[ccp4bb]

[Pavel Afonine]

Just use P1 and "ncs" constraints. What's the problem? Or just keep entire
map and have only symmetry independent copy to work with until finishes,
then make the whole molecule. For real-space refinement it's totally
irrelevant whether you have whole molecule or 1/Nth of it. So.. it isn't
clear what the problem is..
Pavel

On Thu, May 18, 2017 at 10:06 PM, Qingfeng Chen  wrote:

> Hi,
>
> I have an EM map of a tetrameric protein. It was painful to work with this
> map since it is in P1 spacegroup, although 4-fold symmetry was already
> applied during map reconstruction.
>
> I noticed that people used MAPMAN to transform spacegroup, however, it
> seems not working for me. The map remained in P1 spacegroup afterwards.
>
> I used mtz file converted from .mrc and the tetrameric protein model as
> input and choose "run fft to generate simple map". I also specified "output
> map in ccp4 format to cover all atoms in pdb". In "infrequently used
> options", I input P4 in "generate map in spacegroup". Everything else was
> left as default.
>
> Any suggestions will be appreciated.
>
> Thanks!
>

[ccp4bb]

[Dale Tronrud]

   I'm sorry but I'm a little confused by your question.  If your map
already has four-fold symmetry why can't you simply build your model
once in one quarter of the map?  What do you hope to change by
specifying that the space group is P4?

Dale Tronrud

On 5/18/2017 10:06 PM, Qingfeng Chen wrote:
> Hi, 
> 
> I have an EM map of a tetrameric protein. It was painful to work with
> this map since it is in P1 spacegroup, although 4-fold symmetry was
> already applied during map reconstruction. 
> 
> I noticed that people used MAPMAN to transform spacegroup, however, it
> seems not working for me. The map remained in P1 spacegroup afterwards. 
> 
> I used mtz file converted from .mrc and the tetrameric protein model as
> input and choose "run fft to generate simple map". I also specified
> "output map in ccp4 format to cover all atoms in pdb". In "infrequently
> used options", I input P4 in "generate map in spacegroup". Everything
> else was left as default. 
> 
> Any suggestions will be appreciated. 
> 
> Thanks! 

[ccp4bb]

[Qingfeng Chen]

Hi,

I have an EM map of a tetrameric protein. It was painful to work with this
map since it is in P1 spacegroup, although 4-fold symmetry was already
applied during map reconstruction.

I noticed that people used MAPMAN to transform spacegroup, however, it
seems not working for me. The map remained in P1 spacegroup afterwards.

I used mtz file converted from .mrc and the tetrameric protein model as
input and choose "run fft to generate simple map". I also specified "output
map in ccp4 format to cover all atoms in pdb". In "infrequently used
options", I input P4 in "generate map in spacegroup". Everything else was
left as default.

Any suggestions will be appreciated.

Thanks!

[ccp4bb] How to download PROMOTIF v 2.0

[chenzhonghao...@163.com]

Dear all,
 
I want to download PROMOTIF v 2.0 from your ftp server(IP address 
128.40.46.11). 
 However, I can not visit it (ftp://128.40.46.11) because the ftp server was 
shut down.
 Moreover, I also sent emails to g...@uk.ac.ucl.bioc.bsm or 
thorn...@uk.ac.ucl.bioc.bsm. However, both emails were returned because the 
email address were not available.
 
  Would anyone like to tell me how to download it or tell me the right net 
address or email me the problem?
 
 Thanks in advance.
 
best,
 
 
Website: http://www.uoxray.uoregon.edu/local/manuals/promotif/document_2.html

The program is freely available for academic users. Industrial users should 
contact the authors directly. The files can be down loaded from our anonymous 
ftp server (IP address 128.40.46.11). The files are in the /pub/promotif/v2.0 
directory. Please read the LICENSE file, sign it and return it to the authors. 
If you experience problems in accessing the files by ftp contact the authors 
via e-mail at one of the following addresses: g...@uk.ac.ucl.bioc.bsm or 
thorn...@uk.ac.ucl.bioc.bsm .



 Zhonghao Chen

Re: [ccp4bb] NAD dihedral for C2N-C3N-C7N-N7N

[Dale Tronrud]

   I have looked over a number of high resolution models with NAD+ and
NADH in the PDB as well as small molecule structures.  I also have some
familiarity with similar chemistry in the decorations on the edge of
bacteriochlorophyll-a molecules.  The CONH2 group does flip over when
the hydrogen bonding environment calls for it.  It is very hard to tell
the difference between the oxygen atom and the nitrogen atom from the
appearance of the electron density so you always have to check the
hydrogen bonding environment when building an NAD? model.

   I have seen one case where a Ser -> Ala mutation in the protein
caused the group to flip with interesting consequences on the far side
of the co-factor.  My go-to QM person tells me that flipping this group
will change the energies of the molecular orbitals and therefor the
redox potential of the NAD? molecule so this conformational change may
be important to the action of your catalysis.

   I have also seen a number of NAD? models in the PDB where this group
is clearly misorientated.

   As you note, the torsion angle should be close to zero or 180.
However it is unlikely to have exactly those values because there are
non-bonded clashes when everything is in one plane.  Some restraint
libraries inappropriately restrain this group to be co-planar with the
six-membered ring.  As always, check you CIF!

Dale Tronrud


On 5/17/2017 12:46 PM, Jorge Iulek wrote:
> Dear all,
> 
> I came across some difficulty to refine a NAD molecule in a
> structure, specially its amide of the nicotinamide moiety.
> A (very) brief search in deposited structures seems to point that
> not so ever the C2N-C3N-C7N-N7N dihedral is close to either 0 or 180
> degrees, but in most cases it is to one of these, with a preference
> towards 0 degrees. Another search in the literature, and I could not
> find any study on either NAD or even the nicotinamide alone to calculate
> the energy barrier to rotate around this bond (in vacuum, eg).
> My data quality and resolution do not put much confidence on
> B-factor differences, but they seem to indicate that the cited dihedral
> angle should be close to 180 degrees, id est, O7N is "closer" to C2N
> (and, consequently, to N1N) than N7N is. In fact, I have a glutamine
> nearby whose terminal amide is interacting with the nicotinamide amide,
> so my idea is to make one's nitrogen to interact with other's oxygen.
> Concerning b-factor differences for this glutamine, they favor its NE2
> to point to nicotinamide amide, what would imply that the
> C2N-C3N-C7N-N7N dihedral to would be close to 180 degrees rather than 0
> degree.
> Is there any wide study on NAD nicotinamide amide conformation?
> Specially, bound to protein structures?
> Thanks,
> 
> Jorge
> 

[ccp4bb] PhD studentship to study enzymes involved in metabolic diseases

[Elton Zeqiraj]

Dear CCP4 Community,

Please see below for details of a 4-year PhD studentship at the University of 
Leeds with an industry placement at a pharmaceutical company.  

For details on how to apply follow instructions in the link below: 
https://www.findaphd.com/search/ProjectDetails.aspx?PJID=74943=735 


Best wishes,
Elton

**
Elton Zeqiraj, PhD
Sir Henry Dale Fellow
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
Astbury Building, Room 8.109
University of Leeds,
Leeds,
LS2 9JT, 
UK

Tel: +44 113 3433079
email: e.zeqi...@leeds.ac.uk 
web: www.personal.leeds.ac.uk/~fbsez 
**

Please consider the environment.  Do you really need to print this email?



Title: Structure-function, mechanism of action and therapeutic potential of 
enzymes involved in metabolic diseases

Project Description

We are excited to offer a PhD studentship to study enzymes involved in 
metabolic diseases. 
The studentship offers a unique opportunity to experience research at a 
world-class academic institution and a multinational pharmaceutical company. An 
industry placement (minimum of 3 months) at Vertex Pharmaceutical’s site in 
Abingdon (Oxfordshire) will take place in year 3 or 4 of the studentship. 

Techniques you will learn and use: 
- Cryo-electron microscopy and/or X-ray crystallography for determining 
structures of proteins and protein-protein complexes 
- Enzyme substrate kinetic measurements and enzyme inhibition 
- Biophysical techniques such as Fluorescence Polarisation and calorimetry 
- Cell biology and Cellular imaging assays 

The project will suit someone interested in structural biology, enzymology, 
biochemistry and drug discovery. Experience in structural biology is not 
required and suitable training will be provided. 

The Astbury Centre for Structural & Molecular Biology at the University of 
Leeds has excellent facilities for cryo-electron microscopy (two Titian Krios 
300 kV), protein crystallography (crystallisation robots, automatic imaging), 
and protein production with expertise in multi-subunit expression in insect 
cell, yeast and bacterial expression systems. The Astbury Centre is also 
well-equipped with instrumentation for biophysical analysis (e.g. ITC, 
fluorescence, multi-angle light scattering). Additionally, the University 
provides a state-of-the-art infrastructure for mammalian cell culture, 
high-throughput screening, imaging, mammalian genetics, chemical biology and 
proteomics. 

The University of Leeds was named University of the Year 2017 by The Times and 
The Sunday Times’ Good University Guide. 

As well as the stipend below, the successful candidate will receive an annual 
supplement of £2,500 from the industrial partner. 

Informal enquires can be made to Dr Elton Zeqiraj (e.zeqi...@leeds.ac.uk 
).

Funding Notes

To start in Oct 2017. Applicants should have, or be expecting to receive, a 2.1 
Hons degree in a relevant subject. 

This project is eligible for BBSRC funding. We are advertising a range of 
projects and funding will be awarded to the best candidates. The funding covers 
fees at UK/EU level plus a stipend of £14,553 for 4 years. Please note that 
candidates must have been resident in the UK for the last 3 years to be 
eligible for full funding; candidates who have not been resident in the UK are 
eligible for a fees-only studentship. 

https://www.findaphd.com/search/ProjectDetails.aspx?PJID=74943=735 
 

[ccp4bb] Postdoctoral Position in Structural Cell Biology at The Wellcome Centre for Cell Biology, Edinburgh, UK

[ARULANANDAM Jeyaprakash]

A postdoctoral position is available in Jeyapraksh (JP) Arulanandam’s lab at 
the Wellcome Centre for Cell Biology, University of Edinburgh. The JP lab 
(http://jeyaprakash.ed.ac.uk) aims to understand the structural level 
mechanistic details of processes regulating error-free chromosome segregation. 
The successful candidate will combine protein biochemistry and structural 
biology with mammalian cell based assays to dissect the molecular details of 
how specific intermolecular protein interactions achieve stable centromere 
maintenance and accurate chromosome segregation during cell division.

Applicants must have (or will shortly be awarded) a PhD with training in 
biochemistry, structural biology and cell biology. The successful candidate 
will be a highly motivated and enthusiastic individual with an outstanding 
academic track record, good communication skills and the ability to work as a 
team. Applicants with experience in single particle cryoEM, biochemistry and 
mammalian cell based siRNA/CRISPR rescue assays are encouraged to apply.

The post is for 12 months in the first instance with the possibility of 
extension.

Closing Date: 16 June 2017 at 5pm GMT
For further particulars and to apply for this post, please follow  
https://www.vacancies.ed.ac.uk/pls/corehrrecruit/erq_jobspec_version_4.jobspec?p_id=039927

Dr. A. Jeyaprakash Arulanandam
Wellcome Senior Research Fellow
Wellcome Trust Centre for Cell Biology 
University of Edinburgh
Michael Swann Building
Max Born Crescent
Edinburgh EH9 3BF
Tel: +44 131 6507113
Fax: +44 131 6080414
Web: http://jeyaprakash.bio.ed.ac.uk/





-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

Re: [ccp4bb] Revise Your Structure Without Changing the PDB Accession Code and Related Changes to the FTP Archive

[Eleanor Dodson]

This is excellent news - we have a series of related structures solved in
the YSBL - several sets of which are on different crystallographic origins.
It doesnt matter of course crystallographically but is confusing for
comparison..
Now we can alter them to a common origin with redepositing the information..
Eleanor Dodson

On 18 May 2017 at 15:22, Jasmine Young  wrote:

> Hi Ed,
>
> The PDB accession code will be based on the primary data stored in the
> PDB.  Therefore depositors will need to make a new deposition with new PDB
> accession code issued if the atomic coordinates are modified using newly
> processed intensities from the original diffraction images.
>
> More details on the procedure will be made available at a later date.
>
>
> Regards,
>
> Jasmine
>
> ===
> Jasmine Young, Ph.D.
> Biocuration Team Lead
> RCSB Protein Data Bank
> Associate Research Professor
> Center for Integrative Proteomics Research
> Rutgers, The State University of New Jersey
> 174 Frelinghuysen Rd
> Piscataway, NJ 08854-8087
>
> Email: jasm...@rcsb.rutgers.edu
> Phone: (848)445-0103 ext 4920
> Fax: (732)445-4320
> ===
>
> On 5/17/17 12:11 PM, Edward A. Berry wrote:
>
>> Couple of wuestions:
>> What is the procedure for updating an entry? Start a new submission, or
>> mail revised coordinates along with an explanation of changes to
>> deposit@rcsb?
>>
>> "unchanged experimental data" - does this mean the exact same structure
>> factors, or will newly reduced data from the same original diffraction
>> images be acceptable?
>>
>> Thanks,
>> Ed
>>
>> On 05/17/2017 09:28 AM, Jasmine Young wrote:
>>
>>> *Use CAUTION opening attachments or clicking on links in emails - IMT
>>> Help Desk, 4-4115*
>>>
>>>
>>>
>>> The wwPDB is planning to introduce in 2017 a new procedure for the
>>> management by the Depositor of Record (where the Depositor of Record is
>>> defined as the Principal Investigator for the entry) of substantial
>>> revisions to previously released PDB archival entries.
>>>
>>> At present, revised atomic coordinates for an existing released PDB
>>> entry are assigned a new accession code, and the prior entry is obsoleted.
>>> This long-standing wwPDB policy had the unintended consequence of breaking
>>> connections with publications and usage of the prior set of atomic
>>> coordinates, resulting in a non-trivial barrier to submission of atomic
>>> coordinate revisions by our Depositors of Record.
>>>
>>> The wwPDB is introducing a file versioning system that allows Depositors
>>> of Record to update their own previously released entries. Please note, in
>>> the first phase, file versioning will be applied to the atomic coordinates
>>> refined versus unchanged experimental data.
>>>
>>> Version numbers of each PDB archive entry will be designated using a #-#
>>> identifier. The first digit specifies the major version, and the second
>>> designates the minor version. The Structure of Record (i.e., the initial
>>> set of released atomic coordinates) is designated as Version 1-0.
>>> Thereafter, the major version digit is incremented with each substantial
>>> revision of a given entry (e.g., Version 2-0, when the atomic coordinates
>>> are replaced for the first time by the Depositor of Record). “Major version
>>> changes” are defined as updates to the atomic coordinates, polymer
>>> sequence(s), and/or chemical identify of a ligand. All other changes are
>>> defined as “minor changes”. When a major change is made, the minor version
>>> number is reset to 0 (e.g., 1-0 to 1-1 to 2-0). For the avoidance of doubt,
>>> the wwPDB will retain all major versions with the latest minor versions of
>>> an entry within the PDB archive.
>>>
>>> Current wwPDB policies governing the deposition of independently refined
>>> structures based on the data generated by a research group or laboratory
>>> separate from that of the Depositor of Record remain unchanged. Versioning
>>> of atomic coordinates will be strictly limited to substitutions made by the
>>> Depositor of Record.
>>>
>>> Upon introduction of the file versioning system, the wwPDB will revise
>>> each PDB accession code by extending its length and prepending “PDB” (e.g.,
>>> "1abc" will become "pdb_1abc"). This process will enable text mining
>>> detection of PDB entries in the published literature and allow for more
>>> informative and transparent delivery of revised data files. For example,
>>> the atomic coordinates for the second major version of PDB entry 1abc would
>>> have the following form under the new file-naming schema:
>>>
>>> pdb_1abc_xyz_v2-0.cif.gz
>>>
>>> The wwPDB is mindful of the importance of continuity in providing
>>> services and supporting User activities. For as long as practicable, the
>>> wwPDB will continue assigning PDB codes that can be truncated losslessly to
>>> the current four-character style. In the 

Re: [ccp4bb] Revise Your Structure Without Changing the PDB Accession Code and Related Changes to the FTP Archive

[Jasmine Young]

Hi Ed,

The PDB accession code will be based on the primary data stored in the 
PDB.  Therefore depositors will need to make a new deposition with new 
PDB accession code issued if the atomic coordinates are modified using 
newly processed intensities from the original diffraction images.


More details on the procedure will be made available at a later date.


Regards,

Jasmine

===
Jasmine Young, Ph.D.
Biocuration Team Lead
RCSB Protein Data Bank
Associate Research Professor
Center for Integrative Proteomics Research
Rutgers, The State University of New Jersey
174 Frelinghuysen Rd
Piscataway, NJ 08854-8087

Email: jasm...@rcsb.rutgers.edu
Phone: (848)445-0103 ext 4920
Fax: (732)445-4320
===

On 5/17/17 12:11 PM, Edward A. Berry wrote:

Couple of wuestions:
What is the procedure for updating an entry? Start a new submission, 
or mail revised coordinates along with an explanation of changes to 
deposit@rcsb?


"unchanged experimental data" - does this mean the exact same 
structure factors, or will newly reduced data from the same original 
diffraction images be acceptable?


Thanks,
Ed

On 05/17/2017 09:28 AM, Jasmine Young wrote:
*Use CAUTION opening attachments or clicking on links in emails - IMT 
Help Desk, 4-4115*




The wwPDB is planning to introduce in 2017 a new procedure for the 
management by the Depositor of Record (where the Depositor of Record 
is defined as the Principal Investigator for the entry) of 
substantial revisions to previously released PDB archival entries.


At present, revised atomic coordinates for an existing released PDB 
entry are assigned a new accession code, and the prior entry is 
obsoleted. This long-standing wwPDB policy had the unintended 
consequence of breaking connections with publications and usage of 
the prior set of atomic coordinates, resulting in a non-trivial 
barrier to submission of atomic coordinate revisions by our 
Depositors of Record.


The wwPDB is introducing a file versioning system that allows 
Depositors of Record to update their own previously released entries. 
Please note, in the first phase, file versioning will be applied to 
the atomic coordinates refined versus unchanged experimental data.


Version numbers of each PDB archive entry will be designated using a 
#-# identifier. The first digit specifies the major version, and the 
second designates the minor version. The Structure of Record (i.e., 
the initial set of released atomic coordinates) is designated as 
Version 1-0. Thereafter, the major version digit is incremented with 
each substantial revision of a given entry (e.g., Version 2-0, when 
the atomic coordinates are replaced for the first time by the 
Depositor of Record). “Major version changes” are defined as updates 
to the atomic coordinates, polymer sequence(s), and/or chemical 
identify of a ligand. All other changes are defined as “minor 
changes”. When a major change is made, the minor version number is 
reset to 0 (e.g., 1-0 to 1-1 to 2-0). For the avoidance of doubt, the 
wwPDB will retain all major versions with the latest minor versions 
of an entry within the PDB archive.


Current wwPDB policies governing the deposition of independently 
refined structures based on the data generated by a research group or 
laboratory separate from that of the Depositor of Record remain 
unchanged. Versioning of atomic coordinates will be strictly limited 
to substitutions made by the Depositor of Record.


Upon introduction of the file versioning system, the wwPDB will 
revise each PDB accession code by extending its length and prepending 
“PDB” (e.g., "1abc" will become "pdb_1abc"). This process will 
enable text mining detection of PDB entries in the published 
literature and allow for more informative and transparent delivery of 
revised data files. For example, the atomic coordinates for the 
second major version of PDB entry 1abc would have the following form 
under the new file-naming schema:


pdb_1abc_xyz_v2-0.cif.gz

The wwPDB is mindful of the importance of continuity in providing 
services and supporting User activities. For as long as practicable, 
the wwPDB will continue assigning PDB codes that can be truncated 
losslessly to the current four-character style. In the same spirit, 
initial implementation of entry file versioning will appear in a new, 
parallel branch of the PDB archive FTP tree. More details on the new 
FTP tree organization and accessibility of version information will 
be forthcoming. Data files in the current archive location 
ftp://ftp.wwpdb.org/pub/pdb/data/structures/ will continue to use the 
familiar naming style and will contain the latest version in the 
corresponding versioned archive.





--
Regards,

Jasmine

===
Jasmine Young, Ph.D.
Biocuration Team Lead
RCSB Protein 

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

[Clemens Vonrhein]

Dear Michael,

throwing a few other suggestions into the mix (on top of the good
advice you got already from Graeme and Phil):

 * The low resolution I/sigI is quite large and although we have seen
   such values for extremely good crystals collected very carefully on
   very good instruments, it is rather uncommon for "standard"
   (whatever that means) protein crystals that diffract to lowish
   resolution.

   So Phil's suggestion about problems in your error model is quite
   likely - and since refinement programs usually take sigmas into
   account, this might explain why there is no effect when using
   higher-resolution data.

 * You have quite high multiplicity so that you can easily check for
   possible effects of radiation damage, which could also be at play
   here: is there an increase in cell dimensions, a "smiley" like
   shape to Rmerge-vs-Image number plots, visible radiation damage in
   F(early)-F(late) maps ... ?

 * Any ice-rings present? The 3.5A range is known for those ;-)

 * Did you mask your beamstop shadow best/correctly? This shouldn't
   impact the high-resolution limit, but sometimes things can still go
   wrong there.

 * Completeness as calculated in that table is only ever completeness
   of Miller indices and not necessarily observations
   (i.e. significant data) [1].

 * If there is a very large empty detector area (because the
   crystal-detector distance was over-optimistic) and
   integration/scaling is not restricted to something more sensible,
   things can sometimes go wrong in those steps: programs are very
   good in dealing with noise, but if nearly all incoming data is pure
   noise even they can sometimes go off into a wrong minimum (during
   scale or error model parameter refinement).

 * Have a close look at the detailed processing output from the
   program(s)/pipeline(s) you used for processing. Especially if you
   picked up your data directly from the automatic processing options
   available at a synchrotron, you have to be aware that sometimes
   sensible restrictions imposed by hardware, data policy or time
   requirements mean that some programs/pipelines can not necessarily
   be run in recommended default mode (assuming that the defaults are
   chosen by the developers for very good reasons).

   If you processed your data yourself: all programs and pipelines
   should give you plenty of clear warning messages early on that can
   highlight any possible problem mentioned above.

There are some more potential issues one could think of I guess
... see also [2] and [3].

Cheers

Clemens

[1] http://staraniso.globalphasing.org/
[2] http://www.globalphasing.com/autoproc/manual/autoPROC7.html
[3] http://www.globalphasing.com/autoproc/



On Thu, May 18, 2017 at 11:47:30AM +, Michael Jarva wrote:
> 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))  

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

[Keller, Jacob]

It's radiation-damaged. Based on what did you say that it is not?

Also, do you have ice rings or diffuse scattering which might be present in the 
higher-res shells in certain rotation ranges but not others, such as from 
solvent in the loop?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Michael 
Jarva
Sent: Thursday, May 18, 2017 7:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Optimising data processing of a I432 dataset with 75% solvent 
content.

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

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

[Phil Evans]

It is puzzling that in the high res shell (to 3.1A) CC(1/2) is 0.533 (OK), but 
I/sigI is 0.1 (v low). Is the SDcorrection (SDFAC, SDADD) sensible (and 
“Analysis of standard deviations vs. intensity”)? In Aimless the determination 
of SDFAC and SDADD sometimes go haywire - with high multiplicity you could try 
“SDCORRECTION SAMPLESD” to see what it does to mean I/sigI

 Is there a lot of radiation damage (you say not, but if so with the high 
multiplicity you could cut the end of the data)? 

At least the data can’t be anisotropic in the cubic space group!

Probably none of this helps in improving maps. It may be worth looking at 
sharpened maps (from refmac or in coot)

Phil


> On 18 May 2017, at 12:47, Michael Jarva 
> <1295eb3572d0-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> 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
> 
> 

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

[Graeme Winter]

Hi Michael

What integration program did you use? Different programs can sometimes give 
rather different results with very weak data. I'd try everything to see if one 
is better than the others.

It's also worth making sure you are up to date. Certainly there are recent 
changes in xds and dials that are relevant to this problem

Best wishes Graeme

On 18 May 2017, at 12:58, Michael Jarva 
<1295eb3572d0-dmarc-requ...@jiscmail.ac.uk>
 wrote:

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


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[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

Re: [ccp4bb] CYS modification and choice of PEG

[Jose Artur Brito]

Dear Antonio,
we have seen this type of modifications in some of our structures. The 
modification of the cysteine (to cysteine-sulfenic, sulfinic or sulfonic acid) 
usually arises from exposure to oxygen during crystallization. We managed to 
prevent this by either adding TCEP to the protein buffer and/or setting the 
crystallization trays in an anaerobic chamber. This modification is, to the 
best of our knowledge, irreversible.

Have you tried the cysteine in a (CYS+CSU) alternate conformation (meaning that 
some molecules got oxidized and some weren't)?

Regarding the PEG, you can try PEG, PGE or PG4 which are smaller than PE4.

HTH,
Jose