Re: [ccp4bb] [EXT] Re: [ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear Patrick,  

Thank you for the suggestions and for the detailed the statistics!! 

Regards 

Kavya 

On 2024-02-06 23:06, Patrick Shaw Stewart wrote:

> Hi Kavya 
> 
> 1. Make a seed stock from the globules or anything else that you think might 
> be crystalline, and recreen.  In other words, you should add your seed stock 
> to _random screens_ (not optimization experiments).  There could be many 
> conditions that are in the metastable zone of the phase diagram in your 
> normal screens - this method can give you crystals in those conditions. 
> 
> If this works, you'll be in a better position anyway because you'll have more 
> control - by diluting the seed stock, you can control the number of crystals 
> per drop. 
> 
> References: 
> 
>> D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed 
>> matrix-screening method for protein crystallization. _Acta Crystallographica 
>> Section D: Biological Crystallography_, _63_(4), pp.550-554. 
>> 
>> Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, 
>> P.F., 2011. Random microseeding: a theoretical and practical exploration of 
>> seed stability and seeding techniques for successful protein 
>> crystallization. _Crystal Growth & Design_, _11_(8), pp.3432-3441.
> 
> This is how we normally make the seed: 
> 
>> https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf
> 
> 2. Many years ago, we did some data mining of the crystallization conditions 
> in a remark in the PDB.  The concentrations that people reported are below.  
> There were eight reports where over 100 mg/mL was used.  It only goes up to 
> 2004. 
> 
>> https://www.douglas.co.uk/PDB_data.htm [1]
> 
> Good luck, 
> 
> Patrick 
> 
> On Mon, Feb 5, 2024 at 10:27 AM kavyashreem  
> wrote: 
> 
>> Dear All,  
>> 
>> Has anyone worked on a protein which is highly soluble even at 80mg/ml? 
>> 
>> We have one such candidate, which does not precipitate even at 80mg/ml 
>> instead forms phase separated globules in crystallization plate, which 
>> eventually hardens over a period of 1 to 1.5 months (which is florescent 
>> under UV microscope.) 
>> 
>> We tried screening at different pH, but failed to get any hits. 
>> 
>> Since we got few conditions in which the phase separated globules 
>> solidified, we focused on them and expanded with 120mg/ml protein, still 
>> there were not visible precipitates except for the phase separation. This 
>> has been a challenging target so far. We have tried with different 
>> constructs, which unfortunately are not soluble! 
>> 
>> Does POMs help in such cases? Or do you have any other suggestion.  
>> 
>> Thank you  
>> 
>> Regards 
>> 
>> Kavya 
>> 
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> 
> -- 
> 
> patr...@douglas.co.ukDouglas Instruments Ltd.
> Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
> Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek
> 
> http://www.douglas.co.uk
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Re: [ccp4bb] what is isomorphous?

[Robbie Joosten]

Hi Carlos,

In a practical setting you don't have to be very purist. The memory with 
respect to the reflection data is lost if you refine to convergence. Now there 
was are recent discussion on refinement convergence and again you can be quite 
purist here. However, if you go through a few cycles of rebuilding and 
refinement until R and R-free are stable, you are in clear with respect to 
cheating.*

HTH,
Robbie

When working with ligands there a much more severe ways of cheating (oneself).

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Carlos
> Kikuti
> Sent: Thursday, February 8, 2024 00:24
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] what is isomorphous?
> 
> Hello!
> 
> I have to admit my maths is a bit lazy, but this discussion got me stitched 
> up,
> because of a point I believe has not been addressed: the Rfree flags. I've 
> been
> trained to import Rfree flags whenever the crystals have the same space group
> and similar cell dimensions to the search model for molecular replacement -
> this to avoid "cheating" the Rfree validation with reflections that the search
> model has already 'seen'. We often work with series of crystals of the same
> proteins with different ligands, which give groups of very similar unit 
> cells. So
> far my strategy has been to mirror the Rfree flags (using the -ref or -rfree
> keyword in Autoproc) whenever the biggest difference in one of the dimensions
> is 5% - number just out of my instinct, taking in account the Rmerge of ~0.1 
> or
> less in the good cases. Maximum resolutions are between 2.7 and 1.9
> angstroms. Now considering the fact that isomorphism depends on resolution,
> it makes me reconsider the 5% cut-off: this might be fine at the low 
> resolution,
> but what about the higher resolution shells? What would be the best way to
> proceed in these cases, then? Because the level of 'cheating' will also vary 
> with
> resolution...
> 
> Carlos
> 
> On Sun, Dec 31, 2023 at 5:21 PM James Holton   > wrote:
> 
> 
> 
>   Ahh yes. I still have the very helpful email I got from Dame Louise
> Johnson in 2010.  I don't think she would mind my quoting it here:
> 
> 
> 
>   Dear James
> 
>   I was sorry to miss you when you were at Diamond - I was in
> Germany.
> 
>   The story of the two forms of lysozyme crystals goes back to
> about 1964 when
>   it was found that the diffraction patterns from different 
> crystals
> could be
>   placed in one of two classes depending on their intensities.
> This discovery
>   was a big set back at the time and I can remember a lecture
> title being
>   changed from the 'The structure of lysozyme' to 'The structure
> of lysozyme
>   two steps forward and one step back'.  Thereafter the  crystals
> were
>   screened based on intensities of  the (11,11,l) rows to
> distinguish them
>   (e.g. 11,11,4 > 11,11,5 in one form and vice versa in another).
> Data were
>   collected only for those that fulfilled the Type II criteria. 
> (These
>   reflections were easy to measure on the linear diffractometer
> because
>   crystals were mounted to rotate about the diagonal axis). As I
> recall both
>   Type I and Type II could be found in the same crystallisation
> batch .
>   Although sometimes the external morphology allowed
> recognition this was not
>   infallible.
> 
>   The structure was based on Type II crystals. Later a graduate
> student Helen
>   Handoll examined Type I. The work, which was in the early days
> and before
>   refinement programmes, seemed to suggest that the
> differences lay in the
>   arrangement of water or chloride molecules (Lysozyme was
> crystallised from
>   NaCl). But the work was never written up.  Keith Wilson at one
> stage was
>   following this up as lysozyme was being used to test data
> collection
>   strategies but I do not know the outcome.
> 
>   An account of this is given in International Table Volume F
> (Rossmann and
>   Arnold edited 2001) p760.
> 
>   Tony North was much involved in sorting this out and if you
> wanted more info
>   he would be the person to contact.
> 
>   I hope this is helpful. Do let me know if you need more.
> 
>   Best wishes
> 
>   Louise
> 
>   Armed with this advice, I searched the PDB using what I call the
> "Johnson ratio" of F(11,11,4) / F(11,11,5) and found there was a continuous
> spectrum (pasted below). The extrema of this spectrum were 3aw6 and 3aw7
> (circled), which are not only from the same paper, but from the same crystal: 
> a
> dehydration study.  Despite a modest unit cell size change of 0.7%, the 
> R-factor
> between the Fobs of 

Re: [ccp4bb] what is isomorphous?

[Carlos Kikuti]

Hello!

I have to admit my maths is a bit lazy, but this discussion got me stitched
up, because of a point I believe has not been addressed: the Rfree flags.
I've been trained to import Rfree flags whenever the crystals have the same
space group and similar cell dimensions to the search model for molecular
replacement - this to avoid "cheating" the Rfree validation with
reflections that the search model has already 'seen'. We often work with
series of crystals of the same proteins with different ligands, which give
groups of very similar unit cells. So far my strategy has been to mirror
the Rfree flags (using the -ref or -rfree keyword in Autoproc) whenever the
biggest difference in one of the dimensions is 5% - number just out of my
instinct, taking in account the Rmerge of ~0.1 or less in the good cases.
Maximum resolutions are between 2.7 and 1.9 angstroms. Now considering the
fact that isomorphism depends on resolution, it makes me reconsider the 5%
cut-off: this might be fine at the low resolution, but what about the
higher resolution shells? What would be the best way to proceed in these
cases, then? Because the level of 'cheating' will also vary with
resolution...

Carlos

On Sun, Dec 31, 2023 at 5:21 PM James Holton  wrote:

> Ahh yes. I still have the very helpful email I got from Dame Louise
> Johnson in 2010.  I don't think she would mind my quoting it here:
>
> Dear James
>
> I was sorry to miss you when you were at Diamond - I was in Germany.
>
> The story of the two forms of lysozyme crystals goes back to about 1964 when
> it was found that the diffraction patterns from different crystals could be
> placed in one of two classes depending on their intensities.  This discovery
> was a big set back at the time and I can remember a lecture title being
> changed from the 'The structure of lysozyme' to 'The structure of lysozyme
> two steps forward and one step back'.  Thereafter the  crystals were
> screened based on intensities of  the (11,11,l) rows to distinguish them
> (e.g. 11,11,4 > 11,11,5 in one form and vice versa in another). Data were
> collected only for those that fulfilled the Type II criteria. (These
> reflections were easy to measure on the linear diffractometer because
> crystals were mounted to rotate about the diagonal axis). As I recall both
> Type I and Type II could be found in the same crystallisation batch .
> Although sometimes the external morphology allowed recognition this was not
> infallible.
>
> The structure was based on Type II crystals. Later a graduate student Helen
> Handoll examined Type I. The work, which was in the early days and before
> refinement programmes, seemed to suggest that the differences lay in the
> arrangement of water or chloride molecules (Lysozyme was crystallised from
> NaCl). But the work was never written up.  Keith Wilson at one stage was
> following this up as lysozyme was being used to test data collection
> strategies but I do not know the outcome.
>
> An account of this is given in International Table Volume F (Rossmann and
> Arnold edited 2001) p760.
>
> Tony North was much involved in sorting this out and if you wanted more info
> he would be the person to contact.
>
> I hope this is helpful. Do let me know if you need more.
>
> Best wishes
>
> Louise
>
> Armed with this advice, I searched the PDB using what I call the "Johnson
> ratio" of F(11,11,4) / F(11,11,5) and found there was a continuous spectrum
> (pasted below). The extrema of this spectrum were 3aw6 and 3aw7 (circled),
> which are not only from the same paper, but from the same crystal: a
> dehydration study.  Despite a modest unit cell size change of 0.7%, the
> R-factor between the Fobs of these two entries (aka R-iso) is 44%. Its like
> they are different proteins, and a 12% change in relative humidity was all
> it took.  I never did get a chance to tell Louise that it was a dehydration
> effect. It took me too long to figure it out. But, I expect she would have
> found that information delightful.
>
> To weigh in on the OP:
> First: @Doeke, no I am not reviewing your new paper, but I hope whomever
> is is being helpful.
>
> Second: I am with Randy Read that isomorphism means "same shape", and also
> with Bernhard Rupp that "same" is resolution dependent.  Anything is
> "isomorphous" if you stand far enough away from it (like Carl Sagan's "pale
> blue dot").  So, I personally define "isomorphism" in terms of the
> agreement between the structure factors (Fobs). When does it become
> non-isomorphism? I say this is when the changes in Fobs become intolerable.
> What is intolerable? Depends on what you are doing, but in general it is
> good to compare the effect of interest to the existing noise. If the
> changes in Fobs due to the structural shift become larger than SIGFobs,
> then you start having "non-isomorphism".  For the common example of merging
> data from multiple crystals, non-isomorphism becomes intolerable when it is
> large enough to degrade rather than 

[ccp4bb] Post-doc/staff openings: AAV structure, cell entry & gene therapy

[Chapman, Michael S.]

Post-doctoral and staff positions are open for structural studies of AAV, the 
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Appointments at the University of Missouri can be as post-doctoral fellows or 
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Schweitzer Hall, Columbia MO 65211
573.882.4845 | 573.882.9825 (direct) | 
chapma...@missouri.edu




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[ccp4bb] Reminder: School on Single molecule biophysics in cell lysates, 13-17 May 2024, IBT, Vestec, Czech Republic

[Jan Dohnalek]

Just a reminder - the registration to this course is open till *29
February.*

Dear colleagues,

The registration to a specialized course - Basic level school of MOSBRI is
open.
Find all the relevant details on this page
https://www.mosbri.eu/training/basic-level-schools/bls2/

This basic-level school is aimed at biologists, biophysicists, biochemists,
structural biologists, etc., who want to learn a technique enabling high
throughput screening for dynamic parameters of biochemical interactions on
a single molecule level.

The deadline for submission of an application to participate in this course
is: Thursday 29th February 2024.

Jan Dohnalek
MoB-IBT centre of MOSBRI



-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758



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Re: [ccp4bb] program to complete (or to change) side chains to specific rotamers

[Jorge Iulek]

Hi all,

	I answered/thanked Pavel privately for the more involving and expansive 
learning advice on getting better acquainted with cctbx.
	coot without the gui - scripting: I imagined that, after all, in 
general, underlying gui commands are the modules with their 
parameters/input data and then the resulting outputs.
	Thanks James for indications/instructions and Paul later on 
complementing them.

Now, I gotta do work/study.

Jorge


On 2/6/24 16:08, James Holton wrote:

Hey Jorge,

Did you know coot can be scripted and run without the gui?  In general, 
all you need is to record a session and then edit the session file.  A 
shell script for doing something similar to what you want would look 
like this:


#! /bin/tcsh -f
#
#
set pdbfile = wrong.pdb
set mapmtz = refmacout.mtz
set chain = A
set resnum = 123
set TYP = LEU

cat << EOF >! mutate.py
imol_coords = handle_read_draw_molecule("$pdbfile")
imol_map = make_and_draw_map("$mapmtz","FWT","PHWT","",0,0)
set_go_to_atom_chain_residue_atom_name("${chain}",$resnum," CA ")
mutate_and_auto_fit(${resnum},"${chain}",imol_coords,imol_map,"${TYP}")
with_auto_accept([sphere_refine, 3.5])
save_coordinates(0,"coot.pdb")
coot_no_state_real_exit(0)
EOF

# run the program
coot --no-graphics --script mutate.py

A difference here is that instead of picking a particular rotamer, you 
let coot do it for you based on the density.


This might sound like a heavyweight approach, but coot actually spins up 
real fast when it doesn't need to build a graphics window.


I note you did ask for a way to specify a rotamer.  I say try doing that 
in coot and record the session.   However, if that gets too complicated, 
another way to do build-by-rotamer is with these awk programs that I 
wrote long before coot was a thing.  I still find them useful for 
certain tasks. I have created a git repo for them here:

https://github.com/jmholton/build_pdb

HTH,

-James Holton
MAD Scientist



On 2/6/2024 8:24 AM, Jorge Iulek wrote:

Dear all,


As I cannot find by myself, maybe someone can indicate here.
I would like a program, command line - not gui, that I would 
simply indicate residues and the desired rotamer, and so it will 
change the coordinate file to complete/change side chains accordingly, 
say, somehow like I say for Glu251 make it rotamer tp10, and so on.
Less desirable would be to indicate a reference structure for the 
specific rotamer. But I would really prefer that I indicate the 
specific residue and the rotamer I want.

Is there such a program or any ideas for a combination of programs?
Thanks,

Jorge



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Re: [ccp4bb] Calculation of energetics for many mutations

[Krieger, James M]

Hi Nick,

This would probably be a better question for the CCPBioSim mailing list: 
ccpbio...@jiscmail.ac.uk

There are several free energy simulation methods including alchemical ones that 
may help with mutations. Perhaps some docking-based approaches may be useful 
too.

Best wishes
James

From: CCP4 bulletin board  on behalf of Schnicker, 
Nicholas J 
Sent: Tuesday, February 6, 2024 4:55 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Calculation of energetics for many mutations

Hi all,

I'm looking for some suggestions for calculating energetics for an existing 
ligand bound protein structure. There are many mutations/combination of 
mutations within the ligand binding pocket that we'd like to make and then 
calculate energetics for all of them.

Please let me know if you have suggestions for the best software to perform 
this in a high-throughputish manner?

Thanks,
Nick



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Re: [ccp4bb] program to complete (or to change) side chains to specific rotamers

[Paul Emsley]

I agree with James and for the record, the function that it seem to me 
that you want is


set_residue_to_rotamer_name(imol, chain_id, res_no, ins_code, alt_conf, 
rotamer_name)


Paul.



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