Re: [ccp4bb] Yet another "what's my blob" thread

[Roger Rowlett]

Blob one is something possibly distorted by alternate partial occupancy
binding sites near a symmetry axis. Blob 2 could be a chloride ion, or if
that is not enough density, maybe bicarbonate. Blob 1 seems to be attracted
two His residues, of the things in your mixture, Mg(II) seems possible or
Cl-. Blob 2 is less likely to be  metal around those Arg residues, but Cl-
or bicarbonate are possible and well-known in this kind of coordination
environment. Cl- is in your mixture, and bicarbonate can accumulate,
especially at alkaline pH from atmospheric CO2.

Cheers,

Roger Rowlett

On Oct 2, 2017 6:06 PM, "Lucas"  wrote:

I'm in the later stages of solving a structure which contains two
tetramers in the asymetric unit. I found these two blobs (in
equivalent positions on each tetramer) with positively charged
residues around it. Crystallization condition is Magnesium chloride,
Bis-tris and PEG3350. While the second blob looks like a metal, the
first one has a weird shape even though they are expected to have the
same thing. Any ideas?

Lucas

Re: [ccp4bb] Yet another "what's my blob" thread

[Keller, Jacob]

Looks like it's at a symmetry/NCS axis, so that complicates appearances...

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Lucas
Sent: Monday, October 02, 2017 6:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Yet another "what's my blob" thread

I'm in the later stages of solving a structure which contains two tetramers in 
the asymetric unit. I found these two blobs (in equivalent positions on each 
tetramer) with positively charged residues around it. Crystallization condition 
is Magnesium chloride, Bis-tris and PEG3350. While the second blob looks like a 
metal, the first one has a weird shape even though they are expected to have 
the same thing. Any ideas?

Lucas

[ccp4bb] refmac mixed anisotropic refinement

[Abhishek Anan]

Dear all,

How does one exclude water from anisotropic refinement in refmac5.8 under
ccp4-7.0

Here is the relevant section from the com file,

refi -
type REST -
resi MLKF -
meth CGMAT -
bref MIXED anisou residues from 100 A to 200 A

This does not work and the logfile has the following warning,

Data line---

 refi type REST resi MLKF meth CGMAT bref MIXED anisou RESID

 UES 100 A to 200 A

 Unknown subkeyword of REFI

 ? ANIS ?

 Wrong residual option

 Unknown subkeyword of REFI

 ? A?

 Unknown subkeyword of REFI

 ? TO   ?

 Unknown subkeyword of REFI

 ? 200  ?

 Unknown subkeyword of REFI

 ? A?

Is there any other way of defining the residue range or excluding only water?

Thank you for your help,

Abhishek

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Application deadline is October 20th 2017. To apply, please follow the link 
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t...@wehi.edu.au.

Re: [ccp4bb] query of B chain model of protein

[benjamin bax]

Hi,
  Looks like you have some density for same conformation as in A chain.

It could be you have two (or more) conformations for some parts of the 
structure in MolB. It is often tricky to sort out when you have two 
conformations for residues and map is ambiguous (and at 2.8A). 

I would rigid body fit chunks of residues (6-10 residues?) that have density in 
difference map - then prune back things with no density in difference map 
(delete residues and atoms on side-chains outside of density - with delete atom 
in coot).

 Then set occupancy of ‘new’ stuff to 0.3 and do a little tightly restrained 
refinement and calculate new maps (in my experience - if you set occupancy to 
0.5 you are likely to get too much model bias - to see other conformation). 
Quite likely this will not help and it will still be ambiguous. 

[ Alternatively - If you solve another 50 structures of the same thing - in 
different space-groups/cells - you may find different conformations for the 
'missing parts’ appear in a couple of structures - and then you may be able to 
better model them in MolB of this structure]. 

  Ben
 

On 2 Oct 2017, at 15:42, Vikram Dalal  wrote:

Hi all,


I am solving a protein data of 2.6 A. It has two monomers in ASU. It has 377 
residues. I have built residues 26 to 36 and 41 to 377 in A chain while B chain 
has model 42 to 129, 146 to 168 and 189 to 371. Current R factor and R Free is 
23.2  and 28.5, respectively. I have attached electron density (fourier map at 
1.0 contour and difference map at 2.5) figures for 130 to 145, 169 to 188 and C 
terminal region of B chain and superposed model of A chain is shown in green 
color. 

I have tried manually building missing residues of B chain according to A chain 
model, but R free increased to 29. 

Any suggestions will be highly aprreciated. 


Thanks & Regards,







Dr Ben Bax

York Structural Biology Laboratory, 
Department of Chemistry, 
University of York,
York YO10 5DD

ben.d.v@gmail.com

 

 

Re: [ccp4bb] query of B chain model of protein

[Eleanor Dodson]

Not much idea.

I tend to build small amounts at the termini of your current model using
the better model as a guide then re-refine and hope you can keep going..
Eleanor

On 2 October 2017 at 15:42, Vikram Dalal  wrote:

> Hi all,
>
>
> I am solving a protein data of 2.6 A. It has two monomers in ASU. It has
> 377 residues. I have built residues 26 to 36 and 41 to 377 in A chain while
> B chain has model 42 to 129, 146 to 168 and 189 to 371. Current R factor
> and R Free is 23.2  and 28.5, respectively. I have attached electron
> density (fourier map at 1.0 contour and difference map at 2.5) figures for
> 130 to 145, 169 to 188 and C terminal region of B chain and superposed
> model of A chain is shown in green color.
>
> I have tried manually building missing residues of B chain according to A
> chain model, but R free increased to 29.
>
> Any suggestions will be highly aprreciated.
>
>
> Thanks & Regards,
>
>
>
>
>

Re: [ccp4bb] PDB search help

[Gloria Borgstahl]

Our RPA14/32 crystals from 2007 included full length protein for both
subunits (RPA14 and RPA32), but only the central OB fold of RPA32
could be modelled.  The crystals included RPA32(1-270) but only 42-176
could be modelled.  I remember being very frustrated by not being able
to visualize the wHLH domain at the CT or RPA32.  There is disordered
density for the rest due to the flexible linkers.  All of RPA14 could
be modelled.  This happened for all 3 crystal forms we solved...  see
2PI2, 2PQA, and 2Z6K

On Mon, Oct 2, 2017 at 9:22 AM, zaigham mahmood khan
 wrote:
> Hey Rajesh
>
> You may try the following link:
>
> Please enter the protein sequence, and scroll down the "Select standard
> database" tab, and choose "pdb_nr". Once you will "submit" the job, you will
> most likely get what you want to see!
>
> You may pick any pdb ID from the result section, and from pdb.org, you may
> find the residues of that protein that were observed in the electron density
> map as well as whole expression casstte that was attempted to crystallize
> for that particular pdb entry.
>
> Best wishes
>
> -Z
>
>
> Zaigham Mahmood Khan, PhD
>
> Icahn School of Medicine at Mount Sinai
> Department of Oncological Sciences
> 1470 Madison Avenue
> New York
>
> On Wed, Sep 27, 2017 at 6:47 PM, Rajesh
> <1642be9504b8-dmarc-requ...@jiscmail.ac.uk> wrote:
>>
>> Dear BB,
>>
>> Sorry for the off topic.
>>
>> Does anyone know how to search the PDB for the entries that have the
>> density only for part of the protein molecule rather than for the entire
>> length of the protein attempted to crystallize?
>>
>>
>>
>> Thanks,
>> Rajesh..
>
>

Re: [ccp4bb] PDB search help

[zaigham mahmood khan]

Hey Rajesh

You may try the following link:

Please enter the protein sequence, and scroll down the "Select standard
database" tab, and choose "pdb_nr". Once you will "submit" the job, you
will most likely get what you want to see!

You may pick any pdb ID from the result section, and from pdb.org, you may
find the residues of that protein that were observed in the electron
density map as well as whole expression casstte that was attempted to
crystallize for that particular pdb entry.

Best wishes

-Z


Zaigham Mahmood Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York

On Wed, Sep 27, 2017 at 6:47 PM, Rajesh <
1642be9504b8-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear BB,
>
> Sorry for the off topic.
>
> Does anyone know how to search the PDB for the entries that have the
> density only for part of the protein molecule rather than for the entire
> length of the protein attempted to crystallize?
>
>
> Thanks,
> Rajesh..
>

Re: [ccp4bb] Short peptide outliers

[Eleanor Dodson]

That is very pleasing! Ramachandran vindicated yet again..
Eleanor

On 2 October 2017 at 10:31, Meytal Galilee  wrote:

> Dear all,
> Many thanks for your responses.
> Indeed the peptide was wrong handed, flipping the peptide chain fixed all
> my outliers issues!
> Thanks again!
> Meytal
>
> 2017-10-01 23:12 GMT+03:00 Eleanor Dodson :
>
>> That seems strange! You couldn't have built it in the wrong direction
>> could you?
>>
>> Or have bound a L-handed peptide?
>>
>> There are outliers which can be explained by interactions with other
>> features but it would be very very  unlikely that all the residues were
>> outliers
>>
>> Eleanor
>>
>>
>>
> On 1 October 2017 at 17:13, Dale Tronrud  wrote:
>
>> Hi,
>>
>>Bond length and angle targets are defined based on the local
>> chemistry and apply equally to small and large molecules.  The
>> Ramachandran distributions were defined via an examination of,
>> basically, tripeptides.  Your peptide model must be consistent with
>> these prior observations to be considered reliable.  If it is not there
>> is likely something seriously wrong with your interpretation.
>>
>>In addition, your model peptide must make chemically reasonable
>> interactions with its partner.  You didn't describe this aspect of your
>> model, but this is equally critical in the evaluation of the model of a
>> bound ligand.
>>
>>In my opinion the most likely explanation is that multiple
>> conformations of the peptide are binding.  Without seeing the density or
>> being able to examine the data it is hard to generate possibilities.
>>
>> Dale Tronrud
>>
>> On 10/1/2017 2:20 AM, Meytal Galilee wrote:
>> > Hi All,
>> > I have solved a structure of a protein bound to a short peptide (11
>> > residues) at 1.9A.
>> > The peptide fits the map perfectly, however,  all of its residues are
>> > either Ramachandran / bond length / angle outliers.
>> > Fixing any of these issues forces the peptide to misfit the map
>> > dramatically.
>> > Is anyone familiar with short peptides outliers? Are these issues common
>> > / acceptable?
>> > Does anyone have an idea or suggestion?
>> > Many Thanks,
>> > Meytal Galilee
>> >
>>
>
>
>
> --
> Meytal Galilee
>

Re: [ccp4bb] Short peptide outliers

[Meytal Galilee]

Dear all,
Many thanks for your responses.
Indeed the peptide was wrong handed, flipping the peptide chain fixed all
my outliers issues!
Thanks again!
Meytal

2017-10-01 23:12 GMT+03:00 Eleanor Dodson :

> That seems strange! You couldn't have built it in the wrong direction
> could you?
>
> Or have bound a L-handed peptide?
>
> There are outliers which can be explained by interactions with other
> features but it would be very very  unlikely that all the residues were
> outliers
>
> Eleanor
>
>
>
On 1 October 2017 at 17:13, Dale Tronrud  wrote:

> Hi,
>
>Bond length and angle targets are defined based on the local
> chemistry and apply equally to small and large molecules.  The
> Ramachandran distributions were defined via an examination of,
> basically, tripeptides.  Your peptide model must be consistent with
> these prior observations to be considered reliable.  If it is not there
> is likely something seriously wrong with your interpretation.
>
>In addition, your model peptide must make chemically reasonable
> interactions with its partner.  You didn't describe this aspect of your
> model, but this is equally critical in the evaluation of the model of a
> bound ligand.
>
>In my opinion the most likely explanation is that multiple
> conformations of the peptide are binding.  Without seeing the density or
> being able to examine the data it is hard to generate possibilities.
>
> Dale Tronrud
>
> On 10/1/2017 2:20 AM, Meytal Galilee wrote:
> > Hi All,
> > I have solved a structure of a protein bound to a short peptide (11
> > residues) at 1.9A.
> > The peptide fits the map perfectly, however,  all of its residues are
> > either Ramachandran / bond length / angle outliers.
> > Fixing any of these issues forces the peptide to misfit the map
> > dramatically.
> > Is anyone familiar with short peptides outliers? Are these issues common
> > / acceptable?
> > Does anyone have an idea or suggestion?
> > Many Thanks,
> > Meytal Galilee
> >
>



-- 
Meytal Galilee

[ccp4bb] Biophysical Characterisation of Macromolecules and Quantification of Biomolecular Interactions

[Anastassis Perrakis]

Dear all,

I would like to remind you that the deadline for applying to this course is 10 
October 2017

https://www.structuralbiology.eu/events/biophysical-characterisation-of-macromolecules-and-quantification-of-biomolecular-interactions/

You can apply at:

https://www.aanmelder.nl/96461/subscribe

… and don’t forget to write a strong motivation letter as the places are 
limited!

Best regards,

Tassos