[ccp4bb] [Meeting Announcement] Rhine-Knee Regiomeeting 2018 on Structural Biology, Emmetten, Switzerland - Last announcement and call for presentations

[Olieric Vincent (PSI)]

This is the second and last announcement of the Rhine-Knee Regiomeeting 2018 on 
Structural Biology.

http://regiomeeting.eu/meeting-2018

Emmetten, Switzerland
Sep. 26-28, 2018

The deadline for abstract submission and registration is September 7, 2018.


The 32nd Rhine-Knee Regional Meeting on Structural Biology will take place in 
the heart of Switzerland in Emmetten from September 26 to 28. The venue is the 
Seeblick Höhenhotel (http://www.hotelseeblick.ch).

The scientific program includes the 3 keynote speakers:
- Prof. Phil Willmott, Paul Scherrer Institut, Switzerland
- Dr Arjen Jakobi, Kalvi Institute, The Netherland
- Dr. Andy Doré, Heptares Therapeutics, UK

Young researchers who wish to give a presentation are strongly encouraged to 
emphasize the technical and methodological aspects of their work as well as 
encountered problems.

The traditional excursion on the Thursday afternoon will propose a boat ride on 
the lake Lucerne (Vierwaldstättersee) and a hike in one of the most famous 
place in Switzerland, the Rütli meadow.

We thank our sponsors who make this event possible.

Please feel free to forward this announcement to anyone who may be interested 
in participating.

Looking forward to seeing you in September in Emmetten,

Vincent Olieric
regio2...@psi.ch
http://regiomeeting.eu/meeting-2018


---
Paul Scherrer Institute
Dr. Vincent Olieric

Swiss Light Source
WSLA/218
5232 Villigen PSI
Switzerland

+41 (0)56 310 5233
vincent.olie...@psi.ch
www.psi.ch/sls




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Re: [ccp4bb] Protein Purification- reg

[Artem Evdokimov]

Hi Amala,

Depending on the resin you used there may be a conflict with BME and also
50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5
and TCEP instead of BME, or use 30 mM TRIS at pH 8.0

Alternatively your protein is aggregated and does not bind well...

Artem

- Cosmic Cats approve of this message

On Mon, Aug 13, 2018 at 9:49 AM, amala mathimaran 
wrote:

> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa histidine). The
> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
> *E.coli
> *BL21 cells. The expression was good. I am trying to purify a protein
> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
> as buffer A but 250mM imidazole). I eluted the protein in step wise
> gradiant. the protein was less binded in Ni affinity IMAC column because
> eluted fraction contain less amount and the protein remain present in the
> Flow through. Can any one suggest how to increase the binding affinity of
> the protein and how to purify the protein. The protein PI was 6.33
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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[ccp4bb] Protein Purification- reg

[amala mathimaran]

Dear All

I am working with HIS – tag protein in N-terminal (hexa histidine). The
protein from Prokaryotic origin cloned into pET30a+ vector and
expressed in *E.coli
*BL21 cells. The expression was good. I am trying to purify a protein using
Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM NaCl,
15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same as
buffer A but 250mM imidazole). I eluted the protein in step wise gradiant.
the protein was less binded in Ni affinity IMAC column because eluted
fraction contain less amount and the protein remain present in the Flow
through. Can any one suggest how to increase the binding affinity of the
protein and how to purify the protein. The protein PI was 6.33



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[ccp4bb] Post Doc position available in Denmark

[Bjørn Panyella Pedersen]

Postdoctoral position in Structure of Sterol Transporting Membrane 
Proteins at Aarhus University, Denmark.

online posting:
http://www.pedersenlab.dk/source/bp_lab_postdoc_position.pdf


The Pedersen group is looking for a highly skilled and motivated postdoc 
with an interest in working on sterol and hormone transporting membrane 
transporters and with a proven track record in the area of structural 
and/or functional analysis of membrane proteins.

The position will be open from fall 2018, and starting date is 
negotiable. Funding is available for at least 2 years of employment, 
with an automatic extension possible for up to a total maximum of 4 years.

The position:
The position seeks to strengthen ongoing activities in the laboratory of 
Bjørn P. Pedersen on structure, related to the function and mechanism of 
sterol-transporting membrane proteins (pedersenlab.dk). The laboratory's 
interest is the interplay between structure and function of 
transmembrane transport processes with a focus on metabolite uptake 
systems, and the methods uses are primarily crystallography, cryo-EM and 
biochemistry. The group is part of the Section of Structural Biology at 
Aarhus University.

The candidate:
A successful candidate has a relevant Ph.D. degree and a solid and 
documented background in structural biology, biochemistry and/or 
biophysics. Experience with membrane protein expression and purification 
is favored, and the candidate must demonstrate an ability and interest 
to work with membrane proteins with a structural aim. Finally, 
applicants should be ambitious, show strong collaborative skills, and 
should also be able to take initiatives and responsibility within the 
work environment.

Deadline:
All applications must be made online and received by 16/09/2018.



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[ccp4bb] Post Doc position available

[Przemyslaw Grudnik]

We have a job opening for a highly-motivated postdoctoral fellow who holds  
a Ph.D. degree for no longer than five years.
The successful candidate will be involved in projects carried out in the  
newly established Structural Biology Core Facility at Jagiellonian  
University in Krakow, Poland.


Job offer website:  
https://www.fnp.org.pl/assets/Job-offer-1_FIRST-TEAM_S-Glatt.pdf

Laboratory website: http://www.mcb.uj.edu.pl/en/mpl

The ideal candidate should:
- hold a doctoral degree for no longer than 5 years
- have a strong expertise in structural biology (Macromolecular  
Crystallography) and biochemistry
- Have excellent written and oral communication skills in English  
(essential)
- Have good presentation skills (ideally having presented on at least one  
international research conference)

- Enjoy working in a highly collaborative and interdisciplinary environment
- Enjoy scientifically supporting and actively interacting with other  
members of the lab
- Have published at least one publications as first author in an  
international peer reviewed journal


We offer:

- Position for three years with possible extension after the project ends
- Excellent training: In our diverse group you will be able to network  
with international researchers, experience and learn new skills in  
cellular & molecular biology.
- State-of the-art research center with access to the full suite of  
equipment necessary for cell biology, molecular biology,
biochemistry and structural biology research and a network of  
international collaborators in Europe and beyond.


Interested applicants should send a cover letter and CV with the names of  
two references to Sebastian Glatt sebastian.gl...@uj.edu.pl




--
Przemyslaw Grudnik Ph.D.
Structural Biology Core Facility
Malopolska Centre of Biotechnology (MCB)
Jagiellonian University

ul. Gronostajowa 7a 30-387 Krakow, Poland
+48 12 664-61-06



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Re: [ccp4bb] tCNS and space group determination

[Harry Powell]

Hi Marcelo

One thing to try if you have access to a microfocus beamline would be to try to 
collect data from part of the crystal and hope that you hit a single lattice 
with the beam. Sometimes the different lattices are in different parts of the 
physical object in the beam. Recollecting the data on another sample also gives 
you the chance to avoid an icy sample and maybe get a single crystal...

I notice you are using iMosflm to view the image - iMosflm has had a facility 
for processing multiple lattices for many years. It's worth trying, but as Paul 
points out, I wouldn't be optimistic.

Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing) 




On 13 Aug 2018, at 08:59, Paul Adams wrote:

> Hi Marcelo,
> 
>  based on this image it looks like you have multiple (two) lattices, which 
> puts spots very close together. This could be a split crystal as Eleanor 
> suggested, or a feature of the way the crystals grow. It seems unlikely that 
> you’d be able to integrate these lattices well, given how close the spots 
> are. You could try data collection with the detector pushed further back to 
> increase spot separation (not an ideal solution). If the multiple lattices is 
> something you see with all crystals then you may want to work on modifying 
> crystallization conditions. 
>  Sometimes things can be very difficult to figure out. The Dicer structure 
> (see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case where the a 
> and b axes of the orthorhombic lattice were very similar in length and the 
> crystals always grew with a pseudo-merohedral twinning that gave rise to 
> nearly, but not quite, overlapped spots from the two lattices. It doesn’t 
> look like you have a case like this, because the intensity statistics don’t 
> suggest twinning. However, some of the tricks that you might use today on 
> such crystals, such as raster scanning with a microbeam, might be helpful.
> 
>  Cheers,
>   Paul
> 
>> On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
>> 
>> I am sorry. I forgot to attach the image.
>> 
>> Cheers
>> 
>> Marcelo
>> 
>> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato 
>>  escreveu:
>> Dear Eleanor,
>> 
>> Thanks for you answer. 
>> Indeed, there are clear ice rings in the images (example attached). So, I 
>> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 
>> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for 
>> P2 (in two different cells) and P222 data. 
>> I agree that MR seems very good (in all cases), but the final density maps 
>> are always bad. Maybe the data has problems that I am not dealing with. 
>> 
>> Kind regards
>> 
>> Marcelo
>> 
>> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson 
>>  escreveu:
>> This MR looks good to me, but there are serious flaws with the data. Your 
>> secon moment plot from the aimless log has most spectacular spikes which are 
>> always a BAD THING, and the Wilson plot is not very smooth either.. 
>> 
>> As Randy says, try to sort those problems out first.
>> 
>> Then you have this message:
>> 
>> 
>> TRANSLATIONAL NCS:
>> 
>> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
>> A translation of 0.5 along B will generate pseudo-absences along b so you 
>> can be sure whether there is a scre axis or not..
>> 
>> The space group is most likely orthorhombic - these indicators are pretty 
>> convincing for P2/mmm - so I dont know why you have chosen P21 as the 
>> spacegroup? 
>> 
>> 
>> Scores for each symmetry element
>> 
>> Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice 
>> Cell)
>> 
>>  1   0.917   8.18   0.82   61009  0.298 identity
>>  2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l}, 
>> along original k
>>  3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l}, 
>> along original l
>>  4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l}, 
>> along original h
>> 
>> So my suggestions:
>> Sort out data problems
>> 
>> Merge as P2/mmm 
>> 
>> Let MR search select the most likely spacegroup of the 8 possible.
>> 
>> You cant even limit the b axis to be a screw axis .
>> 
>> Your refinement behavior looks OK, but the maps will look bad with spurious 
>> reflections in the list..
>> 
>> Eleanor
>> 
>> 
>> 
>> 
>> 
>> On 10 August 2018 at 19:02, Eleanor Dodson  wrote:
>> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR 
>> solution! 
>> 
>> 
>> 
>> On 10 August 2018 at 18:42, Eleanor Dodson  wrote:
>> Can you attach the refinement log?  
>> 
>> Eleanor
>> 
>> On 10 August 2018 at 16:57, Marcelo Liberato  
>> wrote:
>> Dear Randy, 
>> 
>> Thank you very much for answering. I followed your suggestions but, 
>> unfortunately, I couldn't get a reasonable electron density map after MR and 
>> refinement.
>> 
>> 
>> First I would look at the data to see if you have ice rings, because the 
>> peak 

Re: [ccp4bb] tCNS and space group determination

[Eleanor Dodson]

You could try the dials data processing - it tries to address the problems
of multiple lattices..
Eleanor


On 13 August 2018 at 08:59, Paul Adams  wrote:

> Hi Marcelo,
>
>   based on this image it looks like you have multiple (two) lattices,
> which puts spots very close together. This could be a split crystal as
> Eleanor suggested, or a feature of the way the crystals grow. It seems
> unlikely that you’d be able to integrate these lattices well, given how
> close the spots are. You could try data collection with the detector pushed
> further back to increase spot separation (not an ideal solution). If the
> multiple lattices is something you see with all crystals then you may want
> to work on modifying crystallization conditions.
>   Sometimes things can be very difficult to figure out. The Dicer
> structure (see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case
> where the a and b axes of the orthorhombic lattice were very similar in
> length and the crystals always grew with a pseudo-merohedral twinning that
> gave rise to nearly, but not quite, overlapped spots from the two lattices.
> It doesn’t look like you have a case like this, because the intensity
> statistics don’t suggest twinning. However, some of the tricks that you
> might use today on such crystals, such as raster scanning with a microbeam,
> might be helpful.
>
>   Cheers,
> Paul
>
> > On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
> >
> > I am sorry. I forgot to attach the image.
> >
> > Cheers
> >
> > Marcelo
> >
> > Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato <
> marcelovliber...@gmail.com> escreveu:
> > Dear Eleanor,
> >
> > Thanks for you answer.
> > Indeed, there are clear ice rings in the images (example attached). So,
> I integrated again (P1, P2 and P222) excluding the resolution ranges
> 2.28-2.22 and 3.70-3.64. I am attaching the log files from aimless, MR and
> refmac for P2 (in two different cells) and P222 data.
> > I agree that MR seems very good (in all cases), but the final density
> maps are always bad. Maybe the data has problems that I am not dealing
> with.
> >
> > Kind regards
> >
> > Marcelo
> >
> > Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson <
> eleanor.dod...@york.ac.uk> escreveu:
> > This MR looks good to me, but there are serious flaws with the data.
> Your secon moment plot from the aimless log has most spectacular spikes
> which are always a BAD THING, and the Wilson plot is not very smooth
> either..
> >
> > As Randy says, try to sort those problems out first.
> >
> > Then you have this message:
> >
> >
> > TRANSLATIONAL NCS:
> >
> > Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> > A translation of 0.5 along B will generate pseudo-absences along b so
> you can be sure whether there is a scre axis or not..
> >
> > The space group is most likely orthorhombic - these indicators are
> pretty convincing for P2/mmm - so I dont know why you have chosen P21 as
> the spacegroup?
> >
> >
> > Scores for each symmetry element
> >
> > Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in
> Lattice Cell)
> >
> >   1   0.917   8.18   0.82   61009  0.298 identity
> >   2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1)
> {-h,-k,l}, along original k
> >   3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0)
> {-h,k,-l}, along original l
> >   4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0)
> {h,-k,-l}, along original h
> >
> > So my suggestions:
> > Sort out data problems
> >
> > Merge as P2/mmm
> >
> > Let MR search select the most likely spacegroup of the 8 possible.
> >
> > You cant even limit the b axis to be a screw axis .
> >
> > Your refinement behavior looks OK, but the maps will look bad with
> spurious reflections in the list..
> >
> > Eleanor
> >
> >
> >
> >
> >
> > On 10 August 2018 at 19:02, Eleanor Dodson 
> wrote:
> > Actually Marcelo - Refinement to an R of 41% is pretty good for an MR
> solution!
> >
> >
> >
> > On 10 August 2018 at 18:42, Eleanor Dodson 
> wrote:
> > Can you attach the refinement log?
> >
> > Eleanor
> >
> > On 10 August 2018 at 16:57, Marcelo Liberato 
> wrote:
> > Dear Randy,
> >
> > Thank you very much for answering. I followed your suggestions but,
> unfortunately, I couldn't get a reasonable electron density map after MR
> and refinement.
> >
> >
> > First I would look at the data to see if you have ice rings, because the
> peak in mean intensity and second moment of the intensity at about 2.25A
> resolution suggests an ice ring problem.  If so, you should make sure you
> don't contaminate the data with spurious large intensities.
> >
> > Indeed, the data has ice rings. At first, I required imosflm to remove
> ice rings, but it didn't happened. So, I re-processed the data in different
> space groups removing the ice rings.
> >
> > Second, the statistics (e.g. the second moments plot after tNCS
> correction in Phaser) would be consistent with a scenario in which you have
> 

Re: [ccp4bb] tCNS and space group determination

[Paul Adams]

Hi Marcelo,

  based on this image it looks like you have multiple (two) lattices, which 
puts spots very close together. This could be a split crystal as Eleanor 
suggested, or a feature of the way the crystals grow. It seems unlikely that 
you’d be able to integrate these lattices well, given how close the spots are. 
You could try data collection with the detector pushed further back to increase 
spot separation (not an ideal solution). If the multiple lattices is something 
you see with all crystals then you may want to work on modifying 
crystallization conditions. 
  Sometimes things can be very difficult to figure out. The Dicer structure 
(see Ian MacRae’s paper in Acta D: D63, 993-999, 2007) was a case where the a 
and b axes of the orthorhombic lattice were very similar in length and the 
crystals always grew with a pseudo-merohedral twinning that gave rise to 
nearly, but not quite, overlapped spots from the two lattices. It doesn’t look 
like you have a case like this, because the intensity statistics don’t suggest 
twinning. However, some of the tricks that you might use today on such 
crystals, such as raster scanning with a microbeam, might be helpful.

  Cheers,
Paul

> On Aug 12, 2018, at 9:57 AM, Randy Read  wrote:
> 
> I am sorry. I forgot to attach the image.
> 
> Cheers
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 18:31, Marcelo Liberato 
>  escreveu:
> Dear Eleanor,
> 
> Thanks for you answer. 
> Indeed, there are clear ice rings in the images (example attached). So, I 
> integrated again (P1, P2 and P222) excluding the resolution ranges 2.28-2.22 
> and 3.70-3.64. I am attaching the log files from aimless, MR and refmac for 
> P2 (in two different cells) and P222 data. 
> I agree that MR seems very good (in all cases), but the final density maps 
> are always bad. Maybe the data has problems that I am not dealing with. 
> 
> Kind regards
> 
> Marcelo
> 
> Em sáb, 11 de ago de 2018 às 16:04, Eleanor Dodson 
>  escreveu:
> This MR looks good to me, but there are serious flaws with the data. Your 
> secon moment plot from the aimless log has most spectacular spikes which are 
> always a BAD THING, and the Wilson plot is not very smooth either.. 
> 
> As Randy says, try to sort those problems out first.
> 
> Then you have this message:
> 
> 
> TRANSLATIONAL NCS:
> 
> Translational NCS has been detected at ( 0.000,  0.500,  0.125).
> A translation of 0.5 along B will generate pseudo-absences along b so you can 
> be sure whether there is a scre axis or not..
> 
> The space group is most likely orthorhombic - these indicators are pretty 
> convincing for P2/mmm - so I dont know why you have chosen P21 as the 
> spacegroup? 
> 
> 
> Scores for each symmetry element
> 
> Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice 
> Cell)
> 
>   1   0.917   8.18   0.82   61009  0.298 identity
>   2   0.883   7.85   0.78  100711  0.381 **  2-fold l ( 0 0 1) {-h,-k,l}, 
> along original k
>   3   0.921   8.39   0.84   99542  0.355 *** 2-fold k ( 0 1 0) {-h,k,-l}, 
> along original l
>   4   0.920   8.26   0.83   99218  0.320 *** 2-fold h ( 1 0 0) {h,-k,-l}, 
> along original h
> 
> So my suggestions:
> Sort out data problems
> 
> Merge as P2/mmm 
> 
> Let MR search select the most likely spacegroup of the 8 possible.
> 
> You cant even limit the b axis to be a screw axis .
> 
> Your refinement behavior looks OK, but the maps will look bad with spurious 
> reflections in the list..
> 
> Eleanor
> 
> 
> 
> 
> 
> On 10 August 2018 at 19:02, Eleanor Dodson  wrote:
> Actually Marcelo - Refinement to an R of 41% is pretty good for an MR 
> solution! 
> 
> 
> 
> On 10 August 2018 at 18:42, Eleanor Dodson  wrote:
> Can you attach the refinement log?  
> 
> Eleanor
> 
> On 10 August 2018 at 16:57, Marcelo Liberato  
> wrote:
> Dear Randy, 
> 
> Thank you very much for answering. I followed your suggestions but, 
> unfortunately, I couldn't get a reasonable electron density map after MR and 
> refinement.
> 
> 
> First I would look at the data to see if you have ice rings, because the peak 
> in mean intensity and second moment of the intensity at about 2.25A 
> resolution suggests an ice ring problem.  If so, you should make sure you 
> don't contaminate the data with spurious large intensities.
> 
> Indeed, the data has ice rings. At first, I required imosflm to remove ice 
> rings, but it didn't happened. So, I re-processed the data in different space 
> groups removing the ice rings.  
> 
> Second, the statistics (e.g. the second moments plot after tNCS correction in 
> Phaser) would be consistent with a scenario in which you have pseudosymmetry 
> along with a twin operator that parallels the pseudosymmetry.  If that's 
> true, it's hard to be sure of the symmetry.  For instance, if the structure 
> really is monoclinic, can you be sure you chose the correct axis to be the 
> 2-fold?
>  
> I am not sure. However, I tried two possible axis to be the 2-fold and none 
>