Re: [ccp4bb] Bad density for chains

[Pooja Kesari]

Thank you for the reply.

On Thu, Jan 26, 2017 at 9:07 PM,  wrote:

> Dear Pooja,
>
>
>
> A few remarks:
>
>
>
> -Matthews does not show 4 chains in the asymmetric unit, it
> suggests 4 chains. However, in reality it can be more or less chains,
> although rare, 75% solvent (2 chains) is not unheard of.
>
> -An initial Rfree of 38% is ok, 32% after refinement is a bit
> high
>
> -Disordered loops do exist and you may have to live with them
> (not be able to build them).
>
> -To correct for anisotropy, I suggest the staraniso server from
> global phasing.
>
> -Unless you ran your molecular replacement in P1, Zanuda will
> confirm the space group you choose for MR. So run MR in P1 (if your
> symmetry is not too high) and run Zanuda again. (Have someone) look
> critical at the assigned space group and assess whether another choice
> might also be possible.
>
>
>
> Good luck!
>
> Herman
>
>
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von
> *Pooja Kesari
> *Gesendet:* Donnerstag, 26. Januar 2017 15:12
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] Bad density for chains
>
>
>
> We have a 2.6 A structure showing four chains in an asymmetric unit. Our
> protein is 360 residues around 40 kDa . Mattews shows four chain in an
> assymetric unit (solvent 49% mattews coeff 2.44). The template has about
> 60% homologous with our protein. The molecular replacement against this
> template gave an initial free R of 38.  We did chain tracing and found that
> we have good density (2Fo-Fc) for chain A and B but poor density for C and
> D.
>
>
>
> 1. The density for a particular stretch of 10 amino acids (disordered loop
> region) is absent in all the chains. We could not found density for this
> flexible loop region in any of the already known structures. Any suggestion
> on how can we build this region?
>
>
>
> 2. We did not find density for most of the loop regions in chain C and D
> which were well traced in chain A and B. How can we improving the density
> for these two chains based on chain A and B (Density modification)?
>
>
>
> 3. We analysed the data using phenix xtriage and found that our data shows
> severe anisotropy. Any suggestion of anisotropy correction?
>
>
>
> Pointless and Ctruncate analyses didn't show twinning or NCS.  I have
> checked the space group using Zanuda. We are stuck at a free value of 32.
>
>
>
> On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson 
> wrote:
>
> This is a bit too vague to help much.
>
> How did you solve the structure?
>
> Eleanor
>
>
>
> On 26 January 2017 at 03:50, Pooja Kesari  wrote:
>
> Dear All,
>
> Thank you all for reply.
>
> We have checked the data for twinning.
>
> Our protein is 360 residues around 40 kDa protein.
>
> We have tried TLS refinement.
>
> chain A and B don't superimpose well with chain C and D. (A and B chains
> also share slight difference )
>
> Since we don't have proper density for *some regions*  chain C and D, we
> are not sure whether these chain have similar or different conformations.
>
> We tried anisotropy correction and the model refined a bit.
>
>
>
>
>
> On Wed, Jan 25, 2017 at 10:32 AM, Debanu  wrote:
>
> Hi Pooja,
>
>
>
> Are you positive you have the correct space group and there are no other
> issues like twinning, etc?
>
>
>
> If sure, did you define NCS groups in refinement? TLS refinement? Try
> different refinement programs?
>
>
>
> How big is the molecule? Was it solved by MR or experimental phasing?
>
>
>
> You can try superimposing A/B on C/D and refinement with tight NCS then
> adjust NCS restraints during model adjustments based on local differences
> or also see if phenix autobuild helps.
>
>
>
> Best,
>
> Debanu
>
> --
>
> Debanu Das
>
> Accelero Biostructures
>
>
>
>
> On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:
>
> Dear All,
>
> I have a 2.6 A resolution structure having four chains in an asymmetric
> unit.
>
> The chain A and B have density for almost all residues however we don't
> have proper residue density in chain C and D.What can be tried to build
> chain C and D ?
>
>
>
>
>
> Many Thanks
>
> Pooja
>
>
>
>
>
> --
>
> Thanks & Regards,
> Pooja Kesari
>
> Research Scholar
>
> Department Of Biotechnology
>
> Indian Institute of Technology Roorkee
>
> INDIA
>
>
>
>
>
>
>
>
>
> --
>
> Thanks & Regards,
> Pooja Kesari
>
> Research Scholar
>
> Department Of Biotechnology
>
> Indian Institute of Technology Roorkee
>
> INDIA
>
>
>



-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA

[ccp4bb] Experimental Postdoctoral Position in High Throughput Small Molecule Ligand Screening

[Forness, Jessica D]

Outstanding postdoctoral applicants are sought with the following 
qualifications to work with Dr. Jeffrey Skolnick in the Center for the Study of 
Systems Biology in the School of Biological Sciences at the Georgia Institute 
of Technology:


* Extensive experience in enzyme kinetics studies, enzyme purification or other 
aspects of protein biology and enzyme activity. Experience in handling multiple 
protein systems would be a plus.


* A background in high throughput small molecule ligand screening is strongly 
preferred.


* Experience with or a desire to learn computational biology and molecular 
modeling of protein-ligand interactions.


* The ideal candidate is someone who gets satisfaction out of methods 
development and working through large data sets to see broad-scale patterns.


* A track record of publication in high quality, international journals.


* Ability to work in a team, yet think independently.


To apply, please email your CV to: 
skoln...@gatech.edu

Re: [ccp4bb] wavelength dependence of intensities - Summary

[Tim Gruene]

Dear Loes,

thank you very much for your explanation. As agreed I hereby share the 
conclusive email of your off-list discussion with John Helliwell, and a 
pointer to your article with its explanation  
http://dx.doi.org/10.1107/S1399004715011803:

->
Hi John,

Yes, you are right.  Apart from the cos(theta) contribution, which is 
usually small.
Indeed lambda^3/(2*sin(theta) cos(theta)) is approximately 4 times as 
large when changing the wavelength by a factor 2, and accounting for the 
fact that then theta changes.
So the interference function depends on lambda^3, but for integrated 
reflections obtained by the rotation method the wavelength dependence 
becomes lambda^2.

Greetings,
Loes
<--

Best regards,
Tim


On Thursday 26 January 2017 05:58:31 PM Loes Kroon-Batenburg wrote:
> Dear Tim,
> 
> That is described by the Laue interference function. The crystal
> diffracts in Bragg directions (reflections) at small deviations of the
> Bragg angle and in a small solid angle. They depend on lambda and
> lambda^2 respectively, and integration over these angles brings a factor
> lambda^3 in the diffracted intensity.
> 
> Best wishes,
> Loes
> 
> On 01/26/17 17:15, Tim Gruene wrote:
> > Dear all,
> > 
> > according to Carmelo Giacovazzo's textbook (eq. 3.41 in the 1st edition),
> > the diffraction intensity I(hkl) scales with the wavelength cubed
> > (lambda^3).
> > 
> > 1) is there an easy rationale for this dependency?
> > 2) does it hold over a wide range of energies?
> > 3) does this also hold for neutrons or electrons as radiation source?
> > 
> > Thanks a lot for any helpful comment,
> > Tim
> 
> --
> 
> __
> 
> Dr. Loes Kroon-Batenburg
> Dept. of Crystal and Structural Chemistry
> Bijvoet Center for Biomolecular Research
> Utrecht University
> Padualaan 8, 3584 CH Utrecht
> The Netherlands
> 
> E-mail : l.m.j.kroon-batenb...@uu.nl
> phone  : +31-30-2532865
> fax: +31-30-2533940
> __
-- 
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
Principal Investigator
Biology and Chemistry
OFLC/102
CH-5232 Villigen PSI

Phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A



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Re: [ccp4bb] CCP4BB Digest - 13 Jan 2017 to 14 Jan 2017 (#2017-15)

[pselvam]

Dear All,
thanks for your replies.  I tend to believe that it could be ATP some 
how copurified with the protein. Refinement with ATP, ADP+acetate, did 
not vary too much in the B factors. The main concern is, only one xtal 
was observed at this state  ( mimicked like a ATP bound state and 
changed conformation and space group). All other xtals (around 10 xtals 
were measured) from this plate did not have this change.So it was little 
tough to understand that only this xtal has ATP instead of ADP. We were 
not able to reproduce xtals of similar space group with similar setup 
(with ADP). After tremendous  trials we could grow one xtal with ATP 
which has the P212121 space group and all the observed changes. Is any 
one observed ATP mimicking by compounds other than aluminium/berillyium 
Fluoride?



Thanks
Saravanan

Dear All,
Sorry for the little bit off topic. Is there a possibility for covalent
bond formation between beta phosphate of ADP and acetate molecule both
are coordinated by divalent metal ions? I am working on a Kinase
structure  which was crystallized with ADP and in presence of 1M sodium
acetate. We observed additional density around ADP that fits perfectly
like a gamma phosphate , mimicking like ATP bound state, surrounded and
coordinated by two metal ions(resolution is 1.4A). There is a change in
space group (from I212121 to P212121 ) and further important
conformation changes are observed around ATP binding pocket and distant
region. This is the only xtal we obtained in this space group, and all
other xtals(measured 10 xtals)  from the same plate belong to I212121.

  


Thanks your help and time!

Saravanan
Replies:

Hello Saravanan

At 1.4 Angstrom resolution wouldn't that suggest that you've somehow got 
ATP in there ?  I don't think I understand the other option - were you 
proposing a ADP-O-C(O)2 arrangement to explain the density ?  Surely 
that has a rather different shape, considerably different scattering 
power at the center of the terminal group (C vs P) and probably 
different X-O bond lengths.  All of these should show in the density 
maps at 1.4 Å, although the bond length issue could be quite subtle.


Phil Jeffrey
Princeton

Perhaps it is ATP?

Mark J van Raaij


Remember that 2xADP can disproportionate into ATP + AMP


Hi Saravanan

It sounds to me like you have ATP there, even though you added ADP. Is 
your kinase an inactive mutant? I know kinases can bring nucleotides 
along for the ride during purification.


Regards
Christine



--
Dr. Saravanan Panneerselvam PhD
P11 Beamline Scientist
HASYLAB, DESY
Building 25F, Room 360
Notkestrasse, 85
22607, Hamburg
GERMANY
Phone : 0049 40 8998 2692
Mobile:  0049 40 8998 92692
saravananpann...@gmail.com
saravanan.panneersel...@desy.de