Re: [ccp4bb] RES: [ccp4bb] Se-SAD phasing

[Holton, James M]

It would not surprise me at all if pseudotranslation (aka tNCS) were holding 
you back.  It is like kryptonite to all phasing techniques, MR, MAD, SAD, MIR, 
you name it.  I recently did an analysis of old, never-solved data sets from my 
beamline.  These are MAD/SAD data that don't match anything in the PDB, despite 
being 10 years old or more.  At least 45% of them suffer from 
pseudotranslation.  Another 21% are twinned.

In contrast, if you search the PDB for the word "twin" only 0.5% of all entries 
contain that string.  Might explain why methods development on these two fronts 
has been slow.

I don't suppose you'd be up for crowdsourcing?  As in making your raw data 
publicly available?

-James Holton
MAD Scientist

On 7/18/2019 11:34 AM, Mario Tyago Murakami wrote:
Curiously, in the native data we observed a strong peak of 28%. However, the 
same was not seen in the Semet crystals. There is isomorphism between these 
crystals. Please see below the unit cell parameters. In the previous email I 
sent attached pointless, index.lp from xds and shelxc logs.

Native -> UNIT_CELL_CONSTANTS=   118.082   151.969   166.266  90.000  90.000  
90.000 (tNCS 28% F.C. = 0.5 0.135 0.50)
Semet  -> UNIT_CELL_CONSTANTS=   118.730   151.831   167.070  90.000  90.000  
90.000 (tNCS 8%  F.C. = 0.5 0.140 0.50)


De: Eleanor Dodson 
Enviada em: quinta-feira, 18 de julho de 2019 12:34
Para: Mario Tyago Murakami 

Cc: CCP4BB@JISCMAIL.AC.UK
Assunto: Re: [ccp4bb] Se-SAD phasing

Good advice above..
Someting extra - I would look at information about how the molecules might be 
arranged.
Is there any non-crystallographic translation? ( One with a coordinate 0f say 
z= 0.5 could mean the spacegroup
could be P21212 or P212121 )

Sometimes there is a sub-cell and you can search for fewer sites in that cell..
Eleanor



On Thu, 18 Jul 2019 at 16:28, Mario Tyago Murakami 
mailto:mario.murak...@lnbr.cnpem.br>> wrote:
Dear Andy, Clemens and Michal

The solution for orthrombic SG is very clear. P1 indexing results in a very 
similar Unit cell parameters without appreciable deviations in angles 
<0.1degrees.

It seems more likely the problem pointed by Andy of a lot of sites. I will try 
all the suggestions and hope to come back soon with good news.

Thanks
Mario

Em 18 de jul de 2019 11:44, THOMPSON Andrew 
mailto:andrew.thomp...@synchrotron-soleil.fr>>
 escreveu:

Dear Mario

The data seem very good but you are looking for an awful lot of sites. Keep 
trying with SHELX, but there are some keywords that you might use which may 
help (see extract from SHELX doc). This worked for me for a couple of 
structures with > 100 sites. You should also try with and without Patterson 
seeding.  You may need many thousand trials…. I would also start with the data 
cutoff somewhere between 3.5 - 4 A (to get rid of anisotropy) . You could also 
try manually modifying the Emin value to use just your strongest signal. Don’t 
expect the solution to come out easily - you may have to try different shelx 
runs for days before getting a single solution.

At the same time, you really do need to be absolutely certain of the space 
group ……any tests you can do

Good luck

Andy





For large selenomethionine substructures (which behave more like equal atom ab 
initiostructure solution of small molecules) it may be worth increasing the 
number of Pattersonpeaks used for the Patterson seeding (e.g. PATT 200; the 
default is 100) and adding theinstructions WEED 0.3 (random omit maps) and SKIP 
0.5 (uranium atom removal). Thelatter two are the defaults when PLOP is present 
but are switched off by default if PLOP isabsent. When PATS is used, WEED 
produces a much smaller additional improvement in thehit ratio than when PATS 
is absent. For small substructures (<10 sites), WEED and SKIP cando more harm 
than good by eliminating too many correct sites at once.



De : CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] De la part de 
Mario Tyago Murakami
Envoyé : jeudi 18 juillet 2019 16:19
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] Se-SAD phasing



Dear all,

I am trying to solve the phases of the following SeMet data, but so far 
unsuccessfully. Suggestions are very welcome. Please see below some details 
about the case.



The statistics below is from a merged data from  different kappas of the same 
crystal to increase redundancy. We used the fixed energy 12675 eV since the 
fluorescence detector was not working at the used beamline to get best energy 
values for this crystal.

Xtriage did not indicate any crystallographic pathology, except moderate 
anisotropy.

The unit cells parameters are 118.72   151.82   167.05  90.000  90.000  90.000 
(P212121) containing from 8 to 12 molecules in the asymmetric unit. The protein 
has ~28.5 kDa and 10 Met residues, excluding those from the N- and C-termini, 
proba

[ccp4bb] challenges in structural biology

[Holton, James M]

Hello folks,

I have the distinct honor of chairing the next Gordon Research 
Conference on Diffraction Methods in Structural Biology (July 26-31 
2020).  This meeting will focus on the biggest challenges currently 
faced by structural biologists, and I mean actual real-world 
challenges.  As much as possible, these challenges will take the form of 
friendly competitions with defined parameters, data, a scoring system, 
and "winners", to be established along with other unpublished results 
only at the meeting, as is tradition at GRCs.

But what are the principle challenges in biological structure 
determination today?  I of course have my own ideas, but I feel like I'm 
forgetting something.  Obvious choices are:
1) getting crystals to diffract better
2) building models into low-resolution maps (after failing at #1)
3) telling if a ligand is really there or not
4) the phase problem (dealing with weak signal, twinning and 
pseudotranslation)
5) what does "resolution" really mean?
6) why are macromolecular R factors so much higher than small-molecule ones?
7) what is the best way to process serial crystallography data?
8) how should one deal with non-isomorphism in multi-crystal methods?
9) what is the "structure" of something that won't sit still?

What am I missing?  Is industry facing different problems than 
academics?  Are there specific challenges facing electron-based 
techniques?  If so, could the combined strength of all the world's 
methods developers solve them?  I'm interested in hearing the voice of 
this community.  On or off-list is fine.

-James Holton
MAD Scientist




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Re: [ccp4bb] resolution

[Holton, James M]

Last time I checked phenix.refine did not use sig(F) nor sig(I) in its 
likelihood calculation.  Refmac does, but for a long time it was not the 
default.  You can turn it off with the WEIGHT NOEXP command, or you can 
even run with no "SIGx" at all in your mtz file.  You do this by leaving 
SIGFP out on the LABIN line.  This can sometimes help, but generally not 
by much.

I'll admit I was surprised when I first learned this is the way sigmas 
are treated in modern maximum-likelihood refinement.  But as it turns 
out sig(I) is almost never the dominant source of error in 
macromolecular models, so leaving it out generally goes unnoticed. There 
are also a few cases in the PDB where the sigmas are completely bonkers 
and including them can make things worse.  So, ignoring sigmas is 
perhaps a safe default.

This is not to say that sigmas are completely useless, they play a very 
important role in phasing, where the errors in the intensity differences 
must be correctly propagated in order for phase improvement to have the 
best chance of working. But for refining a native structure against 
intensity or F data, there just isn't much impact. Don't believe me?  
Try it.  Use sftools to change all your sigI values to, say, the 
average.  Then re-run refinement and see how much it changes your final 
stats, if at all.

Leaving out high-angle or otherwise weak data can improve statistics, 
but that is not a reason to leave them out.  What this is telling you is 
that the fine details of the model are still not in agreement with the 
data.  I the case of the OP, I suspect the Fcalc vs Ftrue difference is 
larger than normal.  Something else is wrong.  In such cases I always 
like to look at the real-space representation of Rwork, which is the 
Fo-Fc difference map.  How big is the biggest peak in this map? Is it 
positive or negative? And where is it?

-James Holton
MAD Scientist

On 7/4/2019 11:05 PM, graeme.win...@diamond.ac.uk wrote:
> Pavel,
>
> Please correct if wrong, but I thought most refinement programs used the 
> weights e.g. sig(I/F) with I/F so would not really have a hard cut off 
> anyway? You’re just making the stats worse but the model should stay ~ the 
> same (unless you have outliers in there)
>
> Clearly there will be a point where the model stops improving, which is the 
> “true” limit…
>
> Cheers Graeme
>
>
>
> On 5 Jul 2019, at 06:49, Pavel Afonine 
> mailto:pafon...@gmail.com>> wrote:
>
> Hi Sam Tang,
>
> Sorry for a naive question. Is there any circumstances where one may wish to 
> refine to a lower resolution? For example if one has a dataset processed to 2 
> A, is there any good reasons for he/she to refine to only, say 2.5 A?
>
> yes, certainly. For example, when information content in the data can justify 
> it.. Randy Read can comment on this more! Also instead of a hard cutoff using 
> a smooth weight based attenuation may be even better. AFAIK, no refinement 
> program can do this smartly currently.
> Pavel
>
> 
>
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Re: [ccp4bb] different residues as alternate occupancy?

[Holton, James M]

A classic case of this is crambin.  Residue 25 of 3nir.

-James Holton
MAD Scientist

On 6/24/2019 1:00 AM, Guenter Fritz wrote:
> Dear all,
>
> I am refining a multimer and mass spec data of the sample indicate 
> that there is a mixture of two variants which differ in one amino acid 
> residue. The density that we see is therefore most likely an average 
> of both variants. I have created a pdb file with "alternate residues" 
> each with 0.5 occupancy at this position to use it in refinement.  
> However, the programs detect this as an error in the pdb file.
>
> Has anyone  faced such a problem previously? Any suggestions are very 
> much appreciated.
>
> Thanks a lot and best regards, Guenter
>
> 
>
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Re: [ccp4bb] On estimating Unit Structure Factor distribution

[Holton, James M]

The CCP4 program you are looking for is "cad".  Using the "SCALE" keyword you 
can apply an overall positive or negative B factor to any mtz data set.  
Negative B factors are sharpening, which is what you are trying to do.

There is also another program called "ecalc", which is specifically designed 
for computing normalized structure factors like "U", which is closely related 
to "E".

As for which B factor to apply?  Ostensibly the Wilson B factor and the average 
atomic B factor should be the same number.  The Wilson B, however, is prone to 
systematic errors arising from how you decide which resolution range to fit.  
There are also details about the distribution of B factors in the model and the 
relative abundance of different atomic numbers.  However, I've always found 
these considerations dwarfed by the errors in estimating the Wilson B.  
Depending on what you are trying to accomplish, you might want to raise your B 
factor a bit anyway to reduce the overall noise.


As for F000, another article describing its estimation is here: 
https://doi.org/10.1073/pnas.1302823110

Script from that publication for calculating F000 is here:
https://bl831.als.lbl.gov/END/RAPID/scripts/find_F000.com

This script requires the CCP4 Suite and the Phenix Suite version 1.6 or lower.  
This was the last version of phenix.refine that reported k_sol and B_sol for 
the bulk solvent.  Without these, you cannot calculate the number of electrons 
in the bulk solvent.

Alternately, if you have run refmac you can look through the log for the 
string: "Partial structure1: scale = "
The "scale =" and "B  =" numbers that follow are the k_sol and B_sol for the 
bulk solvent.  You can then provide these numbers on the command line of the 
find_F000.com script above.  Then again, if you have already run refmac it 
might be simpler to provide the MSKOUT command-line parameter to refmac.  This 
will output the bulk solvent as a CCP4 map file.  Once you have that, all you 
need to do is run "mapdump" to report the average value of the electron density 
in this map.  You multiply this by the k_sol value from the log and then 
multiply by the unit cell volume and you now have the number of electrons in 
the bulk solvent.  Add up all the atomic numbers in your PDB file (including 
hydrogen) and you have the rest of the electrons in your unit cell.  The sum of 
all electrons in one unit cell is F000.

HTH

-James Holton
MAD Scientist

On 5/15/2019 5:52 AM, Andre LB Ambrosio wrote:
Dear Pavel, thank you so very much for the prompt feedback.
That would be extremely useful if you could script the Uhkl calculation in 
CCTBX.
With best regards,
Andre.

Em qua, 15 de mai de 2019 às 09:47, Pavel Afonine 
mailto:pafon...@gmail.com>> escreveu:
Hi Andre,

- Is there any macromolecular crystallography software that can compute Uhkl as 
above, or equivalent?

I estimate this can take about 10 minutes to script in CCTBX. I can write a 
script for you, if interested, and send off list.

- If not, would it be more correct to use the Wilson-B or the mean B from the 
final model?

I'd guess mean B from the model is a better estimate.

- How can F(000) be best estimated from the final model, which is not 
necessarily always the most complete or best refined? Should we simply add 
together the number of electrons for all the atoms refined in the asymmetric 
unit (protein + ligands + solvent)?

See my previous email.

Pavel



--
Andre LB Ambrosio



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Re: [ccp4bb] unidentified crescent-shaped electron density

[Holton, James M]

Has anyone ever done the experiment of switching precipitants/cryoprotectants 
and verifying that this crown-of-PEG around lysine goes away when you do?

Just curious,

-James Holton
MAD Scientist

On 4/1/2019 4:26 PM, Paul Emsley wrote:


Agreed. "PG4" - so that you don't have to go searching for it.

On 02/04/19 00:22, Peat, Tom (Manufacturing, Parkville) wrote:
Hello Zhen,

PEG comes as a mixture and the weight given on the bottle is just the average 
molecular weight, so you don’t need any ‘cleavage’ to have smaller molecular 
weight PEGs present in your crystallisation cocktail.
Try putting a small PEG in, it looks like it might fit.
Cheers, tom

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhen Luo
Sent: Tuesday, 2 April 2019 9:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unidentified crescent-shaped electron density

Dear all,

Could you please shed some light on what this crescent-shaped density around 
the lysine side chain might belong to? I now have two unrelated protein 
structures where this kind of density can be found surrounding a lysine side 
chain.


One protein was crystallised in 0.1 M CaCl2 and 20% PEG 3350; the other in 10% 
PEG 2, 20% PEG MME 550, 0.03 M CaCl2/MgCl2, 0.1 M MES/imidazole. Protein 
buffers contained 0.025 M HEPES and 0.15 M NaCl. None of these fitted in well. 
Could it be cleaved PEG?

Any suggestion would be greatly appreciated. Thanks in advance!

Best regards,
Zhen Luo

School of Chemistry and Molecular Biosciences
The University of Queensland, Australia






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Re: [ccp4bb] Refinement

[Holton, James M]

In coot, select.

Validate > Difference Map Peaks > Find Peaks Above 4 r.m.s.d > [Find Peaks]

What do you see?

Always remember, the real-space representation of your Rwork is the Fo-Fc map.  
The biggest peak or valley in this map is usually dominating your Rwork/Rfree.  
If you feel your Rwork/Rfree are too high, it is best to think of something 
sensible to put into your biggest Fo-Fc peak.  If you have a big, negative 
feature in the middle of nowhere, try adjusting your bulk solvent parameters in 
refinement.

It is also a good idea to look at your data quality stats.  If I/sig(I) is 5, 
then I wouldn't expect Rwork/Rfree to ever drop below ~20%.  This is because 
the error in the data is already about 20%.  If your model fits better than 
that, it is probably modelling noise.

If you have no difference features to speak of and your Rwork/Rfree are still 
high, it can be a good thing to prune back your model to include only the atoms 
you are really sure about.  That is, well-ordered main chain, and maybe a few 
strong side chains like disulfides and obvious aromatics.  Refine this heavily 
pruned-model to convergence.  Don't worry about the R factors being high, they 
are supposed to be high when large parts of the model are missing.  By 
"convergence" I mean until the atoms stop moving.  R factors leveling off is 
not "convergence".  If your atoms are still wandering about and changing B 
factors, then run the refinement one more time.  Wait for things to settle.  
This is kind of like running a column, you want to wait until everything has 
equilibrated before you inject something new.  This kind of equilibration is 
the only way to know that the rise or fall in R you see is due to the change 
you just made, as opposed to an after-effect of something you did a few dozen 
cycles ago.  Once that is done, look at the top feature in your Fo-Fc 
difference map.  This tallest peak is the least likely thing in your unit cell 
to be wrong.  Build in this feature.  Then re-refine to convergence again.  
This can take a while, but it is the best way to ensure that you avoid model 
bias of any kind.  Building just one feature at a time is the most conservative 
strategy.  If you're in more of a hurry, you might consider anything above 6 
sigmas to be "safe", but 5 is pushing it, and 4 is dangerous unless there is 
nothing else left in the map.

Happy Building!

-James Holton
MAD Scientist

On 3/23/2019 9:17 PM, StrBio wrote:
ALL.

I have data at 2.4 A in P21 sp gr, helical protein.
Refined to Rwork 29 Rfree 34 with nice density map and all nice statistics 
oither Rfactor (by Phenix). Refmac quit same.
Should I deposit it or look better data?
Any suggestion?





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Re: [ccp4bb] Nitril groups not present on compounds

[Holton, James M]

10% of which beam?

-James Holton
MAD Scientist

On Feb 26, 2019, at 1:38 PM, Maria Håkansson 
mailto:maria.hakans...@saromics.com>> wrote:

Dear all,

We have experienced nitril groups not present in the electron density
after data collection on compounds bound to different proteins.

In one of the projects the compound was tested by MS to check that
the nitril is present in solution before data collection (crystals used to for 
MS analysis).
The nitril should be present since the molecular weight of the compound is the 
same.

Also the crystals have been exposed using 10% of the beam (instead of 100%) to 
try to make
the effect of radiation damage as small as possible. Still the nitril is not 
present.

In both cases the nitril atoms have been modeled with high temperature factors 
but
the electron density is missing, see parts of the compound in the image below 
contoured at 1 sigma level and in a 1.1 Å electron density map.

Has anyone else experienced this?

Best regards,
Maria



Maria Håkansson, PhD, Crystallization Facility Manager
Principal Scientist

SARomics Biostructures AB
Medicon Village
SE-223 81 Lund, Sweden

Mobile: +46 (0)76 8585706
Web: www.saromics.com







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Re: [ccp4bb] Turning off the bulk solvent modelling in Refmac5 to generate Polder maps?

[Holton, James M]

Yes, Dorothee is right.  Don't turn off all the bulk solvent!  That is not a 
polder map.

In refmac you want to use the keyword:
solvent exclude DUM

And then fill the space you want to have no bulk solvent with water atoms with 
residue ID "DUM" and perhaps occupancy set to zero, since you don't want them 
contributing to Fcalc.  You might also want to turn off clashes for these dummy 
atoms using:
vdwrestraints DUMM 0
or
vdwrestraints exlcude

documentation is here:
https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html

Refmac also supports externally-defined solvent, which can be very handy.  All 
you need is structure factors (amplitudes and phases) in the same MTZ file as 
your data.  I like to call them Fsolvent PHIsolvent. Put these on the LABIN 
line with FPART1=Fsolvent PHIP1=PHIsolvent.  You will probably also want to use:
SCPART 1
Tells refmac to optimize the scale and B factor assigned to your bulk solvent.
You probably also want:
SOLVNET NO
This turns off the built-in bulk solvent calculation.  If you don't do this, 
refmac will scale its own default bulk solvent mask alongside your 
manually-provided mask.  Then again, you might want to do that just to see what 
happens.  In fact, you can provide refmac with several "partial structure" 
masks.  All of them will get scaled if you put their number on the SCPART line.

If you want the map that refmac uses by default, use the MSKOUT feature to have 
refmac write it out for you.  You can then scale this map with mapmask to make 
sure it has a range from 0 to 1.  You might also want to use other map masking 
tools on it.  Once you have a solvent map you want to use, you can convert this 
map into structure factors using refmac's "mode sfcalc" feature.  Also 
documented on the link above.  Then you can provide them as FPART1 PHIP1 as 
above.

-James Holton
MAD Scientist

On 2/4/2019 4:13 PM, Dorothee Liebschner wrote:
Hi,

Please note that for polder maps, the bulk solvent is reset locally. Turning 
bulk solvent off entirely most likely deteriorates maps.

Best wishes,

Dorothee

On Mon, Feb 4, 2019 at 3:50 AM Samuel Davis (PG Research) 
mailto:s.w.da...@dundee.ac.uk>> wrote:
Hi,

I'm wondering if anyone knows if it is possible to turn off the bulk solvent 
modelling in Refmac5, for the purpose of generating Polder maps? I know that an 
option for Polder maps is directly implemented in Phenix, but we ideally want 
to use Refmac5, as we have used it for the rest of our refinement and want to 
keep it consistent if possible.

Thanks,

Samuel.

Samuel Davis
MRC 3.5 Year Programme PhD Student
Life Sciences, Biological Chemistry and Drug Discovery, University of Dundee
+44 (0)1382 388325 | swda...@dundee.ac.uk

Scottish University of the Year
The Times / Sunday Times Good University Guide 2016 and 2017

Follow my blog: https://musingsofanearlycareerscientist.wordpress.com

The University of Dundee is a registered Scottish Charity, No: SC015096



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--
Project Scientist, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
1 Cyclotron Road, M/S 33R0345
Berkeley, CA 94720
Tel: (510) 486-5709
Fax: (510) 486-5909
Web: https://phenix-online.org



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Re: [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?

[Holton, James M]

plastic.

Plastic cover slips are no good for UV or polarization, but they are way better 
than glass if you happen to want to try in-situ diffraction. 
(https://doi.org/10.1107/S002188981254)

If you can't afford commercial ones, then you can always cut up some inkjet 
transparency film sheets like McPherson did in the above reference.  Then after 
you've made a few hundred you can ask yourself how much you'd be willing to pay 
somebody else to do it for you.  There is no wrong answer to that question, but 
it will determine which route you take.

-James Holton
MAD Scientist

On 1/31/2019 12:17 AM, Rajnandani Kashyap wrote:
Dear All
I am a PhD student who requires lots of coverslips (!!) for setting up hanging 
drop crystallization. The company sells it for a huge amount. Also there is a 
wide monetary difference between a normal siliconized coverslip and a 22mm 
siliconized circle coverslips. We tried to search for an alternative companies 
but couldn't get any one who sells coverslips with the same dimensions 
(0.19-0.22mm glass thickness and 22 mm glass diameter). Is there any 
alternative company (distribution in India) from where we can buy them for a 
reasonable price?
Thanks in advance for sparing your valuable time and efforts.

Regards
Rajnandani Kashyap
India



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