Re: [ccp4bb] Residual density feature

[Ben Bax]

Dear Colin,
  I think the residual blob usually represents a combination of different 
chemical entities.

I think the correct thing to do is to try to dock all the possible chemical 
entities you have in your crystallisation buffer, purification buffers and 
things that have carried through in an unknown manner from the purification 
into the blob and try and estimate their occupancies (if they give a chemically 
reasonable fit).

If it is a big enough blob you could just try and dock glycerol (if this is in 
your cryo) and water and have them at half occupancy, and have a comment in the 
pdb header.

Regards, Ben


Ben Bax
Senior Scientific Investigator
BioMolecular Sciences UK
RD Platform Technology & Science

GSK
Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
Email   benjamin.d@gsk.com<mailto:benjamin.d@gsk.com>
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Tel   +44 (0) 1438 55 1156

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[cid:image001.png@01D0A754.53FEE6D0]



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Levy
Sent: 14 June 2015 09:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Residual density feature

Dear all,

I am currently working on a structure that contains a residual density feature 
located within the active site. Due to a combination of factors including 
limited occupancy, modest resolution, twinning etc it has not been possible to 
unambiguously identify this feature despite fairly extensive efforts.

What is the best way of dealing with such a feature when depositing the 
structure? Ideally I would like to draw attention to the presence of residual 
density whilst not implying that I have been able to identify it.

Many thanks,

Colin


Manchester
Protein
Structure
Facility

Dr. Colin W. Levy
MIB G034
Tel.  0161 275 5090
Mob.07786 197 554
c.l...@manchester.ac.uk<mailto:c.l...@manchester.ac.uk>




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Re: [ccp4bb] [Fwd: Re: [ccp4bb] FW: New ligand 3-letter code (help-7071)]

[Ben Bax]

Another major problem with the PDB is that it does not seem to believe in the 
existence of different tautomers or protonation states.

For example the ATP analogue AMPPNP can have the nitrogen between the beta and 
gamma phosphates protonated (-P-NH-P) or unprotonated (P-N=P), and there are 
well documented examples of both tautomers in the PDB (NH being a hydrogen bond 
donor and N a hydrogen bond acceptor).
If you look in the CSD you can see that the protonation state  of the nitrogen 
changes the geometry of the P-N-P bond.

However, as I understand it, the PDB considers all tautomeric (and protonated) 
forms of AMPPNP the same. When I tried to deposit a specific AMPPNP tautomer in 
2013, they would not accept it. The PDB also seems to believe, as I understand 
it, that the overall charge on AMPPNP is zero and that the phosphates do not 
carry negative charge.


Ben Bax
Senior Scientific Investigator
BioMolecular Sciences UK
RD Platform Technology & Science

GSK
Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
Email   benjamin.d@gsk.com<mailto:benjamin.d@gsk.com>
Mobile  +44 (0) 7912 600604
Tel +44 (0) 1438 55 1156

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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Martyn 
Symmons
Sent: 22 June 2015 23:39
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [Fwd: Re: [ccp4bb] FW: New ligand 3-letter code 
(help-7071)]

Well the problem is there is a lot more to a ligand than PDB coordinates - 
little things like bond orders... In addition people can publish ligands with 
atoms for which they have no density - so zero-occupancy is allowed too. So who 
should get priority - the group who publishes a ligand first, or the ones who 
actually have density for all the atoms?

These sorts of complications mean we all benefit from peer-review of the 
structure - that is why we put things on hold. And authors should have a chance 
to change their ligand definition based on reviewers'
comments - just as they are allowed to improve the PDB coordinates. So it is a 
worry for them that the PDB might 'publish' the ligand aspect of  their work 
before they have completed the peer-review process.

Maybe you don't believe is peer-review - in reply to which I'd paraphrase what 
people say about democracy - it's pretty bad but better than the alternatives.

But to return to the point I made: what really is the problem with maintaining 
and modifying _separate_ definitions with authors'
_separate_ deposited coordinates (and bond orders) while structures are on hold 
and being reviewed? Journals manage to keep all those submitted papers separate 
in their databases.

cheers
 M.

On Mon, Jun 22, 2015 at 3:12 AM, Edward A. Berry 
mailto:ber...@upstate.edu>> wrote:
>>   I can't imagine a journal doing that can you?  When I work on my
>> supplementary material in a paper I don't expect that the journal
>> will take a bit out and publish it separately to support the work of
>> my competitors. Not out of spite that I was beaten - but because I
>> don't want to take the responsibility for checking their science for them!
>
>
> I don't see the problem here. What about the dozens of authors who
> will benefit from using your ligand in their structure _after_ your
> structure comes out? You don't take responsibility for checking their
> science. Every author gets a copy of his final structure to check
> before it is released and each is responsible for his own.
> The only difference here is whether the competitor got to use it
> first, (which might sting a bit) or only after you had already made it
> your own with the first structure.
>
> I guess the ligand database is the responsibility of the pdb, but they
> depend on first depositors to help set up each ligand, so it is not
> surprising if the type model has coordinates from the first
> depositor's structure (although it would be convenient if they were
> all moved to c.o.m. at 0,0,0). When another group publishes a
> structure with the ligand, they will not be publishing the first
> depositor's coordinates because the ligand will be moved to its
> position in their structure and refined against their data, probably
> with somewhat different restraints.
>
> If the ligand is a top secret novel drug lead that your company is
> developing I guess it would come as a shock to find someone else has
> already deposited it, and it might be good to hasten not the
> publication but protecting of the compound with a patent!
>
> Although Miriam says a new 

Re: [ccp4bb] Refmac automatically handles twinning?

[Ben Bax]

The twin fraction can vary during data collection - if the beam is smaller than 
the crystal
For example you could have crystals which are like two very short ends of 
pencils - with flat ends together (in your case these might pencils could be in 
P3)
In some orientations you could have the beam going through only one pencil (not 
twinned) and some orientations going through both pencils ( 50:50 twin).
This is not yet properly handled in the data processing.



On 29 Mar 2022, at 09:39, Nicholas Keep  wrote:

Just double checking that Refmac automatically handles twinning without the 
need for a keyword or a box click in i2.  I have a crystal in P32 21 that gives 
poor Rfactors and maps (Rfree mid 40s) from what should be an excellent model. 
I have reprocessed in P32, P1 and C2 but all give similar results so it appears 
not to be hidden twinning.  There is no need to click a box or enter a keyword 
for this to be a check for twinning ?

There is severe anisotropy and a few other problems flagged up in iSPYB so 
there may be more complex problems than twinning with these crystals

Best wishes

Nick

-- 
Prof Nicholas H. Keep
Emeritus Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

Dept email n.k...@bbk.ac.uk
Telephone 020-3926-3475  (Will contact me at home if working as well as my 
office)



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Re: [ccp4bb] symmetry possibilities

[Ben Bax]

Hi Jorge,
   I have had some similar cases.
The twin fraction can vary during data collection on the synchrotron
(different areas of the crystal can have different twin fractions).
Sometimes this is not a problem and twin refinement works fine - sometimes
it is a problem and it is hard to get a clear electron density map from the
structure.
 R-factors from twinned structures tend to be lower than 'normal' R-factors
- your ~19/25% would be OK for normal data but ...
If the twin fraction varies as you rotate the crystal and illuminate
different volumes you can sometimes select fewer images to help.

Kind regards, Ben

On Wed, Jul 20, 2022 at 10:12 PM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Dear Jorge,
>
> what you write makes sense to me, and I cannot answer your questions. This
> is just to say that the situation you encounter is not completely uncommon,
> although most crystallographers would abandon such a crystal form, I guess.
> The technical term that describes this 4-fold twinning is "tetartohedral
> twinning" (in contrast to merohedral twinning, which involves two twin
> domains); maybe this helps to find additional pointers.
>
> best wishes,
> Kay
>
> On Wed, 20 Jul 2022 11:10:04 -0300, Jorge Iulek  wrote:
>
> >Dear all,
> >
> >
> >  We had some data collections at a Synchrotron. Crystals are a kind
> >of brush like (lattice strains, to use a term by Dr. Bergfors, though we
> >employed good effort for purification), but we took advantage of the
> >Synchrotron microfocus.
> >
> > Some of the datasets (images) clearly shows more than one lattice
> >(maybe more than two) that, struggling, we managed to process a get a
> >dataset which allowed molecular replacement and then (initial) refinement.
> >
> > But, in a second Synchrotron travel (and after efforts for
> >improving crystals), we got in some cases images with spots "well
> >separated'  "unique lattice" at some of the target spots (radiation on
> >the crystal) in the crystal.
> >
> > We processed these happily to P212121 (though some strange points
> >by pointless and/or xtriage, namely that " the L-test suggests that the
> >data may be twinned,  so the indicated Laue symmetry may be too high").
> >Systematic absences seem to be OK for lower resolution reflections, but
> >at higher resolution there seems to be more of a modulation (if a look
> >at a P222 processing).
> >
> > Anyway, we took, initially, refinement at P212121, nevertheless (I
> >should say not surprisingly), it stuck at 30/31 % R-free, model close
> >(if not at all) to completion. Data resolution is 2.31 A.
> >
> > We went to process these images in P1, and in the three possible
> >P21  (named P21_45, P21_122 and P21_155 - according to approx. axis
> >dimensions) sg's. Then we went to refine (refmac, twin option) the
> >current model (and then due "symmetry copies" produced with pdbset and
> >added to the model to be refined,) in all possible space groups, and
> >*care was taken* to  inherit the former r-free set *and* make the then
> >corresponding twin related reflections to be in the r-free set (to be
> >close to 5% of reflections, but "independent" reflections). It turned
> >out that the R/Rfree values dropped around to ~19/25% for P1 and one
> >(namely, P21_b151) of the P21; higher values for other P21's. As
> >expected, twin domains refine more or less close to 25 (P1) and 50% (any
> >P21), respectively.
> >
> > To mess up a bit more, I made the same study with "another dataset"
> >(another illuminated spot on the - same - crystal). In this case, only
> >the dataset processed in P1 presented "good" R values.
> >
> > I think these observations might correlate to what the "crystal "
> >physically is... a mix of portions genuinely P212121 but mixed, more or
> >less, that in some places with twins in one or more other types,
> >depending on where I focus my beam.
> >
> > Should I look at anything else to establish twin P1 is the best way
> >to refine this structure?
> >
> >
> > Related, and a question I mentioned before in this forum: what if a
> >genuine 2 axis (say , P222 to P2, or even to P1) (I do not mean this is
> >the case here) is ignored such that one would have doubled the number of
> >observations but also doubled the number of parameters to be refined
> >(suppose to exclude NCS in any case). Would one expect R/Rfree values to
> >be similar in both P222 and P2 (or even P1)? How much extra freedom
> >would one have besides the twin domain fractions to refine?
> >
> >
> > Yours,
> >
> >
> >Jorge
> >
> >
> >
> >To unsubscribe from the CCP4BB list, click the following link:
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> >
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Re: [ccp4bb] Regarding the diffraction image

[Ben Bax]

Hi Kavya,
More than one crystal? 
Epitaxial growth (protein crystal growing on salt crystal or vice versa)
Did you try IZIT? 
(https://hamptonresearch.com/product-Izit-Crystal-Dye-33.html).

I have seen salt and protein crystals in the same drop before now - but not 
growing together.
Ben

On 3 Feb 2023, at 22:15, CRAIG A BINGMAN 
<21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote:

Oxidation products of TCEP crystallize in the presence of zinc ions. These 
crystals fluoresce strongly under UV, which seems like a dirty trick, if your 
expectation is that only protein crystals can do that. The brightfield white 
light image of the crystals is also consistent with not-protein crystals, 
because of the high refractive index contrast between the crystals and the 
mother liquor.
 
From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of kavyashreem 
mailto:kavyashr...@instem.res.in>>
Date: Friday, February 3, 2023 at 2:32 AM
To: CCP4BB@JISCMAIL.AC.UK  mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Regarding the diffraction image

Dear all,

We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
condition 10%PEG3350, 50mM Zinc acetate.
Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8. 
Crystal: Crystal:   crystal 
under UV m
<8ef9453e.png>
When we collected the data at an in-house facility, it looked something like 
this:

The minimum resolution spot is around 9Ang and maximum ~2.2Ang.
I have not come across a protein diffraction like this, nor of a salt. When I 
ran the gel for the incubated protein (protein+ligand), there was no 
degradation.
Although, I was sure there is some problem with this image I tried processing, 
which could not be, But indexing showed a unit cell  of 11Ang, 11Ang, 46Ang in 
P3. which was quite expected for two of the axes but not the third.
Can anyone please shed some light on this diffraction image?
How can it happen?
 
Thank you
Regards
Kavya
 
 
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Re: [ccp4bb] RES: [ccp4bb] Rwork and Rfree the same?

[Ben Bax]

Fcalc maps look fantastic. Are you sure they were not using an Fcalc for the 
missing 35% of the data?
  Ben


On 29 Feb 2024, at 21:33, Rafael Marques  wrote:

Sorry for jumping into the post, but I would like the community’s opinion about 
completeness, once this topic was raised here. What could be considered 
reasonable? Recently I have seen a 65% completeness Crystal structure and, 
surprisingly, the electron density map was not that bad for a > 3.2 A 
structure. How such a nice map could have been calculated with such poor 
parameters? I could only think of anisotropy.
 
Best
 

Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
 
Mestre em Física Biomolecular
Universidade de São Paulo
 
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
 
phone: +55 16 99766-0021
 
   "A sorte acompanha uma mente bem treinada"

 
De: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
em nome de Paul Adams mailto:pdad...@lbl.gov>>
Enviado: Wednesday, February 28, 2024 2:58:16 PM
Para: CCP4BB@JISCMAIL.AC.UK  
mailto:CCP4BB@JISCMAIL.AC.UK>>
Assunto: Re: [ccp4bb] Rwork and Rfree the same?
 

By setting wxc (weight of the X-ray term) to 0.1 there is good chance that the 
refinement is dominated by the geometry term and the model isn’t really seeing 
the effect of the X-ray data. I suspect this would result in R-factors that are 
similar. Why they are so low is less clear, but as others have pointed out 38% 
completeness is a problem. It would be good to check if that is 38% overall, or 
just very incomplete in the higher resolution shells. If it is complete at 
lower resolution you might be able to do something with the dataset, but not 
using the default parameterization in refinement programs - you’ll need to 
apply constraints and additional restraints if you can, and look at the 
weighting (by modifying wxc_scale, not wxc).

There is a Phenix mailing list you might want to use as well (I assume you are 
using phenix.refine): https://phenix-online.org/mailman/listinfo/phenixbb 
 

> On Feb 28, 2024, at 8:21 AM, Justin Cruite  > wrote:
> 
> Thanks everyone,
> 
> I agree, 18.4% Rwork and Rfree is too good to be true for a 3.4 Å dataset. 
> The data was processed using autoProc and the staranisano mtz was used for 
> MR. The completeness is only 38%. It could be that the Rfree and Rwork 
> reflection sets are small because of this? What is the best way to check the 
> number of reflections used for Rwork and Rfree? Is this dataset usable at all?
> 
> Thanks!
> 
> Justin
> 
> On Wed, Feb 28, 2024 at 10:21 AM nicfoos  > wrote:
> Hello Justin,
> 
> There is something weird in your results. You mention Rwork/Rfree of 
> 0.1837.
> This means a pretty good refinement and also is very unusual to be 
> obtain for a resolution of 3.37.
> Additionally you should not have Rfree = Rwork.
> I suspect something wrong with you Rfree reflections sets. What size is 
> it ? Is your dataset complet ?
> How did you cut the res. ?
> 
> I hope this may help you.
> 
> Nicolas
> 
> 
> 
> On 2024-02-28 16:10, Justin Cruite wrote:
> > Hi everyone,
> > 
> > What does it mean if your Rwork and Rfree are exactly the same?
> > 
> > I solved a 3.37 Å structure with Phaser-MR and immediately ran 10
> > cycles of refinement with wxc = 0.1. Everything else at default. The
> > Rwork and Rfree are both 0.1837. Is this bad?
> > 
> > Thank you!
> > 
> > Justin
> > 
> > -
> > 
> > To unsubscribe from the CCP4BB list, click the following link:
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--
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Associate Laboratory Director for Biosciences, LBL (https://biosciences.lbl.gov 
) 
Principal Investigator, Computational Crystallography Initiative, LBL 
(http://cci.lbl.gov ) 
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) 
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Re: [ccp4bb] Announcements: Impact of EDMAPS.rcsb.org shutdown and related June 13 Virtual Office Hour on Generating DSN6 and MTZ Files

[Ben Bax]

Hi,
  I am just re-refining an old twinned dataset - which I will
redeposit (refinement is never finished).
The pdb seem to have automagically detwinned my data.
Where do I find my original twinned data (F and SIGF)? (or will I have
to redeposit my original un-detwinned structure factors).
 Thanks, Ben



On Fri, May 31, 2024 at 4:36 PM Ezra Peisach
 wrote:
>
> In fall of 2024, electron density map coefficients will be available in
> the public PDB archive for all X-ray structures. These map coefficients
> will be the same as used in wwPDB Validation Reports.
>
> The new map coefficients files will replace the electron density maps
> and combined map coefficient files calculated and distributed by RCSB
> PDB and used by the NGLviewer at RCSB.org. These data (served by
> EDMAPS.rcsb.org) are calculated using publicly-available coordinate
> files and structure factor files and offered in DSN6 formatted map files
> and MTZ formatted map coefficient files. RCSB PDB plans to shutdown the
> NGL viewer by July 2024
> (https://www.rcsb.org/news/feature/65b42d3fc76ca3abcc925d15) and will no
> longer need the data served by EDMAPS.rcsb.org.
>
> RCSB PDB will be phasing out EDMAPS.rcsb.org:
>
>   *  June 28, 2024: DSN6-formatted map files will no longer be
> provided. EDMAPS.rcsb.org will only serve MTZ files with map coefficients.
>   *  Fall 2024: Electron density map coefficients will be available
> in the public PDB archive for all X-ray structures. At this point,
> EDMAPS.rcsb.org will be shut down, including access to MTZ files with
> map coefficients from this service.
>
> To help users with this transition, RCSB PDB will be holding a Virtual
> Office Hour on Thursday June 13, 2024 from 1:00pm-2:00pm Eastern,
> 10:00am-11am Pacific. Please register online for this event at
> https://go.rutgers.edu/psx4pr63.
>
> Questions about this transition or the Virtual Office Hour can be sent
> to i...@rcsb.org.
>
> 
>
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