Re: [ccp4bb] Refine modified residues based on cryo-EM maps

[Paul Emsley]

On 25/11/2020 03:24, Cheng Zhang wrote:


This question must have been asked before


Correct.


but I couldn't find a good answer online.


the jiscmail archive is a terrible system for archiving our collective 
knowledge. There should be a better way.

I work on a cryo-EM structure with one serine residue covalently linked with a lipid molecule. The map for 
the lipid moiety was there but not good enough to unambiguously place each atom. I tried to manually model 
the lipid moiety to fit the map. But if I refine it in real space, it would move away from the serine 
residue even though I linked it to the residue in the PDB file.


a LINK is necessary but not sufficient.

My question is, how to refine such a 
modified residue in real space without breaking the covalent bond?


This is not a modified residue. A modified residue would require a different 
approach. This is a linked residue.

Is there any way to make a molecular 
topology file for the lipid moiety that also contains restraints for the covalent bond with the modified 
residue?


One would do that one step at a time rather than combined, so the answer to that question may be yes or no. 
You may not need to do step 1 because the residue/compound to which the SER is attached may already be in 
the monomer library.


Also, let's say there is an unnatural amino acid in the protein, how to define this residue so COOT 
or Phenix


Depending on the nature of the unnatural amino acid, this may not need any work at all (it may already exist 
and all you need to do is Replace Residue). But a SER linked to a lipid is not such a case.



can recognize it as an amino acid and the peptide bond restraints still apply 
to it?


Pyrogen does this (FWIW), but I am not sure if Acedrg does.

Anyway, here's a worked example of linking a monomer that already exists in the 
dictionary (PLP) to a LYS:

https://pemsley.github.io/coot/blog/2020/06/30/make-a-link.html

Paul.



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Jon Cooper]

Hello Dale, the statistical rigour you describe is, of course, excellent, but 
in a learning environment, if someone gets a negative result, you have to go 
into overdrive to check that everything has been done correctly, since there is 
a fair chance that human error is the cause. It may be a terrible practice, but 
it would seem to be an important part of the process? Even as a relative 
newcomer to the field (well, since the mid-80's ;-) I have seen many people 
getting nothing in their initial difference maps, even if the ligand is there. 
Frequently it was just the contour level being too high and, depending on how 
far back you go, the solution varied from showing someone how to roll the mouse 
wheel in Coot to having the map recontoured at a computer centre 200 miles away 
and posted back on a magnetic tape, which took about 10 days - a timescale on 
which some people just gave up and did something else! I can't help thinking it 
would be a shame to robotically accept every negative result at face value, not 
least if you're doing something important like curing a pandemic. However, back 
to the original question which I think was whether polder map coefficients 
could be used as refinement targets and I think the answer to that one is 
probably 'no', at least in the X-ray field ;-)

Best wishes, Jon Cooper

 Original Message 
On 24 Nov 2020, 16:02, Dale Tronrud wrote:

> Hi,
>
> To me, this sounds like a very dangerous way to use this tool decide
> if a ligand has bound. I would be very reluctant to modify my map with
> a range of arbitrary parameters until it looked like what I wanted to
> see. The sharpening and blurring of this tool is not guided or limited
> by theory or data.
>
> As you describe it, your choice of map is driven by its agreement
> with your ligand, and the proper way to make this decision is the other
> way around.
>
> The original poster has the problem that their density does not have
> the appearance they desire. They have chosen to run around trying to
> find some way to modify the map to get a variant that does. This is a
> terrible practice, since the final choice of map is being made in a
> fashion that is dominated by bias.
>
> I have no idea what sort of "structural characteristics" have
> convinced this poster of the presence of their ligand despite the
> absence of clear electron density. What other evidence does a
> diffraction pattern give? The map is your best and only source of
> information about your structure that you can get from the diffraction
> pattern. (Mass spec and other experimental techniques could, of course,
> be applied.)
>
> I think we, as a community, could learn a few things from the
> vaccine trial studies that are so much in the news now. In a modern
> clinical trial, to avoid bias in the interpretation of the results, all
> of the statistical procedures are decided upon BEFORE the study is even
> began. This protocol is written down and peer reviewed at the start.
> Then the study is performed and the protocol is followed exactly. If
> the results don't pass the test, the treatment is not supported. There
> is no hunting around, after the fact, for a "better" statistical measure
> until one is found that "works".
>
> This way of handling data analysis in clinical trials was adopted
> after the hard lesson was learned that many trails could be reproduced,
> their results were not.
>
> I would recommend that you decide what sort of map you think is the
> best at showing features of your active site, based on the resolution of
> your data set and other qualities of your project, before you calculate
> your first Fourier transform. If you think a Polder map is the bee's
> knees then calculate a Polder map and live with it. If you are
> convinced of the value of a FEM, or a Buster map, or a SA omit map, or
> whatever, calculate that map instead and live with it.
>
> If you have to calculate twenty different kinds of maps, with
> varying parameters in each, before you find the one that shows the
> density for your ligand; it probably didn't bind.
>
> Dale Tronrud
>
> On 11/24/2020 5:35 AM, John R Helliwell wrote:
>> Dear Nika,
>> A tool I am gaining experience with, but for a challenge like you
>> describe, may help:-
>> In Coot>Calculate you see “Blurring/Sharpening tool”. You are
>> presented with a choice of electron density map (here you would select
>> your Fo-Fc). There is then a slider tool, to the left and to the right,
>> and you can see the impact of negative or positive B factor on your map.
>> Blurring, slide right, may assist your density continuity versus
>> Sharpening, slide left, which may assist the detail of your map. The
>> logic of the tool is that your diffraction data, and of the Fo-Fc
>> differences, can be fine tuned, in or out.
>> Best wishes,
>> John
>>
>> Emeritus Professor John R Helliwell DSc
>>
>>
>>
>>
>>> On 24 Nov 2020, at 11:29, Nika Žibrat  wrote:
>>>
>>> 
>>>
>>> Hello,
>>>
>>>
>>> I have a 

Re: [ccp4bb] Refine modified residues based on cryo-EM maps

[Jon Cooper]

Hello, I think this thread from a few months ago will help.

https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=ind2007=CCP4BB=0=49210

Best wishes, Jon Cooper

 Original Message 
On 25 Nov 2020, 03:24, Cheng Zhang wrote:

> Hi everyone,
>
> This question must have been asked before but I couldn't find a good answer 
> online.
>
> I work on a cryo-EM structure with one serine residue covalently linked with 
> a lipid molecule. The map for the lipid moiety was there but not good enough 
> to unambiguously place each atom. I tried to manually model the lipid moiety 
> to fit the map. But if I refine it in real space, it would move away from the 
> serine residue even though I linked it to the residue in the PDB file. My 
> question is, how to refine such a modified residue in real space without 
> breaking the covalent bond? Is there any way to make a molecular topology 
> file for the lipid moiety that also contains restraints for the covalent bond 
> with the modified residue? Also, let's say there is an unnatural amino acid 
> in the protein, how to define this residue so COOT or Phenix can recognize it 
> as an amino acid and the peptide bond restraints still apply to it?
>
> Thanks!
>
> Cheng
>
> --
>
> -
> Cheng Zhang
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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[ccp4bb] Refine modified residues based on cryo-EM maps

[Cheng Zhang]

Hi everyone,

This question must have been asked before but I couldn't find a good answer
online.

I work on a cryo-EM structure with one serine residue covalently linked
with a lipid molecule. The map for the lipid moiety was there but not good
enough to unambiguously place each atom. I tried to manually model the
lipid moiety to fit the map. But if I refine it in real space, it would
move away from the serine residue even though I linked it to the residue in
the PDB file. My question is, how to refine such a modified residue in real
space without breaking the covalent bond? Is there any way to make a
molecular topology file for the lipid moiety that also contains
restraints for the covalent bond with the modified residue? Also, let's say
there is an unnatural amino acid in the protein, how to define this residue
so COOT or Phenix can recognize it as an amino acid and the peptide bond
restraints still apply to it?

Thanks!

Cheng

-- 
-
Cheng Zhang



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[ccp4bb] Postdoctoral position in drug discovery

[Harp, Joel M]

Postdoctoral position in drug discovery

 An opening is available in the drug discovery laboratory of Dr. Stephen Fesik 
for a post-doc in the field of protein x-ray crystallography.  Responsibilities 
will include protein expression and purification, and all aspects of x-ray 
crystallography including preparation of protein-inhibitor complexes, 
crystallization, home/synchrotron data collection, and structure determination. 
Additional experiences in molecular biology including construct design, 
cloning, site-directed mutagenesis, and experience with recombinant protein 
expression in eukaryotic cells are highly preferred.  The ideal candidate 
should be self-motivated and be able to communicate efficiently in a highly 
collaborative environment. Please send cv to 
jason.p...@vanderbilt.edu




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Re: [ccp4bb] Apple Silicon / XQuartz / X11 / CCP4

[Mailing list]

> On 11 Nov 2020, at 21:04, Alwyn Jones  wrote:
> 
> A greater concern may be lack of support for OpenGL/GLUT

Indeed, big concern.

Probably no other choice than switching to MetalGL : 
https://www.raywenderlich.com/9211-moving-from-opengl-to-metal 

—
Florian






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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[John R Helliwell]

Hello Dale,
Well, warming to your theme, I start with a trust in Coot before a new project.
Secondly, Coot’s blurring and sharpening tool is tethered directly to one’s 
measured diffraction data. 
Thirdly, scrutinising it at a sigma level above 5, Coot’s default, is certainly 
not the same as delving into the lower levels of a map’s sigma (and truly of 
noise).
But, I have just rechecked the Coot manual and see no reference. There are 
several google hits but mainly to cryoEM maps. 
It does seem a free lunch though..
Greetings,
John 


Emeritus Professor John R Helliwell DSc




> On 24 Nov 2020, at 16:02, Dale Tronrud  wrote:
> 
> Hi,
> 
>   To me, this sounds like a very dangerous way to use this tool decide if a 
> ligand has bound.  I would be very reluctant to modify my map with a range of 
> arbitrary parameters until it looked like what I wanted to see.  The 
> sharpening and blurring of this tool is not guided or limited by theory or 
> data.
> 
>   As you describe it, your choice of map is driven by its agreement with your 
> ligand, and the proper way to make this decision is the other way around.
> 
>   The original poster has the problem that their density does not have the 
> appearance they desire.  They have chosen to run around trying to find some 
> way to modify the map to get a variant that does.  This is a terrible 
> practice, since the final choice of map is being made in a fashion that is 
> dominated by bias.
> 
>   I have no idea what sort of "structural characteristics" have convinced 
> this poster of the presence of their ligand despite the absence of clear 
> electron density.  What other evidence does a diffraction pattern give?  The 
> map is your best and only source of information about your structure that you 
> can get from the diffraction pattern.  (Mass spec and other experimental 
> techniques could, of course, be applied.)
> 
>   I think we, as a community, could learn a few things from the vaccine trial 
> studies that are so much in the news now.  In a modern clinical trial, to 
> avoid bias in the interpretation of the results, all of the statistical 
> procedures are decided upon BEFORE the study is even began.  This protocol is 
> written down and peer reviewed at the start. Then the study is performed and 
> the protocol is followed exactly.  If the results don't pass the test, the 
> treatment is not supported.  There is no hunting around, after the fact, for 
> a "better" statistical measure until one is found that "works".
> 
>   This way of handling data analysis in clinical trials was adopted after the 
> hard lesson was learned that many trails could be reproduced, their results 
> were not.
> 
>   I would recommend that you decide what sort of map you think is the best at 
> showing features of your active site, based on the resolution of your data 
> set and other qualities of your project, before you calculate your first 
> Fourier transform.  If you think a Polder map is the bee's knees then 
> calculate a Polder map and live with it.  If you are convinced of the value 
> of a FEM, or a Buster map, or a SA omit map, or whatever, calculate that map 
> instead and live with it.
> 
>   If you have to calculate twenty different kinds of maps, with varying 
> parameters in each, before you find the one that shows the density for your 
> ligand; it probably didn't bind.
> 
> Dale Tronrud
> 
>> On 11/24/2020 5:35 AM, John R Helliwell wrote:
>> Dear Nika,
>> A tool I am gaining experience with, but for a challenge like you describe, 
>> may help:-
>>  In Coot>Calculate you see “Blurring/Sharpening tool”. You are presented 
>> with a choice of electron density map (here you would select your Fo-Fc). 
>> There is then a slider tool, to the  left and to the right, and you can see 
>> the impact of negative or positive B factor on your map. Blurring, slide 
>> right, may assist your density continuity versus Sharpening, slide left, 
>> which may assist the detail of your map. The logic of the tool is that your 
>> diffraction data, and of the Fo-Fc differences, can be fine tuned, in or out.
>> Best wishes,
>> John
>> Emeritus Professor John R Helliwell DSc
 On 24 Nov 2020, at 11:29, Nika Žibrat  wrote:
>>> 
>>> 
>>> 
>>> Hello,
>>> 
>>> 
>>> I have a question about protein-ligand, of which ligand displays an 
>>> ambiguous electron density. I am solving a structure of protein with ligand 
>>>  which was obtained via soaking. Structural characteristics indicate the 
>>> ligand is present however the electron density is quite vague and too small 
>>> for the size of the whole ligand. I did a Polder map which showed much 
>>> larger area of green density. After insertion of my ligand into the green 
>>> density in Polder I ran phenix.refine and there is a lot of red on the spot 
>>> where the ligand is which was to be expected. This leaves me wondering how, 
>>> if even do I incorporate the polder map data into my refine input.
>>> 
>>> 
>>> My question 

Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Dale Tronrud]

Hi,

   To me, this sounds like a very dangerous way to use this tool decide 
if a ligand has bound.  I would be very reluctant to modify my map with 
a range of arbitrary parameters until it looked like what I wanted to 
see.  The sharpening and blurring of this tool is not guided or limited 
by theory or data.


   As you describe it, your choice of map is driven by its agreement 
with your ligand, and the proper way to make this decision is the other 
way around.


   The original poster has the problem that their density does not have 
the appearance they desire.  They have chosen to run around trying to 
find some way to modify the map to get a variant that does.  This is a 
terrible practice, since the final choice of map is being made in a 
fashion that is dominated by bias.


   I have no idea what sort of "structural characteristics" have 
convinced this poster of the presence of their ligand despite the 
absence of clear electron density.  What other evidence does a 
diffraction pattern give?  The map is your best and only source of 
information about your structure that you can get from the diffraction 
pattern.  (Mass spec and other experimental techniques could, of course, 
be applied.)


   I think we, as a community, could learn a few things from the 
vaccine trial studies that are so much in the news now.  In a modern 
clinical trial, to avoid bias in the interpretation of the results, all 
of the statistical procedures are decided upon BEFORE the study is even 
began.  This protocol is written down and peer reviewed at the start. 
Then the study is performed and the protocol is followed exactly.  If 
the results don't pass the test, the treatment is not supported.  There 
is no hunting around, after the fact, for a "better" statistical measure 
until one is found that "works".


   This way of handling data analysis in clinical trials was adopted 
after the hard lesson was learned that many trails could be reproduced, 
their results were not.


   I would recommend that you decide what sort of map you think is the 
best at showing features of your active site, based on the resolution of 
your data set and other qualities of your project, before you calculate 
your first Fourier transform.  If you think a Polder map is the bee's 
knees then calculate a Polder map and live with it.  If you are 
convinced of the value of a FEM, or a Buster map, or a SA omit map, or 
whatever, calculate that map instead and live with it.


   If you have to calculate twenty different kinds of maps, with 
varying parameters in each, before you find the one that shows the 
density for your ligand; it probably didn't bind.


Dale Tronrud

On 11/24/2020 5:35 AM, John R Helliwell wrote:

Dear Nika,
A tool I am gaining experience with, but for a challenge like you 
describe, may help:-
  In Coot>Calculate you see “Blurring/Sharpening tool”. You are 
presented with a choice of electron density map (here you would select 
your Fo-Fc). There is then a slider tool, to the  left and to the right, 
and you can see the impact of negative or positive B factor on your map. 
Blurring, slide right, may assist your density continuity versus 
Sharpening, slide left, which may assist the detail of your map. The 
logic of the tool is that your diffraction data, and of the Fo-Fc 
differences, can be fine tuned, in or out.

Best wishes,
John

Emeritus Professor John R Helliwell DSc





On 24 Nov 2020, at 11:29, Nika Žibrat  wrote:



Hello,


I have a question about protein-ligand, of which ligand displays an 
ambiguous electron density. I am solving a structure of protein with 
ligand  which was obtained via soaking. Structural characteristics 
indicate the ligand is present however the electron density is quite 
vague and too small for the size of the whole ligand. I did a Polder 
map which showed much larger area of green density. After insertion of 
my ligand into the green density in Polder I ran phenix.refine and 
there is a lot of red on the spot where the ligand is which was to be 
expected. This leaves me wondering how, if even do I incorporate the 
polder map data into my refine input.



My question is, how do I continue refining and validating the 
structure in this case?



Thank you,


Nika Žibrat





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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Jon Cooper]

Hello, I was wondering if you are refining the ligand occupancy. Eleanor 
mentioned resolution which is important here. If it's good enough, occupancy 
refinement of the ligand or the fragment will clean the map up, assuming the 
occupancy is much less than one. Sorry, if I'm just saying the obvious...

Best wishes, Jon Cooper
 Original Message 
On 24 Nov 2020, 11:28, Nika Žibrat wrote:

> Hello,
>
> I have a question about protein-ligand, of which ligand displays an ambiguous 
> electron density. I am solving a structure of protein with ligand which was 
> obtained via soaking. Structural characteristics indicate the ligand is 
> present however the electron density is quite vague and too small for the 
> size of the whole ligand. I did a Polder map which showed much larger area of 
> green density. After insertion of my ligand into the green density in Polder 
> I ran phenix.refine and there is a lot of red on the spot where the ligand is 
> which was to be expected. This leaves me wondering how, if even do I 
> incorporate the polder map data into my refine input.
>
> My question is, how do I continue refining and validating the structure in 
> this case?
>
> Thank you,
>
> Nika Žibrat
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[Andrew Mesecar]

Hi Nika,

A couple of other things you could try to improve maps to add to what the
others have suggested.  One, I am assuming that you are refining the
occupancy of the ligand, but if not, that should reduce the negative
density?  2.  Since you mention soaking the ligand, do you have a good
X-ray data set and refined structure of the unliganded protein in the same
space group and about the same resolution?  If so, you could run an
Fo(ligand)-Fo(unliganded) isomorphorous difference map calculation using
the phases of the unliganded structure for a map calculation.  This may
help (or hurt) too.

Best wishes
Andy Mesecar

On Tue, Nov 24, 2020 at 6:30 AM Nika Žibrat  wrote:

> Hello,
>
>
> I have a question about protein-ligand, of which ligand displays an
> ambiguous electron density. I am solving a structure of protein with
> ligand  which was obtained via soaking. Structural characteristics indicate
> the ligand is present however the electron density is quite vague and too
> small for the size of the whole ligand. I did a Polder map which showed
> much larger area of green density. After insertion of my ligand into the
> green density in Polder I ran phenix.refine and there is a lot of red on
> the spot where the ligand is which was to be expected. This leaves me
> wondering how, if even do I incorporate the polder map data into my refine
> input.
>
>
> My question is, how do I continue refining and validating the structure in
> this case?
>
>
> Thank you,
>
>
> Nika Žibrat
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
*Andrew D. Me**secar*
Head, Department of Biochemistry
Walther Professor of Cancer Structural Biology
Deputy Director, Purdue Center for Cancer Research
E-Mail: amese...@purdue.edu
_
*Department of Biochemistry Contact Information:*
175 S. University Street
W. Lafayette, IN 47907-2063
765-494-1607
--
*Research Lab Contact Information:*
Hockmeyer Hall of Structural Biology
Room 311
240 S. Martin Jischke Drive
West Lafayette, IN 47907-1971
765-494-1924
_



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Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density

[John R Helliwell]

Dear Nika,
A tool I am gaining experience with, but for a challenge like you describe, may 
help:-
 In Coot>Calculate you see “Blurring/Sharpening tool”. You are presented with a 
choice of electron density map (here you would select your Fo-Fc). There is 
then a slider tool, to the  left and to the right, and you can see the impact 
of negative or positive B factor on your map. Blurring, slide right, may assist 
your density continuity versus Sharpening, slide left, which may assist the 
detail of your map. The logic of the tool is that your diffraction data, and of 
the Fo-Fc differences, can be fine tuned, in or out. 
Best wishes,
John

Emeritus Professor John R Helliwell DSc




> On 24 Nov 2020, at 11:29, Nika Žibrat  wrote:
> 
> 
> Hello, 
> 
> 
> 
> I have a question about protein-ligand, of which ligand displays an ambiguous 
> electron density. I am solving a structure of protein with ligand  which was 
> obtained via soaking. Structural characteristics indicate the ligand is 
> present however the electron density is quite vague and too small for the 
> size of the whole ligand. I did a Polder map which showed much larger area of 
> green density. After insertion of my ligand into the green density in Polder 
> I ran phenix.refine and there is a lot of red on the spot where the ligand is 
> which was to be expected. This leaves me wondering how, if even do I 
> incorporate the polder map data into my refine input.
> 
> 
> 
> My question is, how do I continue refining and validating the structure in 
> this case? 
> 
> 
> 
> Thank you,
> 
> 
> 
> Nika Žibrat
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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[ccp4bb] Faculty position in cryo-EM at Université de Montréal

[Pascal John]

An Assistant or Associate Professor position has opened in the Department of 
Biochemistry at Université de Montréal:

https://www.umontreal.ca/public/www/documents/offres_emploi_profs/MED_11-20_8_Biochemistry.pdf


Enquiries and applications to:
Lorraine Bidégaré Charette 
mailto:lorraine.bidegare.chare...@umontreal.ca>>



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Re: [ccp4bb] AW: phenix.refine with ligand with ambiguous electron density

[Eleanor Dodson]

Another idea - you dont mention resolution., but possibly the ligand is
very wobbly, and appropriate B values would range widely. Some refinement
defaults restrain the whole "residue" B factors quite tightly to a mean
value. There are ways to relax Bfactor restraints but you will have to read
the manual I guess..
Eleanor

On Tue, 24 Nov 2020 at 12:43, Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Hi Nika,
>
>
>
> Here you need some common sense. The green density in you polder map may
> just be the bulk solvent that was removed from the model to generate the
> polder map. In this case you have to use common sense and ask yourself a
> couple of questions:
>
>- Is the density really from the ligand, of from some other component
>from your crystallization solution? Molecules like tris or hepes love it to
>masquerade for ligands of interest.
>- Could it be that only part of the ligand is visible because the
>other parts are disordered or not present? This happens quite often.
>Instead of the full-ligand, a break-down product or reaction intermediate
>might have bound, or part of the ligand binding site is occupied by crystal
>contact.
>
> In this case I would fit the part of the ligand which has convincing
> electron density, refine and look at the electron density maps to see if
> the fit to the density is convincing and if additional atoms could be added
> to the ligand. If you cannot fit the whole ligand, even after some efforts,
> I would submit a partially fitted model.
>
>
>
> Best,
>
> Herman
>
>
>
>
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *Nika
> Žibrat
> *Gesendet:* Dienstag, 24. November 2020 12:29
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [ccp4bb] phenix.refine with ligand with ambiguous electron
> density
>
>
>
> Hello,
>
>
>
> I have a question about protein-ligand, of which ligand displays an
> ambiguous electron density. I am solving a structure of protein with
> ligand  which was obtained via soaking. Structural characteristics indicate
> the ligand is present however the electron density is quite vague and too
> small for the size of the whole ligand. I did a Polder map which showed
> much larger area of green density. After insertion of my ligand into the
> green density in Polder I ran phenix.refine and there is a lot of red on
> the spot where the ligand is which was to be expected. This leaves me
> wondering how, if even do I incorporate the polder map data into my refine
> input.
>
>
>
> My question is, how do I continue refining and validating the structure in
> this case?
>
>
>
> Thank you,
>
>
>
> Nika Žibrat
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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[ccp4bb] AW: phenix.refine with ligand with ambiguous electron density

[Schreuder, Herman /DE]

Hi Nika,

Here you need some common sense. The green density in you polder map may just 
be the bulk solvent that was removed from the model to generate the polder map. 
In this case you have to use common sense and ask yourself a couple of 
questions:

  *   Is the density really from the ligand, of from some other component from 
your crystallization solution? Molecules like tris or hepes love it to 
masquerade for ligands of interest.
  *   Could it be that only part of the ligand is visible because the other 
parts are disordered or not present? This happens quite often. Instead of the 
full-ligand, a break-down product or reaction intermediate might have bound, or 
part of the ligand binding site is occupied by crystal contact.
In this case I would fit the part of the ligand which has convincing electron 
density, refine and look at the electron density maps to see if the fit to the 
density is convincing and if additional atoms could be added to the ligand. If 
you cannot fit the whole ligand, even after some efforts, I would submit a 
partially fitted model.

Best,
Herman



Von: CCP4 bulletin board  Im Auftrag von Nika Žibrat
Gesendet: Dienstag, 24. November 2020 12:29
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] phenix.refine with ligand with ambiguous electron density


Hello,



I have a question about protein-ligand, of which ligand displays an ambiguous 
electron density. I am solving a structure of protein with ligand  which was 
obtained via soaking. Structural characteristics indicate the ligand is present 
however the electron density is quite vague and too small for the size of the 
whole ligand. I did a Polder map which showed much larger area of green 
density. After insertion of my ligand into the green density in Polder I ran 
phenix.refine and there is a lot of red on the spot where the ligand is which 
was to be expected. This leaves me wondering how, if even do I incorporate the 
polder map data into my refine input.



My question is, how do I continue refining and validating the structure in this 
case?



Thank you,



Nika Žibrat




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Re: [ccp4bb] Diamond-II MX beamlines letters of support

[Hall, Dave (DLSLtd,RAL,LSCI)]

Dear all

Gentle reminder about the deadline of tonight for statements of support for two 
MX beamline projects at Diamond Light Source as part of the Diamond II upgrade 
programme:


  *   A major upgrade (KMX) of I24 will enhance its microfocus capability and 
extend further its serial synchrotron crystallography offering. The KMX 
proposal outline can be found 
here
  *   The I04-1 XChem beamline will be rebuilt as K04 to make a step-change in 
its XChem fragment screening programme. The K04 proposal outline can be found 
here
 .

There are 11 other projects under consideration and only 5 can be funded and 
community support is an essential part of the review process. If you see a need 
in the future for either or both of these proposals to be key to your research 
then please complete the short webform 
here.

Deadline for submission is by 23:59 GMT today (Tuesday 24th November).

Click 
here
 to register support.

Thanks

Dave

From: "ccp4bb@jiscmail.ac.uk"  on behalf of 
"david.h...@diamond.ac.uk" 
Reply to: "david.h...@diamond.ac.uk" 
Date: Thursday, 19 November 2020 at 08:53
To: "ccp4bb@jiscmail.ac.uk" 
Subject: Re: [ccp4bb] Diamond-II MX beamlines letters of support

Dear all

To follow up on Dave and Arnaud’s email (thanks!) the deadline for submissions 
of statements of support has been extended to 23:59 (GMT) on Tuesday 24th 
November.

There are fantastic proposals across the life and physical sciences for 
flagship projects for the Diamond II upgrade and statements of support from the 
community alongside the science cases developed with the user working groups 
are an important part of the review process.

Support statements from our many past, current and future users and supporters 
of the two flagship MX beamline projects outlined below would be greatly 
appreciated. You can be from the future generation of structural biologists 
through to PIs, HoDs, etc from both academia and industry and can submit 
support both as an individual and/or representing your organisation (Dept, 
University, DTP, company, etc) as appropriate.

Your support will help us shape the future science you can do at Diamond.

Statements of support can be submitted via this 
link.

Many thanks

Dave Hall
MX Group Leader
Diamond Light Source

From: "ccp4bb@jiscmail.ac.uk"  on behalf of David Briggs 

Reply to: David Briggs 
Date: Wednesday, 18 November 2020 at 10:10
To: "ccp4bb@jiscmail.ac.uk" 
Subject: [ccp4bb] Diamond-II MX beamlines letters of support

Dear all,

(Apologies for the slightly off-topic cross-post - hopefully, this will be of 
interest to all MX practitioners, not just Diamond light source users.)

As Diamond User Committee MX representatives, we'd just like to remind you that 
the deadline for letters of support for Diamond-II beamline upgrades is this 
Friday, 20th November.

As you are perhaps aware, the Diamond machine is scheduled for a major upgrade 
to a 4th generation source 2025-2027.
Further information about the upgrade can be found 
here.

All MX beamlines will benefit from a brighter, more focused source – however, 
two MX beamlines have been selected as flagship projects for significant 
upgrades.

I04-1 > K04 Ultra XChem.
The I04-1 fixed wavelength beamline used for the XChem fragment screening will 
be completely rebuilt as K04.
Briefly, K04 will allow higher throughput fragment screening (meaning more 
projects and more fragments can be screened) and it is hoped the improved 
characteristics of the Diamond-II beam will allow XChem methodologies to be 
extended to more difficult targets such as transmembrane proteins.

The K04 proposal outline can be found 
here
 [PDF].

I24 > KMX Kinetic micro crystallography.
The I24 microfocus beamline will be upgraded to enhance the capabilities of I24 
and allowing analysis of smaller crystals, radiation-sensitive crystals, and 
expanding the serial synchrotron crystallography (SSX) functionality of the 
beamline. The abilities to conduct room temperature SSX will also be enhanced 
to allow in-crystal kinetic analysis, with various on-line spectroscopic 
(Raman, UV-Vis, XES) detectors built into the 

[ccp4bb] last chance to support - large volume imaging at nm resolutions with x-rays

[Walsh, Martin (DLSLtd,RAL,LSCI)]

Dear all - if you see imaging cellular systems/ tissues organs at subcellular 
resolutions as key to your research questions then please do take a few minutes 
to add your support to our proposal for a dedicated bioimaging beamline at 
diamond for just this! - the beamline will take advantage of the upgrade to the 
diamond machine. This is one proposal out of many (13 in total) and only 5 of 
these will be funded -key to the selection process is showing that there is a 
need and support for this proposal  from the science community generally - we 
have set up a webform for statements of support - these can be concise and are 
encouraged from scientists at all stages of their career who see 
high-throughput large volume nm resolution x-ray bioimaging as important to 
support and develop their research - the deadline is today!

Link to web-form

Link to one-pager with more info: 
tinyurl.com/yxzruh8k


Deadline end-of-day Tuesday 24th November, click 
here to register support
Thanks!
Martin




--
Martin A. Walsh,
Deputy Director Life Sciences &
Group Leader at the Research Complex at Harwell,
Diamond Light Source,
Harwell Science & Innovation Campus,
Didcot OX11 0DE,
UK
T:+44 1235 778518
E:martin.wa...@diamond.ac.uk


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[ccp4bb] phenix.refine with ligand with ambiguous electron density

[Nika Žibrat]

Hello,


I have a question about protein-ligand, of which ligand displays an ambiguous 
electron density. I am solving a structure of protein with ligand  which was 
obtained via soaking. Structural characteristics indicate the ligand is present 
however the electron density is quite vague and too small for the size of the 
whole ligand. I did a Polder map which showed much larger area of green 
density. After insertion of my ligand into the green density in Polder I ran 
phenix.refine and there is a lot of red on the spot where the ligand is which 
was to be expected. This leaves me wondering how, if even do I incorporate the 
polder map data into my refine input.


My question is, how do I continue refining and validating the structure in this 
case?


Thank you,


Nika Žibrat




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