Re: [ccp4bb] Bad density for chains

[Pooja Kesari]

Thank you for the reply.

On Thu, Jan 26, 2017 at 9:07 PM, <herman.schreu...@sanofi.com> 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 <eleanor.dod...@york.ac.uk>
> 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 <pkesar...@gmail.com> 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 <debanu@gmail.com> 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 <pkesar...@gmail.com> 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

Re: [ccp4bb] Bad density for chains

[benjamin bax]

You could try the diffraction anisotropy server: 
http://services.mbi.ucla.edu/anisoscale/.
Sometimes it helps maps become more interpretable. 

 Ben  


On 26 Jan 2017, at 14:11, Pooja Kesari  wrote:

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



Dr Ben Bax

Dept. Biological Chemistry, 
John Innes Centre, 
Norwich Research Park, 
Norwich NR4 7UH, UK

ben.d.v@gmail.com

 

 

[ccp4bb] AW: [ccp4bb] Bad density for chains

[Herman . Schreuder]

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 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> 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 
<pkesar...@gmail.com<mailto:pkesar...@gmail.com>> 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 
<debanu@gmail.com<mailto:debanu@gmail.com>> 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 
<pkesar...@gmail.com<mailto:pkesar...@gmail.com>> 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

Re: [ccp4bb] Bad density for chains

[Eleanor Dodson]

Well - first
Solvent content..

1) there could be 4 molecules, or 3, or 2.. Solvent content varies a lot as
you know. Is there reasonable density for parts of all the chains?

2) Does the MR search clearly show 4 molecules? ie improvement in scores
with extra molecules?

3) Have you checked for sensible NC symmetry - submit your model to the
PISA server and see if it suggests a teramer, or 4 fold symmetry or maybe 2
dimers?
PISA will also suggest buried surface area of each chain - maybe parts of
the C & D chains have nothing to fix them in place
Eleanor



On 26 January 2017 at 14:11, Pooja Kesari  wrote:

> 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
>
>

Re: [ccp4bb] Bad density for chains

[Pavel Afonine]

Hi,

assuming you've exhausted modeling choices and applied proper refinement
strategies, after all Rree=32% is not unheard of at 2.6A resolution (which
actually may be even worse than 2.6A if data set is not 100% complete).
Have a look at R-factors of models in PDB with data resolution around 2.6A:

Histogram of Rwork for models in PDB at resolution 2.50-2.70 A:
 0.119 - 0.145  : 10
 0.145 - 0.171  : 147
 0.171 - 0.197  : 814
 0.197 - 0.223  : 1454
 0.223 - 0.248  : 951
 0.248 - 0.274  : 183
 0.274 - 0.300  : 31
 0.300 - 0.326  : 3
 0.326 - 0.352  : 0
 0.352 - 0.378  : 1
Histogram of Rfree for models in PDB at resolution 2.50-2.70 A:
 0.175 - 0.204  : 51
 0.204 - 0.233  : 420
 0.233 - 0.262  : 1328
 0.262 - 0.291  : 1344
 0.291 - 0.320  : 386
 0.320 - 0.349  : 58
 0.349 - 0.378  : 6
 0.378 - 0.407  : 0
 0.407 - 0.436  : 0
 0.436 - 0.465  : 1
Histogram of Rfree-Rwork for all model in PDB at resolution 2.50-2.70 A:
 0.002 - 0.012  : 27
 0.012 - 0.022  : 137
 0.022 - 0.031  : 360
 0.031 - 0.041  : 645
 0.041 - 0.051  : 750
 0.051 - 0.061  : 754
 0.061 - 0.071  : 498
 0.071 - 0.080  : 251
 0.080 - 0.090  : 114
 0.090 - 0.100  : 58
Number of structures considered: 3594

Sounds like you are not alone...

Pavel


On Thu, Jan 26, 2017 at 6:11 AM, Pooja Kesari  wrote:

> 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
>
>

Re: [ccp4bb] Bad density for chains

[Pooja Kesari]

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

Re: [ccp4bb] Bad density for chains

[Eleanor Dodson]

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
>
>

Re: [ccp4bb] Bad density for chains

[Pooja Kesari]

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

Re: [ccp4bb] Bad density for chains

[Phoebe A. Rice]

Are you sure there really are 4 chains?  50% solvent may be average, but we've 
had crystals with closer to 80%.



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pooja Kesari 
[pkesar...@gmail.com]
Sent: Tuesday, January 24, 2017 10:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Bad density for chains

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

Re: [ccp4bb] Bad density for chains

[Debanu]

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

[ccp4bb] Bad density for chains

[Pooja Kesari]

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