[ccp4bb] Unusual dataset with high Rmerge and extremely low b-factors?

[Michael Jarva]

Hi CCP4BB,

I have some unusual crystal diffraction data I'd like to get your input on.

Almost a year ago I shot some small rods sticking out of a loop, so basically 
no liquid around them - using the microfocus MX2 beamline at the australian 
synchrotron, collected on an EIGER 16M detector.

The crystals diffracted weakly and was seemingly not viable at first glance 
because of high Rmerge/Rpims. See the aimless summary at the bottom of this 
post. This seemed to stem from a low spot intensity at low resolutions 
(I/sd(I)=4.6), but since the CC1/2 was fine I went with it anyway.

Here I also noted an unusually low Mosaicity, 0.05, and Wilson B-factors, 8.02 
Å^2.

Density maps looked great and the build refined easily enough (R/Rfree 
0.1939/0.2259) with a mean B-factor of 19.85, which according to phenix is 
lower than any other structure deposited in that resolution bin. Furthermore, 
the molprobity score is 0.83, and overall real-space correlation CC is 0.855.

So my question is, can I feel comfortable depositing this?

best regards
Michael

Chosen Solution:space group P 1 21 1
Unit cell:44.93   41.90   45.83  90.00  115.57   90.00
Number of batches in file:   1659
The data do not appear to be twinned, from the L-test
Overall InnerShell OuterShell

Low resolution limit   41.34 41.34  2.49

High resolution limit   2.40  8.98  2.40


Rmerge  (within I+/I-) 0.231 0.084 0.782

Rmerge  (all I+ and I-)0.266 0.099 0.983

Rmeas (within I+/I-)   0.323 0.118 1.091

Rmeas (all I+ & I-)0.317 0.118 1.167

Rpim (within I+/I-)0.225 0.084 0.759

Rpim (all I+ & I-) 0.171 0.063 0.623

Rmerge in top intensity bin0.079- -

Total number of observations   19901   362  2067

Total number unique 6054   126   611

Mean((I)/sd(I))  2.7   4.6   1.0

Mn(I) half-set correlation CC(1/2) 0.958 0.991 0.570

Completeness98.6  98.2  97.3

Multiplicity 3.3   2.9   3.4

Mean(Chi^2) 0.48  0.33  0.50


Anomalous completeness  81.7  92.2  75.1

Anomalous multiplicity   1.5   1.8   1.9

DelAnom correlation between half-sets -0.003 0.041 0.045

Mid-Slope of Anom Normal Probability   0.704   - -


The anomalous signal appears to be weak so anomalous flag was left OFF


Estimates of resolution limits: overall

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.67A

   from Mn(I/sd) >  2.00: limit =  2.87A


Estimates of resolution limits in reciprocal lattice directions:

  Along0.96 a* - 0.28 c*

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.40A  == maximum 
resolution

  Along k axis

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.86A

  Along   -0.17 a* + 0.99 c*

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.98A


Anisotropic deltaB (i.e. range of principal components), A^2:  8.62


Average unit cell:44.93   41.90   45.83   90.00  115.57   90.00

Space group: P 1 21 1

Average mosaicity:   0.05


Minimum and maximum SD correction factors: Fulls   1.27   1.28 Partials   0.00  
 0.00





Michael Jarva, PhD
ACRF Chemical Biology Division

The Walter and Eliza Hall Institute of Medical Research
1G Royal Parade
Parkville Victoria 3052
Australia

Phone: +61 3 9345 2493
Email: jarv...@wehi.edu.au | Web: http://www.wehi.edu.au/

The ACRF Chemical Biology Division is supported by the

Australian Cancer Research Foundation


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You must not disclose, forward, print or use it without the permission of the 
sender.

The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
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the continuing connection to country and community.
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Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

[Michael Jarva]

Thank you to Tomas, Bill, and Andrew for their tips and insights! It was really 
all I hoped for and more.


The eLife paper with its downloadable text file of mined pi-pi stacking is 
really useful and is easily adapted into fast visual inspection with pymol. 
Problem now is having more examples to look at than I have time for!



cheers

michael



From: Tomas Malinauskas 
Sent: Sunday, November 11, 2018 3:11 AM
To: Michael Jarva
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

Dear Michael,

On Sat, Nov 10, 2018 at 9:56 AM Michael Jarva  wrote:
>
> Dear ccp4 community,
>
>
> I have recently been working with a structure that has an Asparagine that 
> makes a planar stacking connection with a Tryptophan ring 
> (pep_ASN-TRP_v2.png), that seem to be a true pi-stacking interaction and I'd 
> like to find more examples of this.

You could check the following paper:
https://elifesciences.org/articles/31486
Pi-Pi contacts are an overlooked protein feature relevant to phase separation

They have a list of examples (including Asn-Trp pi-pi stacking) in PDB
(Figure 1, data source):
Pi-Pi contact annotations for the full PDB set.
Text file listing the pi-pi contacts observed across our non-redundant
PDB set, with contact types shown by residue annotations where single
amino acid names refer to sidechains and pairs of amino acids refer to
the backbone peptide bond between residue i and residue i + 1.
https://doi.org/10.7554/eLife.31486.005

Best wishes,
Tomas

Dr. Tomas Malinauskas
University of Oxford
Wellcome Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford OX3 7BN
United Kingdom
to...@strubi.ox.ac.uk
tomas.malinaus...@gmail.com





From: William G. Scott 
Sent: Sunday, November 11, 2018 1:54 AM
To: Michael Jarva
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

Hi Michael:

It always makes me happy to see that there are people who care about this.

3.3 to 3.4 Å should be an ideal distance for this, and, as you note, the lone 
pair
residing on the (sp^3-hybridized) nitrogen would have to be oriented for
favorable overlap, which is a bit harder to deduce from your figure.

The other rotomer would place oxygen at that position. Because it is 
double-bonded
to the gamma carbon, the lone pairs are oriented differently, and the pi-bond 
would
be approximately parallel to the plane containing the tryptophan, which would 
give
a nice pi-stacking interaction similar to what is seen with adjacent base pairs 
in nucleic acids.

Expectation bias, as well as having partial charges turned on during 
refinement, might
influence the rotomeric state of examples from the PDB, so be careful of social 
consensus.
With classical electostatics, N has a partial positive charge, and O has a 
partial negative
charge.  (If you think about it, all pi-stacking interactions, from the point 
of view of classical
electrostatics, would be somewhat repulsive.)

Good luck with this, and let us know of the outcome.


Bill



William G. Scott
Director, Program in Biochemistry and Molecular Biology
Professor, Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
University of California at Santa Cruz
Santa Cruz, California 95064
USA

http://scottlab.ucsc.edu<http://scottlab.ucsc.edu/>

> On Nov 10, 2018, at 1:45 AM, Michael Jarva  wrote:
>
> Dear ccp4 community,
>
> I have recently been working with a structure that has an Asparagine that 
> makes a planar stacking connection with a Tryptophan ring 
> (pep_ASN-TRP_v2.png), that seem to be a true pi-stacking interaction and I'd 
> like to find more examples of this.
>
> I've found a few other examples in the literature but they are mostly amide 
> hydrogen-pi interactions (4PTI_ASN-TYR_v2.png and 1N4W_ASN-FAD_v2.png), which 
> can be seen by the way the Asn is dipping down into the pi-cloud. One 
> potential exception is of a DNA-binding protein where the orientation is more 
> planar (3HXQ_GLN-DNA_v2.png).
>
> So, I'm looking for examples of Asparagine or Glutamine pi-stacking and am 
> sure there are more of them out there and would greatly appreciate any 
> examples.
>
> best regards
> Michael
>
> Michael Jarva, PhD
> ACRF Chemical Biology Division
> The Walter and Eliza Hall Institute of Medical Research
> 1G Royal Parade
> Parkville Victoria 3052
> Australia
> Phone: +61 3 9345 2493
> Email: jarv...@wehi.edu.au | Web: http://www.wehi.edu.au/
> The ACRF Chemical Biology Division is supported by the
> Australian Cancer Research Foundation
>
> ___
>
> The information in this email is confidential and intended solely for the 
> addressee.
> You must not disclose, forward, print or use it without the permission

Re: [ccp4bb] blast pdb for a very short sequence (3 amino acids)

[Michael Jarva]

With the vast number of sequences available I doubt you’ll get any meaningful 
results with only 3 amino acids…
However, I believe the full list of sequences in the PDB is available here for 
download:
ftp://ftp.rcsb.org/pub/pdb/derived_data/pdb_seqres.txt

Using this you could run your own local scripts to do customised searches.

Cheers
Mike

From: CCP4 bulletin board > 
on behalf of Xiao Lei >
Reply-To: Xiao Lei >
Date: Thursday, 26 October 2017 at 14:40
To: "CCP4BB@JISCMAIL.AC.UK" 
>
Subject: [ccp4bb] blast pdb for a very short sequence (3 amino acids)

Dear CCP4BB members,

Sorry this post is off-topic. I am asking is there a way to blast pdb for a 
very short sequence like 2 or 3 amino acids?  I tried this for a positive 
control in pdb database but returned that "Your search parameters were adjusted 
to search for a short input sequence."  and "No significant similarity found. ".

Re: [ccp4bb] PDB ligand 3-letter code for TCEP?

[Michael Jarva]

Finding the ligand you’re looking for in the PDB can be a bit tricky.
I believe you’re looking for TCE: http://www4.rcsb.org/ligand/TCE
Which I found by searching for its IUPAC name: 
3,3',3''-phosphanetriyltripropanoic acid

If that fails, you can usually find the SMILES string for your ligand and then 
search for that at using: http://www.rcsb.org/pdb/ligand/chemAdvSearch.do

Cheers
michael

From: CCP4 bulletin board > 
on behalf of Alex Lee >
Reply-To: Alex Lee >
Date: Thursday, 19 October 2017 at 11:19
To: "CCP4BB@JISCMAIL.AC.UK" 
>
Subject: [ccp4bb] PDB ligand 3-letter code for TCEP?

Hi All,

I tried to look for the 3-letter ligand for reducing reagent TCEP in PDB but I 
could not find one.  I even search with "Tris(2-carboxyethyl)phosphine 
hydrochloride" in PDB but got right hit. How do I need to to to pull the TCEP 
code out?

[ccp4bb] Another troublesome dataset (High Rfree after MR)

[Michael Jarva]

To add to the current anisotropic discussion I recently got a dataset I’m 
unable to refine and I’m hoping I could get some help on figuring out if 
there’s anything I can do.

I get a clear cut solution with Phaser using the same protein as search model 
and got a TFZ of >16, LLG >200, and a packing that makes sense, so I don’t 
doubt the solution. However, the maps look terrible, more like something I 
would expect from a 3.65Å dataset rather than the 2.65Å it supposedly is.

The dataset merges well in I4122 to 2.65Å with an overall Rmerge of 5% and a 
CC1/2 of >0.5 in the outer shell (see the bottom for full summary). There is 
some minor radiation damage but I could cut out most of it due to the high 
symmetry.

Xtriage reports no indication of twinning, but does say that the data is 
moderately anisotropic, so I ran the unmerged data through the StarAniso 
server, which reported the ellipsoidal resolution limits to be 2.304, 2.893, 
and 3.039. Refining with the anisotropically truncated data improves the maps 
somewhat, but I am still unable to get the Rfree below 38%. I tried using both 
phenix.refine and buster with similar results.

I’ve considered the choice of space group and tried I41, F222, I212121 , and 
C2, but with the same results, and Zanuda tells me the same thing.
Lastly, there is some minor ice rings, so my last try was to exclud the ice 
ring resolutions, but this made little to no difference.

Normally I would just write this off as the data being bad but this time all 
the statistics tell me this should be doable so I’m curious what has gone wrong.

Cheers
Michael Jarva



Summary data forProject: XDSproject Crystal: XDScrystal Dataset: 
XDSdataset


   Overall  InnerShell  OuterShell

Low resolution limit   34.87 34.87  2.78

High resolution limit   2.65  8.79  2.65


Rmerge  (within I+/I-) 0.052 0.026 1.595

Rmerge  (all I+ and I-)0.057 0.030 1.805

Rmeas (within I+/I-)   0.062 0.031 1.924

Rmeas (all I+ & I-)0.063 0.033 1.993

Rpim (within I+/I-)0.032 0.017 1.042

Rpim (all I+ & I-) 0.025 0.014 0.817

Rmerge in top intensity bin0.030- -

Total number of observations   19931   566  2681

Total number unique 3597   114   471

Mean((I)/sd(I)) 11.3  42.3   0.8

Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.575

Completeness97.9  93.1  99.6

Multiplicity 5.5   5.0   5.7


Anomalous completeness  92.4  92.1  96.8

Anomalous multiplicity   3.0   3.0   3.0

DelAnom correlation between half-sets  0.176 0.258 0.051

Mid-Slope of Anom Normal Probability   1.078   - -


Average unit cell:   82.39   82.39   69.73   90.00   90.00   90.00

Space group: I 41 2 2

Average mosaicity:   0.10

[ccp4bb] Optimising data processing of a I432 dataset with 75% solvent content.

[Michael Jarva]

Dear all,

I have a dataset that have two very interesting properties: a) It's in I432, 
and b) has a whooping 75% solvent content.
You might think that the solvent content obviously is a big red flag, and so 
did I, but I have phased this successfully with just one monomer, and the 
packing result does makes a lot of sense. The resulting maps contain no extra 
umodelled blobs, and trying to phase it with an additional molecules does not 
give a good solution.

The problem I have is that the diffraction intensity/Rmerge plummets/explodes 
around the 3.5Å mark (I assume because of the high solvent content) to such an 
extent that even though I have little radiation damage, 100% completeness in 
high resolution shells, and very high redundancy, any attempt to merge the 
dataset at a higher resolution has so far given no improvement to the maps.

I'm hoping that there might be a few tricks out there I can apply to the spot 
finding/integration/scaling steps have it merge in a even slightly higher 
resolution than I currently have been able to do.
Although I have a feeling that the only thing I can do is to grow another, much 
bigger, crystal…

many thanks for any feedback
/michael

See below for sample outputs from aimless:

   Overall  InnerShell  OuterShell
Low resolution limit   43.50 43.50  3.32
High resolution limit   3.10  8.78  3.10

Rmerge  (within I+/I-) 0.079 0.01021.891
Rmerge  (all I+ and I-)0.081 0.01122.502
Rmeas (within I+/I-)   0.084 0.01123.102
Rmeas (all I+ & I-)0.084 0.01123.169
Rpim (within I+/I-)0.027 0.004 7.335
Rpim (all I+ & I-) 0.020 0.003 5.450
Rmerge in top intensity bin0.010- -
Total number of observations   34917  1495  6448
Total number unique 2057   112   362
Mean((I)/sd(I)) 18.3 130.9   0.1
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.533
Completeness99.9  97.4 100.0
Multiplicity17.0  13.3  17.8

  Overall  InnerShell  OuterShell
Low resolution limit   43.50 43.50  3.84
High resolution limit   3.50  8.58  3.50

Rmerge  (within I+/I-) 0.052 0.011 2.422
Rmerge  (all I+ and I-)0.056 0.012 2.659
Rmeas (within I+/I-)   0.055 0.011 2.553
Rmeas (all I+ & I-)0.058 0.013 2.738
Rpim (within I+/I-)0.017 0.004 0.804
Rpim (all I+ & I-) 0.014 0.003 0.644
Rmerge in top intensity bin0.010- -
Total number of observations   24596  1690  6071
Total number unique 1462   120   343
Mean((I)/sd(I)) 25.8 132.0   1.0
Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.771
Completeness99.8  97.6 100.0
Multiplicity16.8  14.1  17.7