Re: [ccp4bb] Running CCP4_Blend on Mac

[Ravikumar]

Hi David and James,

Thank you for your kind response. Blend is working now.

Ravikumar.

On Fri, Mar 6, 2020 at 1:15 PM David Waterman  wrote:

> Dear Ravi Kumar
>
> If I remember correctly, Blend requires the path to the executable Rscript
> rather than R. I can't check this now but I can look into it on Monday if
> you are still having trouble.
>
> Best wishes
> David
>
> On Fri, 6 Mar 2020, 21:02 Reddiravikumar Kumar, 
> wrote:
>
>> Hi all,
>>
>> I am trying to run Blend program from CCP4interface (7.0.078) to
>> merge multiple datasets (9). I am running the CCP4 on Mac mojave 10.14.6 OS
>> and installed R program in /usr/local/bin/R. In the CCP4 interface, the
>> same path is added under system administration/configure interface/external
>> programs/add a program/. When i start the blend program, it is showing
>> Dependency error: BLEND requires R. Am I missing something here.
>> Please advise me running blend through ccp4 interface?
>>
>>
>> Thank you,
>> --
>> ravi kumar
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>>
>

-- 
ravi kumar



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Re: [ccp4bb] Flexible C terminus

[Zachary A. Wood]

Hi Chitra,

Sometimes disorder ‘is’ the functional state of a peptide segment, and in those 
cases, if you see the segment ordered in your crystal structure, it is not 
physiologically relevant; rather it is a result of crystal packing. Sometimes 
disordered segments undergo a folding when they bind a ligand, or they are 
targets for post-translational modifications, and in some cases they can 
actually generate an entropic force which can modify the structural ensemble of 
the protein (https://www.nature.com/articles/s41586-018-0699-5). In the latter 
case, the protein had a disordered 30 residue C-terminus that used entropic 
force to change the structure of the folded portion of the protein to enhance 
ligand affinity.

Best regards,

Z


***
Zachary A. Wood, Ph.D.
Associate Professor and Graduate Coordinator
Department of Biochemistry & Molecular Biology
University of Georgia
Life Sciences Building, Rm A426B
120 Green Street
Athens, GA  30602-7229
Office: 706-583-0304
Lab:706-583-0303
FAX: 706-542-1738
***


From: CCP4 bulletin board  on behalf of Debanu Das 

Reply-To: "debanu@gmail.com" 
Date: Thursday, March 12, 2020 at 12:53 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Flexible C terminus

[EXTERNAL SENDER - PROCEED CAUTIOUSLY]
Hi Chitra,

To add to the discussion, I can offer an example about obtaining the structure 
of a flexible/disordered N-term of a membrane protein.

Previous structures of the full-length multidrug efflux transporter AcrB (12 TM 
helices in each protein ~1000 residues, forms a trimer, so 36 TM helices) were 
missing the first 6 residues in the N-term in the cytoplasm but we could 
determine this structure and look at some interesting sequence-structure 
implications of these first 6 residues in our structure:

"Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å resolution 
reveals the N-terminal region with conserved amino acids
Debanu Das,* Qian Steven Xu,* Jonas Y. Lee, Irina Ankoudinova, Candice Huang, 
Yun Lou, Andy DeGiovanni, Rosalind Kim, and Sung-Hou Kim"

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2023878/

I think there should also be examples of C-term or N-term 
expression/purification tags that are ordered in some crystal forms of a target 
but disordered in other crystal forms/structures of the same target/homologs.

Best,
Debanu
--
Debanu Das

On Thu, Mar 12, 2020 at 5:02 AM chitra latka 
mailto:chitra.la...@gmail.com>> wrote:
Dear Klemens,

I am going to setup the crystallisation of the entire protein anyhow. I hope I 
get lucky :)

Thanks
Chitra

On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild 
mailto:klemens.w...@bzh.uni-heidelberg.de>> 
wrote:
On 12.03.20 08:53, chitra latka wrote:
Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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Dear Chitra

I would nevertheless try. Sometimes flexible termini fold back either in cis or 
in trans (crystal packing, a case I just had Yesterday) and you might learn sth 
important for biological regulation if you are lucky. At the same time I would 
truncate the terminus and crystallize the globular domain in parallel.

Good luck

Klemens


--
Regards
Chitra



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Re: [ccp4bb] Flexible C terminus

[Debanu Das]

Hi Chitra,

To add to the discussion, I can offer an example about obtaining the
structure of a flexible/disordered N-term of a membrane protein.

Previous structures of the full-length multidrug efflux transporter AcrB
(12 TM helices in each protein ~1000 residues, forms a trimer, so 36 TM
helices) were missing the first 6 residues in the N-term in the cytoplasm
but we could determine this structure and look at some interesting
sequence-structure implications of these first 6 residues in our structure:

"Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å
resolution reveals the N-terminal region with conserved amino acids
Debanu Das,* Qian Steven Xu,* Jonas Y. Lee, Irina Ankoudinova, Candice
Huang, Yun Lou, Andy DeGiovanni, Rosalind Kim, and Sung-Hou Kim"

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2023878/

I think there should also be examples of C-term or N-term
expression/purification tags that are ordered in some crystal forms of a
target but disordered in other crystal forms/structures of the same
target/homologs.

Best,
Debanu
--
Debanu Das

On Thu, Mar 12, 2020 at 5:02 AM chitra latka  wrote:

> Dear Klemens,
>
> I am going to setup the crystallisation of the entire protein anyhow. I
> hope I get lucky :)
>
> Thanks
> Chitra
>
> On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild <
> klemens.w...@bzh.uni-heidelberg.de> wrote:
>
>> On 12.03.20 08:53, chitra latka wrote:
>>
>> Dear All,
>>
>> I am working on a protein that has flexible C terminus. None of the
>> available structures even in homologs have density for C term region
>> (around 20 odd residues). All the available pdb entries have missing
>> density for these 20 residues at C terminus.
>>
>> I am going to try my luck crystallising the entire protein in hope of
>> getting density for C term residues as well (Fingers crossed).
>>
>> Has anyone faced a similar problem where they have managed to get density
>> for a flexible terminus successfully?
>>
>> Any suggestions would be appreciated.
>>
>> Cheers !
>>
>> Chitra Latka
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> Dear Chitra
>>
>> I would nevertheless try. Sometimes flexible termini fold back either in
>> cis or in trans (crystal packing, a case I just had Yesterday) and you
>> might learn sth important for biological regulation if you are lucky. At
>> the same time I would truncate the terminus and crystallize the globular
>> domain in parallel.
>>
>> Good luck
>>
>> Klemens
>>
>
>
> --
> Regards
> Chitra
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] [3dem] Which resolution?

[Petrus Zwart]

Hi Jacob,

On Thu, Mar 12, 2020 at 9:13 AM Keller, Jacob 
wrote:

> I would think the most information-reflecting representation for
> systematic absences (or maybe for all reflections) would be not I/sig but
> the reflection's (|log|) ratio to the expected intensity in that shell
> (median intensity, say).


Xtriage does something like this as part of its space group assignment
algorithm. A choice of space group implies assigning reflections the label
acentric, centric or absent. Each of these have their own prior
distribution, which can be convoluted with a gaussian to compute a
likelihood for that specific space group hypothesis. It provides a decent
way of assigning space groups in an automated manner.


> (...)
>


> Maybe more generally, should refinement incorporate weighting for these
> deviant spots? Or maybe it already does, but my understanding was that
> I/sig was the most salient for weighting.
>

The best option is to have a decent likelihood function that takes into
account the (almost) full uncertainty of the observation into
consideration, as described by Read & Pannu (https://bit.ly/2W6qmVR)
including various numerical /mathematical approaches to compute this ( Read
& McCoy https://bit.ly/2Qa6b5I;  Perpendicular Pronoun & Perryman
https://bit.ly/2TKjJXH ).

P






> JPK
>
> +
> Jacob Pearson Keller
> Research Scientist / Looger Lab
> HHMI Janelia Research Campus
> 19700 Helix Dr, Ashburn, VA 20147
> Desk: (571)209-4000 x3159
> Cell: (301)592-7004
> +
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Kay
> Diederichs
> Sent: Tuesday, March 10, 2020 2:48 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] [3dem] Which resolution?
>
> I'd say that it depends on your state of knowledge, and on their I and
> sigma.
>
> - if you know the space group for sure before you do the measurement of
> the systematic absences, their I and sigma don't matter to you (because
> they don't influence your mental model of the experiment), so their
> information content is (close to) zero.
> - if the space group is completely unknown, some groups of reflections
> (e.g. h,k,l = 0,0,2n+1) can only be considered "potentially systematic
> absences". Then both I and sigma matter. "small" or "high" I/sigma for each
> member of such a group of reflections would indeed add quite some
> information in this situation, so an information content of up to 1 bit
> would be justified. "intermediate" I/sigma (say, 0.5 to 2) would be closer
> to zero bit, since it does not let you safely decide between "yes" or "no"
> (the recent paper by Randy Read and coworkers relates I and sigma to bits
> of information, but not in the context of decision making from potentially
> systematic absent reflections).
>
> So it is not quite straightforward, I think.
>
> best wishes,
> Kay
>
> On Tue, 10 Mar 2020 01:26:03 +0100, James Holton  wrote:
>
> >I'd say they are 1 bit each, since they are the answer to a yes-or-no
> >question.
> >
> >-James Holton
> >MAD Scientist
> >
> >On 2/27/2020 6:32 PM, Keller, Jacob wrote:
> >> How would one evaluate the information content of systematic absences?
> >>
> >> JPK
> >>
> >> On Feb 26, 2020 8:14 PM, James Holton  wrote:
> >> In my opinion the threshold should be zero bits.  Yes, this is where
> >> CC1/2 = 0 (or FSC = 0).  If there is correlation then there is
> >> information, and why throw out information if there is information to
> >> be had?  Yes, this information comes with noise attached, but that is
> >> why we have weights.
> >>
> >> It is also important to remember that zero intensity is still useful
> >> information.  Systematic absences are an excellent example.  They
> >> have no intensity at all, but they speak volumes about the structure.
> >> In a similar way, high-angle zero-intensity observations also tell us
> >> something.  Ever tried unrestrained B factor refinement at poor
> >> resolution?  It is hard to do nowadays because of all the safety
> >> catches in modern software, but you can get great R factors this way.
> >> A telltale sign of this kind of "over fitting" is remarkably large
> >> Fcalc values beyond the resolution cutoff.  These don't contribute to
> >> the R factor, however, because Fobs is missing for these hkls. So,
> >> including zero-intensity data suppresses at least some types of
> >> over-fitting.
> >>
> >> The thing I like most about the zero-information resolution cutoff is
> >> that it forces us to address the real problem: what do you mean by

[ccp4bb] EM postdoc position at AstraZeneca UK

[Debreczeni, Judit]

Dear all,

I would like to remind you that we are still looking to fill a postdoc position 
at AstraZeneca (Cambridge UK) in collaboration with Prof Ashok Venkitaraman's 
group at the MRC Cancer Unit. This is an opportunity for a talented and 
self-motivated structural biologist to undertake biophysical and cryoEM studies 
of macromolecular complexes between BRCA2 and other tumour suppressor proteins.

To learn more about the position and to apply please follow this link.
https://careers.astrazeneca.com/job/cambridge/postdoc-fellow-brca2-complexes-by-em/7684/15093657

Closing date: end of March 2020.

Please do not reply to this message, applications need to be submitted through 
the above portal to be considered.

many thanks
Judit

---
Judit Debreczeni
Associate Principal Scientist
SBF, Discovery Sciences
AstraZeneca

AstraZeneca
Darwin (310) Building
Cambridge Science Park
Milton Road
Cambridge
CB4 0WG
UK




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Re: [ccp4bb] [3dem] Which resolution?

[Keller, Jacob]

I would think the most information-reflecting representation for systematic 
absences (or maybe for all reflections) would be not I/sig but the reflection's 
(|log|) ratio to the expected intensity in that shell (median intensity, say). 
Thus, when the median intensity is 1000 counts, and one observes a spot of 1 
count, this would be quite information-rich even though its I/sig would be 
really small.

This approach would also reflect the lesser information contained in twinned 
data, where deviations from mean intensities are smaller, even though I/sig be 
quite large.

Regarding spots that are not systematic absence candidates, isn't it true that 
a very weak spot (e.g. 10 counts) of I/sig = 2 might contain more information 
than a strong spot (1000 counts) of I/sig = 20 in the same shell, if the median 
counts in the shell were 1000?

I used to hear rumors that maps calculated from the 1000 strongest spots were 
almost tantamount to using all reflections; maybe these flaky maps would be 
improved by using instead the 1000 reflections which deviate most from expected 
intensities? (i.e., both stronger and weaker.)

Maybe more generally, should refinement incorporate weighting for these deviant 
spots? Or maybe it already does, but my understanding was that I/sig was the 
most salient for weighting.

I guess the general idea is that the more unexpected the value is, the more it 
captures something unique about the thing being measured, thus more information.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board  On Behalf Of Kay Diederichs
Sent: Tuesday, March 10, 2020 2:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [3dem] Which resolution?

I'd say that it depends on your state of knowledge, and on their I and sigma.

- if you know the space group for sure before you do the measurement of the 
systematic absences, their I and sigma don't matter to you (because they don't 
influence your mental model of the experiment), so their information content is 
(close to) zero.
- if the space group is completely unknown, some groups of reflections (e.g. 
h,k,l = 0,0,2n+1) can only be considered "potentially systematic absences". 
Then both I and sigma matter. "small" or "high" I/sigma for each member of such 
a group of reflections would indeed add quite some information in this 
situation, so an information content of up to 1 bit would be justified. 
"intermediate" I/sigma (say, 0.5 to 2) would be closer to zero bit, since it 
does not let you safely decide between "yes" or "no" (the recent paper by Randy 
Read and coworkers relates I and sigma to bits of information, but not in the 
context of decision making from potentially systematic absent reflections). 

So it is not quite straightforward, I think.

best wishes,
Kay

On Tue, 10 Mar 2020 01:26:03 +0100, James Holton  wrote:

>I'd say they are 1 bit each, since they are the answer to a yes-or-no 
>question.
>
>-James Holton
>MAD Scientist
>
>On 2/27/2020 6:32 PM, Keller, Jacob wrote:
>> How would one evaluate the information content of systematic absences?
>>
>> JPK
>>
>> On Feb 26, 2020 8:14 PM, James Holton  wrote:
>> In my opinion the threshold should be zero bits.  Yes, this is where
>> CC1/2 = 0 (or FSC = 0).  If there is correlation then there is 
>> information, and why throw out information if there is information to 
>> be had?  Yes, this information comes with noise attached, but that is 
>> why we have weights.
>>
>> It is also important to remember that zero intensity is still useful 
>> information.  Systematic absences are an excellent example.  They 
>> have no intensity at all, but they speak volumes about the structure.  
>> In a similar way, high-angle zero-intensity observations also tell us 
>> something.  Ever tried unrestrained B factor refinement at poor 
>> resolution?  It is hard to do nowadays because of all the safety 
>> catches in modern software, but you can get great R factors this way.
>> A telltale sign of this kind of "over fitting" is remarkably large 
>> Fcalc values beyond the resolution cutoff.  These don't contribute to 
>> the R factor, however, because Fobs is missing for these hkls. So, 
>> including zero-intensity data suppresses at least some types of 
>> over-fitting.
>>
>> The thing I like most about the zero-information resolution cutoff is 
>> that it 

Re: [ccp4bb] Flexible C terminus

[Tanner, John J.]

Dear Chitra,

Try adding a ligand to the crystallization. This worked for us with ALDH7A1:

https://www.ncbi.nlm.nih.gov/pubmed/26260980

ALDH7A1 is an example of an enzyme with a flexible C-terminus (11 residues). 
The conformation of the C-terminus depends on the presence of a ligand in the 
substrate site. With the product bound, the C-terminus adopts the “in” 
conformation (4ZUL). Without the product bound, the C-terminus adopts either 
the “out” conformation (4ZUK, chains A-F,H) or is disordered (4ZUK, chain G).

In this case, the flexibility of the C-terminus is essential for catalytic 
activity:.

https://www.ncbi.nlm.nih.gov/pubmed/29045138

Good luck!

Jack

John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S. College Ave.
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 Mar 12, 2020, at 8:01 AM, Oganesyan, Vaheh 
mailto:vaheh.oganes...@astrazeneca.com>> wrote:

Hi Chitra Latka,

By far the best approach is to find what protein is interacting with the one 
you have the structure and try co-crystallizing them together. At least there 
will be some more biology (science) involved in what you will be doing. You may 
get lucky and get different packing of your full length protein and get some 
sort of structure for last 20 aa. Then what?

Regards,

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of chitra latka
Sent: Thursday, March 12, 2020 3:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Flexible C terminus

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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Re: [ccp4bb] Flexible C terminus

[Oganesyan, Vaheh]

Hi Chitra Latka,

By far the best approach is to find what protein is interacting with the one 
you have the structure and try co-crystallizing them together. At least there 
will be some more biology (science) involved in what you will be doing. You may 
get lucky and get different packing of your full length protein and get some 
sort of structure for last 20 aa. Then what?

Regards,

From: CCP4 bulletin board  On Behalf Of chitra latka
Sent: Thursday, March 12, 2020 3:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Flexible C terminus

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus

[Schreuder, Herman /DE]

Dear Chitra,

Crystallizing both proteins together is a very good idea: If you get a 
structure, it will be an interesting one, even if the C-terminus remains 
invisible.

Concerning the flexible C-terminus: is it the linker linking the second domain 
to the first one? In that case it might just be a bait, attracting a protease 
to get the second domain chopped off. In that case, chances of getting it 
structured may be slim and if you get it structured, it might be a crystal 
packing artifact. However, as Klemens mentioned, you should nevertheless try!

If this flexible C-terminus gets cleaved by a protease, you may also try to get 
some cocrystal structure with the protease. There are quite a few tricks 
available to achieve this.

Best, Herman



Von: chitra latka 
Gesendet: Donnerstag, 12. März 2020 12:59
An: Schreuder, Herman /DE 
Cc: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is chitra.la...@gmail.com

Dear Herman,

Its a two domain protein and the second domain gets chopped off and stabilises 
the other domain by binding near the flexible C - terminus of first domain. I 
am trying to crystallise both the proteins together. Literature review doesn't 
report binding of the protein to any other protein. So, it kind of has the 
ligand or another domain to stabilise but eve then density of C-terminus 
residues remain missing.

Thanks
Chitra Latka










On Thu, Mar 12, 2020 at 4:08 PM Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:
Dear Chitra,

There usually is a reason the C-terminus is disordered. Either it needs to bind 
a ligand to get ordered, or it needs to bind to some other protein. You have to 
check the literature. If the C-terminus binds a ligand, you have to add this 
ligand to your crystallization experiment. If it binds to some other protein, 
you could try cocrystallization of both proteins, or just try to cocrystallize 
the other protein with only the 20 residues or so of the C-terminus.

Best, Herman

Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von chitra latka
Gesendet: Donnerstag, 12. März 2020 08:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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--
Regards
Chitra



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Re: [ccp4bb] Flexible C terminus

[chitra latka]

Dear Klemens,

I am going to setup the crystallisation of the entire protein anyhow. I
hope I get lucky :)

Thanks
Chitra

On Thu, Mar 12, 2020 at 5:12 PM Klemens Wild <
klemens.w...@bzh.uni-heidelberg.de> wrote:

> On 12.03.20 08:53, chitra latka wrote:
>
> Dear All,
>
> I am working on a protein that has flexible C terminus. None of the
> available structures even in homologs have density for C term region
> (around 20 odd residues). All the available pdb entries have missing
> density for these 20 residues at C terminus.
>
> I am going to try my luck crystallising the entire protein in hope of
> getting density for C term residues as well (Fingers crossed).
>
> Has anyone faced a similar problem where they have managed to get density
> for a flexible terminus successfully?
>
> Any suggestions would be appreciated.
>
> Cheers !
>
> Chitra Latka
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> Dear Chitra
>
> I would nevertheless try. Sometimes flexible termini fold back either in
> cis or in trans (crystal packing, a case I just had Yesterday) and you
> might learn sth important for biological regulation if you are lucky. At
> the same time I would truncate the terminus and crystallize the globular
> domain in parallel.
>
> Good luck
>
> Klemens
>


-- 
Regards
Chitra



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Re: [ccp4bb] [EXTERNAL] [ccp4bb] Flexible C terminus

[chitra latka]

Dear Herman,

Its a two domain protein and the second domain gets chopped off and
stabilises the other domain by binding near the flexible C - terminus of
first domain. I am trying to crystallise both the proteins together.
Literature review doesn't report binding of the protein to any other
protein. So, it kind of has the ligand or another domain to stabilise but
eve then density of C-terminus residues remain missing.

Thanks
Chitra Latka










On Thu, Mar 12, 2020 at 4:08 PM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Dear Chitra,
>
>
>
> There usually is a reason the C-terminus is disordered. Either it needs to
> bind a ligand to get ordered, or it needs to bind to some other protein.
> You have to check the literature. If the C-terminus binds a ligand, you
> have to add this ligand to your crystallization experiment. If it binds to
> some other protein, you could try cocrystallization of both proteins, or
> just try to cocrystallize the other protein with only the 20 residues or so
> of the C-terminus.
>
>
>
> Best, Herman
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *chitra
> latka
> *Gesendet:* Donnerstag, 12. März 2020 08:54
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [EXTERNAL] [ccp4bb] Flexible C terminus
>
>
>
> *EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk
>
>
>
> Dear All,
>
>
>
> I am working on a protein that has flexible C terminus. None of the
> available structures even in homologs have density for C term region
> (around 20 odd residues). All the available pdb entries have missing
> density for these 20 residues at C terminus.
>
>
>
> I am going to try my luck crystallising the entire protein in hope of
> getting density for C term residues as well (Fingers crossed).
>
>
>
> Has anyone faced a similar problem where they have managed to get density
> for a flexible terminus successfully?
>
>
>
> Any suggestions would be appreciated.
>
>
>
> Cheers !
>
>
>
> Chitra Latka
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>


-- 
Regards
Chitra



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Re: [ccp4bb] [3dem] Which resolution?

[Gergely Katona]

Hi,

I think through bond and through space B factor (+ sphericity) restraints 
primarily exist for pragmatic reasons: they are needed to maintain the 
numerical stability of the refinement. That is a separate issue from making 
physical sense. If one finds consistent B-factor similarity in atoms connected 
through bonds and near each other that lends support to a physical 
interpretation. Unfortunately that is not the case: a CG atom in a lysine 
residue tend to have higher Biso than a CB atom and lower Biso than a CD atom 
even though they are both CB and CD are covalently bonded to CG and they are 
physically close to one other. That tells me that the position of the atom in a 
residue predicts their relative B factors than bonding connectivity. Spherical 
targets also work against evidence as the probability of finding perfectly 
isotropic atoms in a protein structure is close to zero in high resolution 
structures. It is enough take a look at the excellent PARVATI home page at the 
fraction of atoms at anisotropy=1: 
http://skuld.bmsc.washington.edu/parvati/parvati_survey.html

We did not try to put atoms into predetermined groups, but used clustering 
analysis of the ADP tensors in a set of high resolution structures.  We found 
that ADPs of covalently bonded atoms are rarely the most similar. Only in 
weakly defined parts of the structure bonding connectivity has strong 
predictive power, but I wonder if that is not entirely the effect of 
restraints. 

What is really surprising is that ADPs of chemical similar atoms have a 
tendency to be the most similar even though they are located in completely 
different parts of the structure. And that includes similarity in displacement 
directions in the absence of obvious symmetry.
Different crystal structures have different disorder and resolution in you 
analysis is shown to have a role. Therefore restraints might need to be 
tailored to the actual type of disorder (for example using TLS or not). I agree 
that when it comes to physically relevant ADP restraints, our toolbox may also 
be incomplete. 

Best wishes,

Gergely

-Original Message-
From: CCP4 bulletin board  On Behalf Of Bohdan Schneider
Sent: March 12, 2020 11:05
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [3dem] Which resolution?

Hello,

B-factors actually do have a physical meaning which is at least to some extent 
reflected by the crystal structures as refined. This can be demonstrated at 
higher resolution structures: when we created three tiers of structures, better 
than 1.9 Å, 1.9-2.4 Å, and 2.4-3.0 Å, structures in the first one showed 
distinct distributions of B factors for the amino acid side chain/main chain 
atoms inside and outside the protein, for DNA bases, and phosphates, and for 
water at the interface, and on the biomolecule surface. The distinction is less 
clear for the
1.9-2.4 Å structures and is lost completely below that resolution limit.

We think that the distributions for the high resolution structures can be 
developed into meaningful set of constraints and/or validation criteria.

If interested, you can read more in our open access paper Acta Cryst. 
(2014). D70, 2413–2419 (doi:10.1107/S1399004714014631).

Best regards,

Bohdan, bs.structbio.org

On 2020-03-11 16:41, Gerard DVD Kleywegt wrote:
>>> If this is the case, why can't we use model B factors to validate 
>>> our structure? I know some people are skeptical about this approach 
>>> because B factors are refinable parameters.
>>>
>>> Rangana
>>
>> It is not clear to me exactly what you are asking.
>>
>> B factors _should_ be validated, precisely because they are refined 
>> parameters that are part of your model.   Where have you seen 
>> skepticism?
> 
> Rangana said that B-values should not be used *to validate 
> structures*, NOT that B-values themselves shouldn't be validated themselves.
> 
> I suppose I am at least in part to blame for the former notion and the 
> reason for this (at least circa 1995 when the Angry Young Men from 
> Uppsala first starting harping on about this) was that B-values 
> tend(ed) to be error sinks which could "absorb" all sorts of errors 
> and phenomena in addition to modelling atomic displacement (e.g., 
> unresolved disorder, unresolved NCS differences, incorrect restraints, 
> incorrect atom types modelled, partial ocupancies, etc.).
> 
> --Gerard
> 
> **
>     Gerard J. Kleywegt
> 
>    http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
> **
>     The opinions in this message are fictional.  Any similarity
>     to actual opinions, living or dead, is purely coincidental.
> **
>     Little known gastromathematical curiosity: let "z" be the
>     radius and "a" the thickness of a pizza. Then the volume
>      of that pizza is 

Re: [ccp4bb] Red trapezoids in Coot

[Joël Bloch]

Dear Paul (both),

Thanks a lot! Now it worked!

The Cys is at the active site/ binding pocked of a designer enzyme, which 
actually originally was drafted into the scaffold of a carbohydrate binding 
enzyme.

Best wishes,
Joël

Joël Bloch
Postdoctoral fellow
Group Prof. Locher
Institute of Molecular Biology and Biophysics
ETH Zurich, HPK G18
Otto-Stern-Weg 5
CH-8093 Zürich, Switzerland
phone: +41 ( 0 ) 44 63 - 36839
e-mail : joel.bl...@mol.biol.ethz.ch

On 12 Mar 2020, at 11:10, Paul Emsley  wrote:

On 12/03/2020 09:14, Joël Bloch wrote:
Is there any way in Coot 0.8.9.2 to remove/hide the red trapezoids that are 
indicating planar bonds?
I couldn’t find anything on that, neither in the manual nor FAQ.

(Good for you for reading the documentation)

The red trapezoids represent cis peptides that are in front of a residue that 
is not a proline. They are quite rare (but less so in carbohydrate binding 
proteins (why should that be? is that what you have?)).

Calculate -> Scripting -> Python
set_draw_cis_peptide_markups(0)

Paul.



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Re: [ccp4bb] Red trapezoids in Coot

[Paul Bond]

Hi Joël,

You can go to Calculate -> Scripting -> Python and enter:

set_draw_cis_peptide_markups(0)

Kind regards,
Paul

On Thu, 12 Mar 2020 at 09:58, Joël Bloch  wrote:

> Hi Dave
>
> Thanks a lot for the hint. The Cis-bond should be correct, as it is a
> buried glycine residue in a crystal structure at 0.95 Å resolution.
>
> But it would be great, if there would be a way to simply hide the cis-bond
> warning. I would like to generate a supplementary figure with a broad view
> at active site electron density, which in this particular case looks much
> clearer in Coot than in PyMol
>
> Best wishes,
> Joël
>
> On 12 Mar 2020, at 10:34, David Briggs  wrote:
>
> 
> Hi Joel,
>
> The red trapezoids are indicative of cis-peptide bonds that are
> exceptionally rare, and usually indicate incorrect tracing of the backbone
> (unless you have good enough data to unambiguously decide that a
> cis-peptide bond is correct).
>
> I'm afraid I don't know how to get rid of them, other than correcting the
> backbone.
>
> There is a Cis<>Trans button you can add to coot which might correct the
> issue for you (right-click on the icon bar, select "Manage Buttons", and
> add the appropriate button)
>
> HTH
>
> Dave
>
> --
>
> Dr David C. Briggs
>
> Senior Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> about.me/david_briggs
>
> --
> *From:* CCP4 bulletin board  on behalf of Joël
> Bloch 
> *Sent:* 12 March 2020 09:14
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Red trapezoids in Coot
>
> Dear CCP4 community,
>
> Is there any way in Coot 0.8.9.2 to remove/hide the red trapezoids that
> are indicating planar bonds?
>
> I couldn’t find anything on that, neither in the manual nor FAQ.
>
> Thanks a lot for your help!
>
> Best wishes,
> Joël
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
>
> https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1data=02%7C01%7C%7C5f83fa21c789422339b408d7c6676029%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637196019617460410sdata=suEVs%2BAbwYkXyo1W8C14kWP70duWVWZSnEVB23exFek%3Dreserved=0
>
> The Francis Crick Institute Limited is a registered charity in England and
> Wales no. 1140062 and a company registered in England and Wales no.
> 06885462, with its registered office at 1 Midland Road London NW1 1AT
>
>
> --
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Re: [ccp4bb] Red trapezoids in Coot

[Paul Emsley]

On 12/03/2020 09:14, Joël Bloch wrote:


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

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



(Good for you for reading the documentation)

The red trapezoids represent cis peptides that are in front of a residue that is not a proline. They are 
quite rare (but less so in carbohydrate binding proteins (why should that be? is that what you have?)).


Calculate -> Scripting -> Python
set_draw_cis_peptide_markups(0)

Paul.



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Re: [ccp4bb] [3dem] Which resolution?

[Bohdan Schneider]

Hello,

B-factors actually do have a physical meaning which is at least to some 
extent reflected by the crystal structures as refined. This can be 
demonstrated at higher resolution structures: when we created three 
tiers of structures, better than 1.9 Å, 1.9-2.4 Å, and 2.4-3.0 Å, 
structures in the first one showed distinct distributions of B factors 
for the amino acid side chain/main chain atoms inside and outside the 
protein, for DNA bases, and phosphates, and for water at the interface, 
and on the biomolecule surface. The distinction is less clear for the 
1.9-2.4 Å structures and is lost completely below that resolution limit.


We think that the distributions for the high resolution structures can 
be developed into meaningful set of constraints and/or validation criteria.


If interested, you can read more in our open access paper Acta Cryst. 
(2014). D70, 2413–2419 (doi:10.1107/S1399004714014631).


Best regards,

Bohdan, bs.structbio.org

On 2020-03-11 16:41, Gerard DVD Kleywegt wrote:
If this is the case, why can't we use model B factors to validate our 
structure? I know some people are skeptical about this approach 
because B factors are refinable parameters.


Rangana


It is not clear to me exactly what you are asking.

B factors _should_ be validated, precisely because they are refined 
parameters

that are part of your model.   Where have you seen skepticism?


Rangana said that B-values should not be used *to validate structures*, 
NOT that B-values themselves shouldn't be validated themselves.


I suppose I am at least in part to blame for the former notion and the 
reason for this (at least circa 1995 when the Angry Young Men from 
Uppsala first starting harping on about this) was that B-values tend(ed) 
to be error sinks which could "absorb" all sorts of errors and phenomena 
in addition to modelling atomic displacement (e.g., unresolved disorder, 
unresolved NCS differences, incorrect restraints, incorrect atom types 
modelled, partial ocupancies, etc.).


--Gerard

**
    Gerard J. Kleywegt

   http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
**
    The opinions in this message are fictional.  Any similarity
    to actual opinions, living or dead, is purely coincidental.
**
    Little known gastromathematical curiosity: let "z" be the
    radius and "a" the thickness of a pizza. Then the volume
     of that pizza is equal to pi*z*z*a !
**



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Re: [ccp4bb] Red trapezoids in Coot

[Joël Bloch]

Hi Dave

Thanks a lot for the hint. The Cis-bond should be correct, as it is a buried 
glycine residue in a crystal structure at 0.95 Å resolution.

But it would be great, if there would be a way to simply hide the cis-bond 
warning. I would like to generate a supplementary figure with a broad view at 
active site electron density, which in this particular case looks much clearer 
in Coot than in PyMol

Best wishes,
Joël

On 12 Mar 2020, at 10:34, David Briggs  wrote:


Hi Joel,

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

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

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

HTH

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Joël Bloch 

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

Dear CCP4 community,

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

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

Thanks a lot for your help!

Best wishes,
Joël



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Re: [ccp4bb] Red trapezoids in Coot

[David Briggs]

Hi Joel,

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

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

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

HTH

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Joël Bloch 

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

Dear CCP4 community,

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

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

Thanks a lot for your help!

Best wishes,
Joël



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[ccp4bb] Red trapezoids in Coot

[Joël Bloch]

Dear CCP4 community,

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

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

Thanks a lot for your help!

Best wishes,
Joël



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Re: [ccp4bb] Flexible C terminus

[Klemens Wild]

On 12.03.20 08:53, chitra latka wrote:

Dear All,

I am working on a protein that has flexible C terminus. None of the 
available structures even in homologs have density for C term region 
(around 20 odd residues). All the available pdb entries have missing 
density for these 20 residues at C terminus.


I am going to try my luck crystallising the entire protein in hope of 
getting density for C term residues as well (Fingers crossed).


Has anyone faced a similar problem where they have managed to get 
density for a flexible terminus successfully?


Any suggestions would be appreciated.

Cheers !

Chitra Latka




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Dear Chitra

I would nevertheless try. Sometimes flexible termini fold back either in 
cis or in trans (crystal packing, a case I just had Yesterday) and you 
might learn sth important for biological regulation if you are lucky. At 
the same time I would truncate the terminus and crystallize the globular 
domain in parallel.


Good luck

Klemens




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Re: [ccp4bb] [3dem] Which resolution?

[Nave, Colin (DLSLtd,RAL,LSCI)]

All good points in the contributions from Alexis and Randy.

To confirm Alexis’ interpretation of my previous post, my main concern about 
the ½ bit threshold for “nominal” resolution was that it is in danger of being 
adopted independently of the requirement (for example by those doing x-ray 
imaging of biological cells). I might be approaching the issue rather 
differently from others as I was interested in the data needed to identify a 
feature with a particular size and contrast in the presence of noise and 
background.

Randy convincingly argues that there is information in the data down to a 0.1 
or 0.01 bit threshold and one should use this data if one has it. One issue 
when collecting data is how to position a detector of limited size. The signal 
to background ratio can generally be improved by putting the detector far away 
but the “actual” resolution of the data might then be compromised if the edge 
of the detector corresponds to a 0.5 bit threshold. However, this set up might 
be the best way  for identifying a bound substrate in an electron density map 
even though it compromises any subsequent refinement. Is there a difference 
between an “optical resolution” and a “refinement resolution” from the point of 
view of interpretability? Is this related to Frank’s “need to turn to 
interpretability?” Frank – do you mean interpreting an electron density map of 
some flavour, using machine learning or otherwise?

My conclusion with all this is that we need to define our use of some of the 
terms such as resolution, noise, background, interpretability etc. Doing this 
would mean that new terms would be required as any meaningful definition for 
any term would be likely limit its use.  Most of what is below comes from the 
discussions on this email thread, for example in distinguishing between noise 
and parts of electron density maps which can’t be interpreted by means of a 
model. Here is a first attempt. The list is far too long but probably 
insufficient


1.   Resolution PDB – Like it or not I think James has a point. “I 
personally think that the resolution assigned to the PDB deposition should 
remain the classical I/sigI > 3 at 80% rule”. We can of course discuss the 
details but his point is a valid one.


2.   Resolution PDB refinement - Can we get the PDB to accept an additional 
term defined by  both the resolution and corresponding information content of 
the data actually used for refinement?



3.   Resolution Instrument –  For a telescope it is defined by the aperture 
of the lens. For a MX instrument it is defined by the Bragg resolution at the 
edge of the detector. John Spence recently reminded me that “according to the 
old definitions, resolution should be a property of an instrument (eg a 
microscope) , not the sample.” This is of course a harsh definition but 
nevertheless a useful one.  However, see point 13 below.


4.   Noise in the data– This is a property of the rawest measurement one 
has and corresponds to the best one can do to estimate the Poisson statistics 
for photon counting. I hope Dale, on the “question of density” topic would 
accept this as noise. The manufacturers of detectors often put an imperfect 
model of the detector between the raw data and the user of the instrument so 
the estimate of the intensity and noise is already model dependent. Ideally 
noise can still be given from the intensity (no. of photons in each detector 
pixel). In some respects this “raw” data is the only thing one is certain of.


5.   Background in the data – This is a part of the data one is not 
primarily interested in (e.g. air scatter, solvent scatter). The data reduction 
programs have a model for the background in order to subtract it, taking in to 
account the noise in the data as defined above. This model will not be perfect 
- does the background vary linearly across a spot if this is the model? 
(Similarly the profiles for any profile fitting will not be perfect).



6.   Integrated intensities – derived with individual error estimates by 
the integration programs.



7.   Other systematic errors in the data. This could include radiation 
damage during data collection. The result would be that metrics such as CC1/2, 
FSC etc. derived from the integrated intensity measurements will be worse than 
that expected from the error estimates.


8.   Noise in the electron density maps – This is a function of the noise 
in the data (Parseval’s theorem). This can be improved by exposing for longer. 
See also background in the electron density maps.


9.   Background in the electron density maps – This is essentially parts of 
the maps which one cannot model. It is not noise. However, the systematic 
errors in the data (point 4) could contribute to it. Recent examples in this 
thread include overlapping disordered waters merging in to bulk solvent. If one 
can interpret it in terms of a model, it is no longer background. One can have 
a standard 

[ccp4bb] AW: [EXTERNAL] [ccp4bb] Flexible C terminus

[Schreuder, Herman /DE]

Dear Chitra,

There usually is a reason the C-terminus is disordered. Either it needs to bind 
a ligand to get ordered, or it needs to bind to some other protein. You have to 
check the literature. If the C-terminus binds a ligand, you have to add this 
ligand to your crystallization experiment. If it binds to some other protein, 
you could try cocrystallization of both proteins, or just try to cocrystallize 
the other protein with only the 20 residues or so of the C-terminus.

Best, Herman

Von: CCP4 bulletin board  Im Auftrag von chitra latka
Gesendet: Donnerstag, 12. März 2020 08:54
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Flexible C terminus


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Dear All,

I am working on a protein that has flexible C terminus. None of the available 
structures even in homologs have density for C term region (around 20 odd 
residues). All the available pdb entries have missing density for these 20 
residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of getting 
density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density for a 
flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka




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[ccp4bb] Flexible C terminus

[chitra latka]

Dear All,

I am working on a protein that has flexible C terminus. None of the
available structures even in homologs have density for C term region
(around 20 odd residues). All the available pdb entries have missing
density for these 20 residues at C terminus.

I am going to try my luck crystallising the entire protein in hope of
getting density for C term residues as well (Fingers crossed).

Has anyone faced a similar problem where they have managed to get density
for a flexible terminus successfully?

Any suggestions would be appreciated.

Cheers !

Chitra Latka



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1