Dear All,
 
If a protein contains 6 subunits, 4 subunits from the same sequence (subunit A, 
B, C, D all from the same sequence), each of the 2 other subunits from 2 
diffrent sequences (subunit E from the second sequence, subunit F from the 
third sequence), in this situation should I use NCS restraint or not?
 
If my protein contains 2 subunits, both of the 2 subunits composed of the 
eaxctly same sequence, however supposing the 2 subunits have a little diffrent 
conformation, in this situation should we use NCS retraint or not?
 
Smith
 
 

Re: [ccp4bb] Problem in optimization

[Smith Liu]

Additive screening









At 2015-04-11 18:00:21, 高艺娜 gaoy...@cau.edu.cn wrote:
Dear all
I have tried a variety of methods on regular optimization of the crystal ,but  
all have failed,the 17 kDa protein crystal still have many nucleation and poor 
diffraction also poor resolution(the best 3.5－4.0Å）
The crystallization conditions are:
0.1 M Tris pH-6.5, 
0.2 M Sodium acetate
1.8 M Ammonium sulphate

Could some one suggest me how to trouble shoot this problem?

Best wishes,

Re: [ccp4bb] on building a helix fragment by coot

[Smith Liu]

Dear Rhys,

 
My difficulty is, suppose I have 20 residues (just an example), the first 8 
sidechains fit the dnsity, the last 8 fit the density, however for the middle 4 
residues, the map can hold 5 sidechains or hold 3 sidechains, not exactly 4 
sidechains. This is caused by the poor resolution. But my boss needs me to get 
a good fit.
 
Can you give more advices?
 
Smith
 
 









At 2015-04-01 13:01:38, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote:
Hi Smith,

What resolution are you working at and what kind of phases do you have 
(experimental?, good/bad?)?
I've had the most success using the place helix function, then trimming it 
back so it fits the denisty. Once the helix is place you can try rigid body 
fitting it so it is orientated correctly for adding sidechains.
If your resolution and or phases are poor however it can be difficult to fit 
fragments into density.

Good luck,

Rhys

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Smith Liu 
[smith_liu...@163.com]
Sent: 01 April 2015 05:58
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] on building a helix fragment by coot

Dear All,

If I need to build a fragment of helix to a fragment of density by coot, 
should I use baton build method or should I use place Helix here function? I 
have tried both, but I find it is difficult to place the sidechains to the map 
mosition which looks like where shold be occupied by the sidechains.

I am looking forward to getting your message on how to correctly buld the 
helix.

Best regards.

Smith

[ccp4bb] on building a helix fragment by coot

[Smith Liu]

Dear All,
 
Caused by poor resolution, I can only build a series of Ala into my helix 
density (for sure the density is for helix). Then I tried to use the dock 
sequence in the extension of the Coot to change to my target sequence. Coot 
says it was not confidence on the alignment, thus it did not change the Alas 
into my target sequence.
 
In the Molecule to be sequenced in the coot dock sequence, if we paste the 
sequence, show we still need to input the pir file? For the sequenc we paste, 
it should be my target sequence rather than the series of Alas , right? Why in 
this graphical interface there are Sequence closest fragment and Sequnece 
all fragments!?
 
I ask these questions in order to avoid my failling to do something which Coot 
can do.
 
I am looking forward to getting your reply.
 
Smith







 Forwarding messages 
From: Smith Liu smith_liu...@163.com
Date: 2015-04-01 12:58:01
To: lists...@jiscmail.ac.uk,CCP4BB@JISCMAIL.AC.UK ccp4bb@jiscmail.ac.uk
Subject: on building a helix fragment by coot

Dear All,
 
If I need to build a fragment of helix to a fragment of density by coot, should 
I use baton build method or should I use place Helix here function? I have 
tried both, but I find it is difficult to place the sidechains to the map 
mosition which looks like where shold be occupied by the sidechains.
 
I am looking forward to getting your message on how to correctly buld the helix.
 
Best regards.
 
Smith

[ccp4bb] on building a helix fragment by coot

[Smith Liu]

Dear All,
 
If I need to build a fragment of helix to a fragment of density by coot, should 
I use baton build method or should I use place Helix here function? I have 
tried both, but I find it is difficult to place the sidechains to the map 
mosition which looks like where shold be occupied by the sidechains.
 
I am looking forward to getting your message on how to correctly buld the helix.
 
Best regards.
 
Smith

[ccp4bb] on refinement by coot

[Smith Liu]

Dear All,
 
Suppose there is a 10-residue helix, there are 2 of the 10 Calphas do not 
position so well so the sidechains of the 2 Calphas do not align with the 
density. Is any Coot commands which can refine the 2 Calpha positions and the 
corresponding sidechains? By the way, if I use Wincoot, will you let me know 
how to use Wincoot by inputing command?
 
Smith

[ccp4bb] on openning the PDB file and the mtz file by Coot

[Smith Liu]

Dear All,
 
When we by Coot open the PDB fle and the mtz file from the same refinement, the 
protein backbone (and the sidechains) and the electron density always fit 
automatically. Will you please tell me the mechanism of the Coot how the PDB 
file automatically fit the  mtz file in its graphical window?
 
Suppose I have a PDB file and a mtz file (PDB file from protein A, mtz file 
from protein B, which is a  homology protein of protein A) which are not from 
the same refinment (thus not fit automatically in Coot), will you please tell 
me what modification I should make on the files in order to have the Coot to 
fit the protein bakbone (and the sidechains) and the electron density?
 
I am looking forward to getting your reply.
 
Smith

[ccp4bb] on Baton build

[Smith Liu]

Dear All,
 
In coot when I try to build a peptide by baton method, I find the starting 
point of the baton starts from somewhere in the sidechain rather than from the 
Calpha position. In addition, when I try to changing the starting residue by 
Baton-build params, the starting point of the Baton does not change residue at 
all.
 
Will you please tell me how to setting the issue?
 
Smith

Re: [ccp4bb] how to recover my data

[Smith Liu]

Thanks Ian. I got it from the bin folder you mentioned. A moment ago I have 
failed to localize it from the CCP4 graphical interface.
 
Smith







At 2015-03-07 21:13:58, Ian Tickle ianj...@gmail.com wrote:

Hi Smith


Not sure what you mean, the current version of CCP4 (as have all previous 
versions) certainly does contain mtzdump:


 which mtzdump
/software/CCP4-6.5.0/ccp4-6.5/bin/mtzdump


The limited output is intentional: mtzdump by default only lists the first 10 
reflexions, and you have exactly 10 in your list.  If you want more then supply 
an NREF value, e.g.


echo nref 9 |mtzdump HKLIN my.mtz |less


But mtzdump (or any MTZ utility for that matter) is not going to help you with 
a corrupted file, since the MTZ read routines assume that the file has a 
strictly defined format which has most likely been modified when it was 
transferred from the damaged disk, and is therefore now unreadable by any 
program that assumes the defined format.


What does the technician who transferred the data have to say about it?  He is 
the only person who can possibly tell you how the file has been corrupted.  
What you need is a hex dump of the file (e.g. using 'hexdump') and someone who 
understands a) the correct format and b) exactly how it was corrupted.  Then it 
may be possible to write a bespoke program tor recover your data.  But that is 
not going to be easy: it will require someone with some expertise in 
programming.

Attempting to read the file with random programs has even less chance of 
working than I have of winning the lottery!  As others have suggested there are 
much easier ways of recovering your data (such as reprocessing the images).


Cheers


-- Ian





On 7 March 2015 at 06:29, Smith Liu smith_liu...@163.com wrote:

Dear All,
 
The current version of CCP4 does not contain mtzdump. I use UCLA MBI — mtzdump 
to process it. For a mtz file, the output says there was no valid reflection 
data. For another mtz file, it only gave about 50 lines information (not a new 
mtz file). As for the original mtz is very large, I think at least some message 
in the mtz file has been missing or has not been recovered by UCLA MBI — 
mtzdump.
 
Please feel free for further advise.
 
Smith
 
 
Follwing: output from the UCLAMBI-mtzdump



mtzdump - dump data from an MTZ reflection data file. A CCP4 program
Your mtzdump Results
Cell Dimensions : (obsolete - refer to dataset cell dimensions above)

  130.3260  130.3260  360.3400   90.   90.   90. 

 *  Resolution Range :

0.30.22676 (180.170 -  2.100 A )

 * Sort Order :

  1 2 3 0 0

 * Space group = 'I 4 2 2' (number 97)



 
| |
|
1 ASC  0  62  0  100.00 32.5 32.5 180.17   2.10   H  H
| |
|
2 NONE 0  43  0  100.00 13.4 13.4 180.17   2.10   H  K
| |
|
3 NONE 0 171  0  100.00 64.4 64.4 180.17   2.10   H  L
| |
|
4 NONE0.019.0 0  100.00 9.51 9.51 180.17   2.10   I  
FreeR_flag
| |
|
5 NONE   65.8466807.5 47890   47.08 14985.84 14985.84  41.21   2.10   J  IMEAN
| |
|
6 NONE   22.7 37273.6 47890   47.08  1161.73  1161.73  41.21   2.10   Q  
SIGIMEAN
| |
|
7 NONE   65.8466807.5 50197   44.54 15601.05 15601.05  41.21   2.10   K  I(+)
| |
|
8 NONE   36.9 37273.6 50197   44.54  1723.38  1723.38  41.21   2.10   M  SIGI(+)
| |
|
9 NONE   65.8466807.5 53778   40.58 16626.28 16626.28  41.21   2.10   K  I(-)
| |
|
10 NONE   36.9 37273.6 53778   40.58  1568.40  1568.40  41.21   2.10   M  
SIGI(-)
| |
|
11 NONE   67.3  6770.8 47890   47.08  1006.23  1006.23  41.21   2.10   F  F
| |
|
12 NONE5.6   446.6 47890   47.0876.6976.69  41.21   2.10   Q  SIGF
| |
|
13 NONE -514.8   537.4 56085   38.03 0.2187.57  41.21   2.10   D  DANO
| |
|
14 NONE0.0   380.1 56085   38.0391.6391.63  41.21   2.10   Q  
SIGDANO
| |
|
15 NONE   67.3  6770.8 50197   44.54  1032.56  1032.56  41.21   2.10   G  F(+)
| |
|
16 NONE8.2   446.6 50197   44.5490.1390.13  41.21   2.10   L  
SIGF(+)
| |
|
17 NONE   67.3  6770.8 53778   40.58  1075.89  1075.89  41.21   2.10   G  F(-)
| |
|
18 NONE7.6   446.6 53778   40.5878.6778.67  41.21   2.10   L  
SIGF(-)
| |
|
19 NONE 0   2  47890   47.08  0.4  0.4  41.21   2.10   Y  ISYM
| |
|
| |


LIST OF REPLECTIONS

 ===

0   0   28.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0   4   10.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0   69.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0

Re: [ccp4bb] how to recover my data

[Smith Liu]

Dear All,

 
For the corrupted mtz file, if we open it by notepad, it would be messy code or 
unreadble code.
 
In addition, not all the mtz files in the damaged hardware were corrupted, some 
mts files are still readable by Coot.
 
Do you have any suggestions to correct the unreadbale mtz files to readable?
 
Smith








At 2015-03-07 22:51:12, Zhijie Li zhijie...@utoronto.ca wrote:

Smith,
 
As Ian said, mtzdump can give you some clues, but will not solve your problem.
 
The file that mtzdump could not read apparently has changed length due to some 
sort of insertion. The other file that mtzdump could process has the correct 
length and the stats look reasonable, so it might be OK, although we still can 
not rule out the possibility that a few numbers might have been changed in the 
dataset. Have you tried to do anything with the latter file, such as a few 
rounds of refinement? What did you see?
 
If you send me the two mtz I can take a look and make some guesses. If the 
problem was as simple as introduction of some 0D 0A insertion/deletion it can 
be fixed. If you can consult the technician who recovered the data and tell us 
what software he used it would be helpful.
 
Zhijie
 
 
 
From:Smith Liu
Sent: Saturday, March 07, 2015 9:24 AM
To:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to recover my data
 
Thanks Ian. I got it from the bin folder you mentioned. A moment ago I have 
failed to localize it from the CCP4 graphical interface.
 
Smith





 
At 2015-03-07 21:13:58, Ian Tickle ianj...@gmail.com wrote:

Hi Smith


Not sure what you mean, the current version of CCP4 (as have all previous 
versions) certainly does contain mtzdump:


 which mtzdump
/software/CCP4-6.5.0/ccp4-6.5/bin/mtzdump


The limited output is intentional: mtzdump by default only lists the first 10 
reflexions, and you have exactly 10 in your list.  If you want more then supply 
an NREF value, e.g.


echo nref 9 |mtzdump HKLIN my.mtz |less


But mtzdump (or any MTZ utility for that matter) is not going to help you with 
a corrupted file, since the MTZ read routines assume that the file has a 
strictly defined format which has most likely been modified when it was 
transferred from the damaged disk, and is therefore now unreadable by any 
program that assumes the defined format.


What does the technician who transferred the data have to say about it?  He is 
the only person who can possibly tell you how the file has been corrupted.  
What you need is a hex dump of the file (e.g. using 'hexdump') and someone who 
understands a) the correct format and b) exactly how it was corrupted.  Then it 
may be possible to write a bespoke program tor recover your data.  But that is 
not going to be easy: it will require someone with some expertise in 
programming.

Attempting to read the file with random programs has even less chance of 
working than I have of winning the lottery!  As others have suggested there are 
much easier ways of recovering your data (such as reprocessing the images).


Cheers


-- Ian

 
 
On 7 March 2015 at 06:29, Smith Liu smith_liu...@163.com wrote:

Dear All,
 
The current version of CCP4 does not contain mtzdump. I use UCLA MBI — mtzdump 
to process it. For a mtz file, the output says there was no valid reflection 
data. For another mtz file, it only gave about 50 lines information (not a new 
mtz file). As for the original mtz is very large, I think at least some message 
in the mtz file has been missing or has not been recovered by UCLA MBI — 
mtzdump.
 
Please feel free for further advise.
 
Smith
 
 
Follwing: output from the UCLAMBI-mtzdump



mtzdump - dump data from an MTZ reflection data file. A CCP4 program
Your mtzdump Results
Cell Dimensions : (obsolete - refer to dataset cell dimensions above)

  130.3260  130.3260  360.3400   90.   90.   90. 

 *  Resolution Range :

0.30.22676 (180.170 -  2.100 A )

 * Sort Order :

  1 2 3 0 0

 * Space group = 'I 4 2 2' (number 97)



 
| |
|
1 ASC  0  62  0  100.00 32.5 32.5 180.17   2.10   H  H
| |
|
2 NONE 0  43  0  100.00 13.4 13.4 180.17   2.10   H  K
| |
|
3 NONE 0 171  0  100.00 64.4 64.4 180.17   2.10   H  L
| |
|
4 NONE0.019.0 0  100.00 9.51 9.51 180.17   2.10   I  
FreeR_flag
| |
|
5 NONE   65.8466807.5 47890   47.08 14985.84 14985.84  41.21   2.10   J  IMEAN
| |
|
6 NONE   22.7 37273.6 47890   47.08  1161.73  1161.73  41.21   2.10   Q  
SIGIMEAN
| |
|
7 NONE   65.8466807.5 50197   44.54 15601.05 15601.05  41.21   2.10   K  I(+)
| |
|
8 NONE   36.9 37273.6 50197   44.54  1723.38  1723.38  41.21   2.10   M  SIGI(+)
| |
|
9 NONE   65.8466807.5 53778   40.58 16626.28 16626.28  41.21   2.10   K  I(-)
| |
|
10 NONE   36.9 37273.6 53778   40.58  1568.40  1568.40  41.21   2.10   M  
SIGI(-)
| |
|
11 NONE   67.3  6770.8 47890   47.08  1006.23  1006.23  41.21   2.10   F  F
| |
|
12 NONE5.6   446.6 47890   47.08

Re: [ccp4bb] how to recover my data

[Smith Liu]

Dear All,
 
The current version of CCP4 does not contain mtzdump. I use UCLA MBI — mtzdump 
to process it. For a mtz file, the output says there was no valid reflection 
data. For another mtz file, it only gave about 50 lines information (not a new 
mtz file). As for the original mtz is very large, I think at least some message 
in the mtz file has been missing or has not been recovered by UCLA MBI — 
mtzdump.
 
Please feel free for further advise.
 
Smith
 
 
Follwing: output from the UCLAMBI-mtzdump



mtzdump - dump data from an MTZ reflection data file. A CCP4 program
Your mtzdump Results
Cell Dimensions : (obsolete - refer to dataset cell dimensions above)

  130.3260  130.3260  360.3400   90.   90.   90. 

 *  Resolution Range :

0.30.22676 (180.170 -  2.100 A )

 * Sort Order :

  1 2 3 0 0

 * Space group = 'I 4 2 2' (number 97)



 
| |
|
1 ASC  0  62  0  100.00 32.5 32.5 180.17   2.10   H  H
| |
|
2 NONE 0  43  0  100.00 13.4 13.4 180.17   2.10   H  K
| |
|
3 NONE 0 171  0  100.00 64.4 64.4 180.17   2.10   H  L
| |
|
4 NONE0.019.0 0  100.00 9.51 9.51 180.17   2.10   I  
FreeR_flag
| |
|
5 NONE   65.8466807.5 47890   47.08 14985.84 14985.84  41.21   2.10   J  IMEAN
| |
|
6 NONE   22.7 37273.6 47890   47.08  1161.73  1161.73  41.21   2.10   Q  
SIGIMEAN
| |
|
7 NONE   65.8466807.5 50197   44.54 15601.05 15601.05  41.21   2.10   K  I(+)
| |
|
8 NONE   36.9 37273.6 50197   44.54  1723.38  1723.38  41.21   2.10   M  SIGI(+)
| |
|
9 NONE   65.8466807.5 53778   40.58 16626.28 16626.28  41.21   2.10   K  I(-)
| |
|
10 NONE   36.9 37273.6 53778   40.58  1568.40  1568.40  41.21   2.10   M  
SIGI(-)
| |
|
11 NONE   67.3  6770.8 47890   47.08  1006.23  1006.23  41.21   2.10   F  F
| |
|
12 NONE5.6   446.6 47890   47.0876.6976.69  41.21   2.10   Q  SIGF
| |
|
13 NONE -514.8   537.4 56085   38.03 0.2187.57  41.21   2.10   D  DANO
| |
|
14 NONE0.0   380.1 56085   38.0391.6391.63  41.21   2.10   Q  
SIGDANO
| |
|
15 NONE   67.3  6770.8 50197   44.54  1032.56  1032.56  41.21   2.10   G  F(+)
| |
|
16 NONE8.2   446.6 50197   44.5490.1390.13  41.21   2.10   L  
SIGF(+)
| |
|
17 NONE   67.3  6770.8 53778   40.58  1075.89  1075.89  41.21   2.10   G  F(-)
| |
|
18 NONE7.6   446.6 53778   40.5878.6778.67  41.21   2.10   L  
SIGF(-)
| |
|
19 NONE 0   2  47890   47.08  0.4  0.4  41.21   2.10   Y  ISYM
| |
|
| |


LIST OF REPLECTIONS

 ===

0   0   28.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0   4   10.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0   69.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0   8   18.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0  103.00   ? ? ? ? ?  
  ? ? ? ? ? ?  
  ? ? ? ?  
0   0  12   17.00   3090.57268.91   3090.57268.91   3090.57
   268.91554.30 24.36  0.00  0.00554.30
24.36554.30 24.36  1.00
0   0  149.00  23242.80   1362.28  23242.80   1362.28  23242.80
  1362.28   1522.33 44.83  0.00  0.00   1522.33
44.83   1522.33 44.83  1.00
0   0  165.00  29139.17   1697.95  29139.17   1697.95  29139.17
  1697.95   1704.50 49.90  0.00  0.00   1704.50
49.90   1704.50 49.90  1.00
0   0  18   12.00478.99 57.42478.99 57.42478.99
57.42217.65 13.29  0.00  0.00217.65
13.29217.65 13.29  1.00
0   0  203.00 113229.90   6443.99 113229.90   6443.99 113229.90
  6443.99   3358.69 96.10  0.00  0.00   3358.69
96.10   3358.69 96.10  1.00
 end, thus very limited information output



At 2015-03-06 15:56:19, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Smith,

an mtz-file is a binary file and none of the programs you list is
suitable to open it in a meaningful manner.

Why don't you follow the