[ccp4bb]

[Pavel Afonine]

Yes, that is what I have been doing. Build one subunit and assemble into
> tetramer before realspace refinement (with "ncs" constraints). I used
> tetramer for refinement because I want the distances between the inter
> subunits interaction partners to be considered. The problem is, whenever I
> want to make some manual adjustments of the model afterwards, I have to
> break it into monomer and repeat the whole process again. I assume if the
> map is in P4 spacegroup, I only need to modify one subunit of this protein
> and changes will be automatically applied to the other subunits. Am I
> right?
>

So you have 4 copies and NCS group consisting of one master (or reference)
copy and three related by NCS symmetry. Then you make changes in master
copy and when you run refinement with NCS constraints the changes will be
propagated onto the related copies by applying NCS constraints. This is how
this works in phenix.real_space_refine. So no need to make identical
changes in all 4 copies or go to P4.



> Another question is, when I tried to refine my model using phenix
> realspace refinement (energy minimization and adp), the statistics
> generally become better (map CC, rotamer outlier etc), however, the
> ramachandron statistics become worse, with increase number of outliers
> (from below 1% to around 5%) and allowed (from several percent to 10-20%).
> Do you know what could be the cause? I know I am asking the right person...
>

This is not expected (about worsening Ramachandran plot outliers). I'm sure
we can solve this problem if you send me model and map files (and
resolution) off list (if files too large to send by email please use
Dropbox or similar file sharing tools).

Pavel

[ccp4bb]

[Qingfeng Chen]

Sorry for the confusion. I just want to avoid the hassle to go back and
forth between one subunit and tetramer. My understanding is that I only
need to modify one subunit of this protein and changes will be
automatically applied to the other subunits if the map is changed to p4
spacegroup. I have seen people do that using MAPMAN in a couple of papers
and just want to know what I am doing wrong.

On Fri, May 19, 2017 at 12:15 AM, Dale Tronrud 
wrote:

>
>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]

Yes, that is what I have been doing. Build one subunit and assemble into
tetramer before realspace refinement (with "ncs" constraints). I used
tetramer for refinement because I want the distances between the inter
subunits interaction partners to be considered. The problem is, whenever I
want to make some manual adjustments of the model afterwards, I have to
break it into monomer and repeat the whole process again. I assume if the
map is in P4 spacegroup, I only need to modify one subunit of this protein
and changes will be automatically applied to the other subunits. Am I
right?

Another question is, when I tried to refine my model using phenix realspace
refinement (energy minimization and adp), the statistics generally become
better (map CC, rotamer outlier etc), however, the ramachandron statistics
become worse, with increase number of outliers (from below 1% to around 5%)
and allowed (from several percent to 10-20%). Do you know what could be the
cause? I know I am asking the right person...

On Fri, May 19, 2017 at 12:33 AM, Pavel Afonine  wrote:

> 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!
>>
>
>

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

[Victor Lamzin]

There are theories that the NAD carboxamide group in an enzyme active 
site should be out of the nicotine plane by 20-30 degrees, to help 
develop a partial positive charge on the C4 atom. This also helps 
distorting the planarity of the nicotine ring to ease the catalytic 
transformation of NAD to NADH. A while ago we published a paper on a 
related topic, the enzymatic activation of NADH: Meijers et al 
https://www.ncbi.nlm.nih.gov/pubmed/11134046


Victor Lamzin


On 19/05/2017 00:44, Dale Tronrud wrote:

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

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

[Kay Diederichs]

Dear Michael,

as said in this thread, careful re-integration (or other programs) might give 
you better data. If using XDS, you could try RELRAD=7 (or even 10 if the 
background is very low; the default is 5) in INTEGRATE  (this as yet 
undocumented option is in the latest XDS and determines the radius of the 
background area relative to that of the peak; 
BEAM_DIVERGENCE/BEAM_DIVERGENCE_E.S.D.) Of course, all other optimiatzion 
options should be tried.. 

On the other hand (and again as others already said), some of the stats look 
strange - the CC1/2 of 53% asks for extending the resolution, whereas the low 
 of 0.1 says it won't help much. You can evaluate radiation damage 
with the R_d plot from XDSSTAT. This is most easily produced and interpreted in 
XDSGUI - if the red fit line intersects the green level then this means that 
after this many frames, radiation damage starts dominating all other sources of 
error.

Most importantly - which problem do you really need to solve? 3.5A structures 
are not unheard of, and can be published and deposited. Another 0.1A higher 
resolution may not help much. Is there a problem in refinement or map 
interpretation? What is current Rwork/Rfree overall and in the highest shell?

best,

Kay


On Thu, 18 May 2017 11:47:30 +, 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 3.5� 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
>
>

[ccp4bb] Webinar: Is the X-ray diffraction theory we use correct?

[Jonathan Agbenyega]

Is the X-ray diffraction theory we use correct?

Join Dr Paul Fewster, former Head of Research at PANalytical and 
Co-editor for IUCr Journals to find out more. This event will take place 
on Tuesday, May 30, 2017 4:00 PM – 5:00 PM BST, 5:00 PM – 6:00 PM CEST, 
11:00 AM – 12:00 midday EDT, 8:00 AM – 9.00 AM PDT


The theory of X-ray diffraction from crystals has been established for 
over 100 years; although it is still used, it cannot account for some of 
the experimental data. The theory combined with measured data can 
sometimes lead to the wrong structural model. In this webinar you will 
hear about a new theory that includes the diffraction from crystals in 
all directions, which explains the diffraction from polycrystalline 
materials and the data collected in serial crystallography without the 
need for complex structural requirements.


The webinar will be followed by a Q+A session, where you can quiz Paul 
directly.


You can read more about Paul’s work in two papers published in Acta 
Crystallographica Section A: Foundations and Advances


Fewster, P.F. (2014)./Acta Cryst/. A*70*, 257-282 



Fewster, P.F. (2016). /Acta Cryst/. A*72*, 50-54 
__


Register now to reserve your place.

https://attendee.gotowebinar.com/register/4267568948471460099

If you have any questions, please contact Dr Jonathan Agbenyega, 
j...@iucr.org 


With best wishes

Jonathan



--
Jonathan K. Agbenyega PhD MRSC
Business Development Manager
International Union of Crystallography
5 Abbey Square, Chester CH1 2HU, England
tel: +44 (0) 1244 342878
email: j...@iucr.org
http://www.iucr2017.org/

[ccp4bb] Electronic Laboratory Notebook

[Sebastiano Pasqualato]

Dear all,
another enquiry for the great community!

We are considering the idea of moving to electronic laboratory notebooks rather 
than paper ones.

Are you happily using one and would warmly suggest its implementation?
Our department does not only deal with biochemical experiments, but performs a 
lot of genomics (big data analysis) and mouse genetics experiments, so 
experience of Notebooks used in departments that also have those activities 
would be a plus.
We will consider free and paid softwares, if you have information on the costs 
that would be also very much appreciated!

Thanks a lot,
ciao
Sebastiano


-- 
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit

[ccp4bb] info on BLI instrumentation

[Sebastiano Pasqualato]

Dear all,
is anybody using BLI instruments such as the ForteBio Octet in their 
lab/department?

A colleague in a neighbour institute is looking for information on their 
performance, overall and in comparison with SRP instruments.
Any advice with respect to:
- how these instruments really perform;
- easy of use;
- cost of performing experiments;
- biological/biochemical problems that can addressed
will be highly appreciated!

thanks a lot in advance,
best,
ciao,
Sebastiano


-- 
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit

[ccp4bb] Job posting: Research Engineer BioMAX

[Uwe Müller]

Research engineer BioMAX
Lunds universitet, MAX IV, Diffraction & Scattering

Lund University was founded in 1666 and is repeatedly ranked among the world’s 
top 100 universities. The University has 42 000 students and 7 400 staff based 
in Lund, Helsingborg and Malmö. We are united in our efforts to understand, 
explain and improve our world and the human condition.

MAX IV is a national large-scale research laboratory providing scientists from 
Sweden and the rest of the world with instruments for research in areas such as 
materials science, structural biology, chemistry and nanotechnology. Fully 
developed it will receive more than 2 000 scientists annually. 200 people are 
currently employed at the MAX IV Laboratory.

BioMAX is the first macromolecular crystallography beamline at the new ultimate 
storage ring MAX IV. The beamline has been designed to deliver an energy 
tunable, high brilliant, microfocused x-ray beam at micrometer size and to 
support the majority of techniques in the field for crystals down to the 
micrometer range. A fast x-ray detector in combination with a high capacity 
sample changer is enabling very efficient data collection schemes.

BioMAX has been recently set into operation. Nevertheless, to reach the 
anticipated performance level within the coming year, substantial commissioning 
and optimization tasks are still pending.

In order to develop, to operate and to maintain BioMAX, we are now seeking to 
hire a research engineer with a background and interest in X-ray synchrotron 
radiation based techniques and instrumentation.

 Tasks

• You will participate in the commissioning of the beamline and its end-station.
• You will share responsibilities for operation, maintenance and further 
development of the beamline in close collaboration with the team of scientists 
and engineers of the MX-group and MAX IV staff.
• You will develop strategies for diagnostics and controls of the BioMAX 
technical infrastructure.
• You will share responsibility for the support of external users during their 
beamtime.
• You will contribute to the design, the purchase and the installation of new 
beamline and experimental station components to ensure the availability of a 
competitive and reliable beamline environment.
• You will work on the design of the new micro-focusing beamline MicroMAX.

Note: The amount and nature of the participation in user support will depend on 
the interests and the profile of the successful candidate.

Qualifications

The following qualifications are required:

• You hold a university degree in engineering or natural sciences in subjects 
of relevance for BioMAX.
• You have a solid background and knowledge in mechatronics.
• You have a solid experience in the planning, design/construction/purchase, 
operation and maintenance of complex scientific equipment in particular such 
equipment that is of relevance for BioMAX.
• You have experience with programing / interfacing of hardware.
• You have a solid knowledge and experiences in instrumentation controls 
hardware and software.
• Experience with programing / interfacing of hardware.
• You have a demonstrated ability to work independently.
• You have excellent command of English, well-developed communication skills 
and team working skills.

The following qualifications are considered an asset:

• Experience in the design, commissioning, and use x-ray hardware.
• Demonstrated knowledge of X-ray instrumentation and associated techniques.
• Experience with vacuum and cryogenics technologies.
• Experience of developing, assembling, running, and maintaining equipment for 
x-ray techniques.
• Interest/experience in scientific use of techniques of interest for BioMAX 
(i.e. experiments, data analysis, publication / presentation of results).

Experience with support of user groups at a synchrotron radiation facility or a 
similar user-oriented research facility is a further merit.

Probationary period may be applied.



Lund University welcomes applicants with diverse backgrounds and experiences. 
We regard gender equality and diversity as a strength and an asset.
We kindly decline all sales and marketing contacts.
Type of employment  Permanent position
Contract type   Full time
First day of employment As per agreement
Salary  Monthly salary
Number of positions 1
Working hours   100 %
CityLund
County  Skåne län
Country Sweden
Reference numberPA2017/1219
Contact

  *   Uwe Müller, +46 73065268


Union representative

  *   OFR/S:Fackförbundet ST:s kansli, 046-222 93 62, 
s...@st.lu.se
  *   SACO:Saco-s-rådet vid Lunds , 046-222 93 64, 
kan...@saco-s.lu.se


Published   18.Apr.2017
Last application date   31.May.2017 11:59 PM CET


Uwe Mueller
Group manager diffraction & scattering beamlines
BioMAX beam line manager

MAX IV Laboratory
Lund University
P.O. Box 118, SE-221 00 Lund, Sweden
Visiting address: Fotongatan 2, 225 94 Lund
Mobile: