Re: [ccp4bb] structure with missing density

[Pavel Afonine]

Hi Deepanshu,

how complete the data set? What's completeness across resolution zones?
Systematically missing reflections can have systematic impact in real space
(maps). I've seen entire ligands or domains disappear due to missing
low-resolution data.

Just another check-point to consider..

Good luck!
Pavel

On Wed, Oct 10, 2018 at 10:59 AM Deepanshu Choudhary <
deepanshui...@gmail.com> wrote:

> Dear all,
> I am working on a protein complex. After many efforts, I obtained crystals
> from one of the refinement screen (which took few weeks to grow) and I got
> a 3 Å dataset at synchrotron. I scaled the dataset in P21 spacegroup (which
> is also confirmed by Zanuda and Pointless). There is no twinning detected.
> I solved the phase using molecular replacement with a model of over 90%
> sequence identity. After several rounds of refinement with Refmac, the
> Rfree is 0.307 and Rfactor of 0.23.
> The density looks good and I can see everything that's important. But one
> of the proteins has missing density in 2 of its beta strands (corresponding
> to ~15%) and its not appearing upon several rounds of refinement. Also, the
> B factors are higher for this protein. The missing beta strands are not at
> the interface of the complex.
> I ran some crystals on the gel and did silver staining to find both the
> proteins and no degradation products. I doubt that there is any proteolytic
> cleavage because the protein is unlikely to remain folded if those beta
> strands are chopped out.
> I want to ask if such a structure with missing density and high B-factors
> (>100) can be deposited. Is it possible that some parts of the lattice
> don't have this protein with missing density which is resulting in high B
> factors?
> I would appreciate your efforts if someone can send me few references
> describing such type of structures. I would also welcome any other
> suggestions and recommendations.
>
> Thanks and regards,
> Deepanshu
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] structure with missing density

[Ditlev Egeskov Brodersen]

Dear Deepanshu,

Is the primary sequence in the neighbourhood of your missing strands conserved? 
Perhaps that part differs in your structure relative to the search model. Have 
you tried omitting the region from MR and rerunning refinement without to see 
if some new density pops up?

If not, although this doesn’t sound like a classical flexible region, it may 
still be disordered. If you really can’t build it, you can of course leave it 
out of the structure and deposit anyway. The rest of the structure might be 
very relevant for other people.

I also note that your refinement R factor is a bit low relative to the Rfree, 
which is indicating overfitting. Do you use individual B factors? You probably 
should use grouped Bs at 3 Å.

Best,

Ditlev

---

Ditlev E. Brodersen
Lektor, Associate Professor
Department of Molecular Biology and Genetics, Aarhus University
Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark

Phone:  +45 21669001
Lab home page: www.bioxray.au.dk/~deb
Educational IT portal: curriculearn.dk

Sent from my iPhone

Den 10. okt. 2018 kl. 04.59 skrev Deepanshu Choudhary 
mailto:deepanshui...@gmail.com>>:

Dear all,
I am working on a protein complex. After many efforts, I obtained crystals from 
one of the refinement screen (which took few weeks to grow) and I got a 3 Å 
dataset at synchrotron. I scaled the dataset in P21 spacegroup (which is also 
confirmed by Zanuda and Pointless). There is no twinning detected. I solved the 
phase using molecular replacement with a model of over 90% sequence identity. 
After several rounds of refinement with Refmac, the Rfree is 0.307 and Rfactor 
of 0.23.
The density looks good and I can see everything that's important. But one of 
the proteins has missing density in 2 of its beta strands (corresponding to 
~15%) and its not appearing upon several rounds of refinement. Also, the B 
factors are higher for this protein. The missing beta strands are not at the 
interface of the complex.
I ran some crystals on the gel and did silver staining to find both the 
proteins and no degradation products. I doubt that there is any proteolytic 
cleavage because the protein is unlikely to remain folded if those beta strands 
are chopped out.
I want to ask if such a structure with missing density and high B-factors 
(>100) can be deposited. Is it possible that some parts of the lattice don't 
have this protein with missing density which is resulting in high B factors?
I would appreciate your efforts if someone can send me few references 
describing such type of structures. I would also welcome any other suggestions 
and recommendations.

Thanks and regards,
Deepanshu



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] structure with missing density

[Deepanshu Choudhary]

Dear all,
I am working on a protein complex. After many efforts, I obtained crystals
from one of the refinement screen (which took few weeks to grow) and I got
a 3 Å dataset at synchrotron. I scaled the dataset in P21 spacegroup (which
is also confirmed by Zanuda and Pointless). There is no twinning detected.
I solved the phase using molecular replacement with a model of over 90%
sequence identity. After several rounds of refinement with Refmac, the
Rfree is 0.307 and Rfactor of 0.23.
The density looks good and I can see everything that's important. But one
of the proteins has missing density in 2 of its beta strands (corresponding
to ~15%) and its not appearing upon several rounds of refinement. Also, the
B factors are higher for this protein. The missing beta strands are not at
the interface of the complex.
I ran some crystals on the gel and did silver staining to find both the
proteins and no degradation products. I doubt that there is any proteolytic
cleavage because the protein is unlikely to remain folded if those beta
strands are chopped out.
I want to ask if such a structure with missing density and high B-factors
(>100) can be deposited. Is it possible that some parts of the lattice
don't have this protein with missing density which is resulting in high B
factors?
I would appreciate your efforts if someone can send me few references
describing such type of structures. I would also welcome any other
suggestions and recommendations.

Thanks and regards,
Deepanshu



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Structure with missing density

[Liang Tong]

Hello Rajesh
  We serendipitously encountered three cases where a contaminating fungus
led to proteolysis of the proteins, which was required for their
crystallization. We identified the fungus by sequencing a little bit of its
genome, and found that it is Penicillium!

  Here are two of the papers -

Mandel CR, Gebauer D, Zhang H, Tong L. (2006). A serendipitous discovery
that in situ proteolysis is essential for the crystallization of yeast
CPSF-100 (Ydh1p). *Acta Cryst*. *F62*, 1041-1045.

Bai Y, Auperin TC, Tong L. (2007). The use of in situ proteolysis in the
crystallization of murine CstF-77. *Acta Cryst*. *F63*, 135-138.


best regards
Liang



Liang Tong, William R. Kenan, Jr. Professor and Chair
Department of Biological Sciences
Columbia University, New York, NY 10027
(212) 854-5203
lt...@columbia.edu
http://tonglab.biology.columbia.edu




On Fri, Mar 9, 2018 at 3:25 PM, Rajesh <
1642be9504b8-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear BB,
>
> I apologize for the off topic. But I strongly believe this is the right
> place to ask my question.
>
> We are working on a protein that is 300 amino acids in length. After many
> efforts, we could solve the structure at 1.60 Å resolution. It took
> almost 10 months to get this crystal and we could not reproduce it. The
> maps are interpretable and we could model only till residue 190 and we
> could not see any density for the rest of the molecule. My question is-
> Does anyone has encountered such a structure with lots of missing density?
> I would appreciate your efforts if someone can send me few references
> describing such type of structures. Is there any chance that microbes can
> cleave the protein in the crystal drops?
>
>
>
>
>
> Thanks,
>
> Rajesh..
>
>

Re: [ccp4bb] Structure with missing density

[Edward Snell]

Dear Rajesh,

We had a similar problem with a yeast glutaminyl-tRNA synthetase. In eukaryotic 
systems these have an appended N-terminal domain not present in prokaryotes. 
When the full length protein was crystallized the last ~200 residues were 
missing from the structure. A packing diagram (highly recommended) showed large 
solvent channels. We re-expressed the full length protein with a His tag on the 
N-terminus, purified with a Ni affinity column, and then recrystallized, 
re-determined the structure and missing residues, followed by anti-his antibody 
detection on dissolved crystals – the resides were in the crystal but not 
structurally resolved. We ran a lot of gels which gave similar data but not as 
conclusive as the antibody approach. The N terminal was present but flexible in 
the large solvent channels. We used SAXS to look at the full length protein and 
detected the N-terminal determining that it was globular with a flexible 
linker. We cut the linker off and the N-terminal domain was crystallized on its 
own resulting in crystals within minutes of setting up. We used the SAXS 
envelope to position the various components of the full length protein and 
determined that the N-terminal domain is structurally similar to the GAtB 
subunit of GatCAB.

The full story is described in two papers, Nucleic Acids Res. 2012 
Apr;40(8):3723-31. doi: 10.1093/nar/gkr1223 and J Mol Biol. 2013 Jul 
24;425(14):2480-93. doi: 10.1016/j.jmb.2013.03.043. It took a while but 
provided a very biologically interesting result.

I would note a remarkable development in SAXS analysis related to these early 
studies that now allows the direct determination of electron density from a 
scattering curve rather than bead models or envelopes that were originally 
used: Grant, Thomas D. (2018). Ab initio electron density determination 
directly from solution scattering data. Nature Methods. 
http://dx.doi.org/10.1038/nmeth.4581. And shamelessly, also note the 
crystallization facility involved, http://getacrystal.org.

Yours is a smaller protein, but I hope this example might be of some use to 
answering your question,

Best wishes,

Eddie

Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
BioInnovations Chaired Professorship, University at Buffalo, SUNY
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
[cid:image001.png@01D3B7BC.B5187550]
Heisenberg was probably here!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rajesh
Sent: Friday, March 9, 2018 3:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Structure with missing density


Dear BB,

I apologize for the off topic. But I strongly believe this is the right place 
to ask my question.

We are working on a protein that is 300 amino acids in length. After many 
efforts, we could solve the structure at 1.60 Å resolution. It took almost 10 
months to get this crystal and we could not reproduce it. The maps are 
interpretable and we could model only till residue 190 and we could not see any 
density for the rest of the molecule. My question is- Does anyone has 
encountered such a structure with lots of missing density? I would appreciate 
your efforts if someone can send me few references describing such type of 
structures. Is there any chance that microbes can cleave the protein in the 
crystal drops?





Thanks,

Rajesh..

Re: [ccp4bb] Structure with missing density

[Thomas Cleveland]

Hi,

Long crystallization times and irreproducibility together with missing
residues points strongly to some slow proteolysis in your crystal drops
that eventually generated a crystallizable fragment. A lot of people
actually do this on purpose by adding proteases to generate stable
fragments. Here's

one example paper that came up from quick googling but there are lots of
others you can find in the literature.

Tom



On Fri, Mar 9, 2018 at 3:25 PM, Rajesh <
1642be9504b8-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear BB,
>
> I apologize for the off topic. But I strongly believe this is the right
> place to ask my question.
>
> We are working on a protein that is 300 amino acids in length. After many
> efforts, we could solve the structure at 1.60 Å resolution. It took
> almost 10 months to get this crystal and we could not reproduce it. The
> maps are interpretable and we could model only till residue 190 and we
> could not see any density for the rest of the molecule. My question is-
> Does anyone has encountered such a structure with lots of missing density?
> I would appreciate your efforts if someone can send me few references
> describing such type of structures. Is there any chance that microbes can
> cleave the protein in the crystal drops?
>
>
>
>
>
> Thanks,
>
> Rajesh..
>
>

[ccp4bb] Structure with missing density

[Rajesh]

Dear BB,

I apologize for the off topic. But I strongly believe this is the right place 
to ask my question.

We are working on a protein that is 300 amino acids in length. After many 
efforts, we could solve the structure at 1.60 Å resolution. It took almost 10 
months to get this crystal and we could not reproduce it. The maps are 
interpretable and we could model only till residue 190 and we could not see any 
density for the rest of the molecule. My question is- Does anyone has 
encountered such a structure with lots of missing density? I would appreciate 
your efforts if someone can send me few references describing such type of 
structures. Is there any chance that microbes can cleave the protein in the 
crystal drops?

 

 

Thanks,

Rajesh..