Re: [ccp4bb] Crystallizing a tough target

[Savvas Savvides]

Dear Kavya,

we encountered this issue for a protein complex at 95 mg/mL involving Human 
Serum Albumin (HSA) and a designed protein binder (Alphabody) as described in 
Pannecoucke et al. 2021 Sci Adv 7 (13), DOI:10.1126/sciadv.abe1682.
The breakthrough in that case came from crystallization experiments at 
different temperatures (14C, 21C and 37C), with 37C coming out as the clear 
winner.

best wishes
Savvas


——
Prof. Savvas Savvides
VIB Center for Inflammation Research
Ghent University, Dept. of Biochemistry & Microbiology
Technologiepark 71, 9052 Ghent, Belgium

Email: savvas.savvi...@ugent.be<mailto:savvas.savvi...@ugent.be> ; 
savvas.savvi...@irc.vib-ugent.be<mailto:savvas.savvi...@irc.vib-ugent.be>
Phone: +32 (0)472 928 519 (mobile) ; +32 (0)9 331 36 60 (office)
Web:https://www.irc.ugent.be/index.php/groups/savvides-unit

On 5 Feb 2024, at 11:27, kavyashreem  wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




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Re: [ccp4bb] Dry shipper in limbo

[Savvas Savvides]

Dear Kevin,

A similar incident from just two weeks ago, in Europe in our case, was resolved 
by travelling to the regional FedEx package collection/distribution center to 
recover the dry-shipper in person. This involved gaining access to the FedEx 
center and talking to the right people.

I would absolutely not wait this out because your shipment is probably 
somewhere in the corner of a distribution center used as a seat by employees 
taking a break. I can attest to the fact that they do have such corners where 
“problematic" shipments are piled up!

Despite what you were told on the phone or could deduce from any online 
tracking systems (in our case the dry-shipper was not even trackable), there is 
still a (very good) chance that your shipment might still be at a regional 
collection/distribution center.

The key is to get to any information about where this place might be, talk to 
the local people, and find out what the actual route of the shipment might have 
been after getting picked up from your lab. The routes often involve 
intermediate collection/distribution sites.

To their credit, and in contrast to the general FedEx customer service, the 
people at the regional FedEx distribution center (including the shift manager) 
were very empathic and helpful, and used means that go above and beyond SOPs to 
help. This included sharing photos of the shipment via WhatsApp with 
drivers/employees of the three previous shifts etc… Despite the volumes of work 
they handle, drivers and other courier employees actually do remember unusually 
looking shipments, such as a shipping-case containing a dry-shipper!

Best of luck and wishes,
Savvas




On 28 Jul 2023, at 08:15, Savvas Savvides  wrote:



Best wishes,
Savvas

Begin forwarded message:

From: "Dr. Kevin M Jude" mailto:kj...@stanford.edu>>
Date: 28 July 2023 at 05:16:39 CEST
To: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
Subject: [ccp4bb] Dry shipper in limbo
Reply-To: "Dr. Kevin M Jude" mailto:kj...@stanford.edu>>


My first adventure in international crystallography is off to an inauspicious 
start. On Monday, I sent a dry shipper “overnight” from California to 
Saskatchewan, but it has been stuck in the Memphis FedEx facility for a few 
days. I’ve gotten several conflicting explanations of the status from FedEx on 
the phone, but the most likely seems that it has “pre-cleared” customs and yet 
has not yet made it to Calgary. It’s not clear whether anyone actually knows 
where the shipping case is, since I was asked to give a physical description of 
it. Is there anything else I can do from a few thousand miles away, or do I 
just have to wait this out?

--
Kevin Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305
Phone: (650) 723-6431



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Re: [ccp4bb] Structure prediction - waiting to happen

[Savvas Savvides]

Dear Rams,

I salute you for sharing this.

Just a week ago, I also received a remark along these lines on a declined grant 
application. The remark was the only unfavourable point, which suggested that 
it must have weighed disproportionally towards the negative outcome. This was a 
two-stage evaluation process and the grant was cut in stage-1 where it was 
evaluated by a small group of evaluators, none of whom was a structural 
biologist/biochemist. Stage-2 would have involved peer review by international 
experts.

Despite my initial disbelief about what this remark might have caused and upon 
reflection, I realized that it might be time to become proactive in future 
applications in anticipation of the apparent growing trend towards such remarks 
and perceptions.

I think that a generalized form of preemptive text might not serve the purpose 
well, but perhaps well-articulated statements specific to the proposed 
biological problem at hand (perhaps aided by illustrations demonstrating the 
inability of structure prediction to address the problem at hand) might be the 
better way to go. Even though many of us who teach courses in experimental 
structural biology and structural bioinformatics at undergraduate and graduate 
levels are already actively addressing many of these issues, there is a much 
bigger and far more senior scientific population out there that makes important 
decisions on science policy/funding/infrastructures/evaluations/recruitment/etc 
that are not getting such educational exposure.

The following resources provide good material and starting points to reflect 
and elaborate upon.

The article by Perrakis and Sixma in EMBO Reports 
https://www.embopress.org/doi/full/10.15252/embr.202154046

The recent comment paper in Nature Methods by Thomas Jane
https://doi.org/10.1038/s41592-022-01760-4

A correspondence in Science by Moore, Hendrickson, Henderson and Brunger
https://www.science.org/doi/10.1126/science.abn9422?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed


Best wishes
Savvas


----
Savvas Savvides
VIB Center for Inflammation Research
Ghent University, Dept. of Biochemistry & Microbiology
Technologiepark 71, 9052 Ghent, Belgium

Email: savvas.savvi...@ugent.be<mailto:savvas.savvi...@ugent.be> ; 
savvas.savvi...@irc.vib-ugent.be<mailto:savvas.savvi...@irc.vib-ugent.be>
Phone: +32 (0)472 928 519 (mobile) ; +32 (0)9 331 36 60 (office)
Web: https://savvideslab.sites.vib.be/en#/

On 1 Apr 2023, at 16:57, Subramanian, Ramaswamy  wrote:

Ian,

Thank you.  This is not an April fools..
Rams
subra...@purdue.edu



On Apr 1, 2023, at 10:46 AM, Ian Tickle  wrote:

 External Email: Use caution with attachments, links, or sharing data 


Hi Ramaswamy

I assume this is an April Fool's but it's still a serious question because many 
reviewers who are not crystallographers or electron microscopists may not fully 
appreciate the difference currently between the precision of structures 
obtained by experimental and predictive methods, though the latter are 
certainly catching up.  The answer of course lies in the mean co-ordinate 
precision, related to the map resolution.

Quoting 
https://people.cryst.bbk.ac.uk/~ubcg05m/precgrant.html<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpeople.cryst.bbk.ac.uk%2F~ubcg05m%2Fprecgrant.html&data=05%7C01%7CSavvas.Savvides%40ugent.be%7Cec42b9fc865d4c73dc2908db32c16a7c%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C638159578530258708%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=vtJ3sWUBKSJBRADTzD0xzrzluAdccTcGJE8L5DqsDlo%3D&reserved=0>
 :

"The accuracy and precision required of an experimentally determined model of a 
macromolecule depends on the biological questions being asked of the structure. 
 Questions involving the overall fold of a protein, or its topological 
similarity to other proteins, can be answered by structures of fairly low 
precision such as those obtained from very low resolution X-ray crystal 
diffraction data [or AlphaFold].  Questions involving reaction mechanisms 
require much greater accuracy and precision as obtained from well-refined, 
high-resolution X-ray structures, including proper statistical analyses of the 
standard uncertainties (s.u.'s) of atomic positions and bond lengths.".

According to 
https://www.nature.com/articles/s41586-021-03819-2<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41586-021-03819-2&data=05%7C01%7CSavvas.Savvides%40ugent.be%7Cec42b9fc865d4c73dc2908db32c16a7c%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C638159578530258708%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=qPH8CNx0jecLhVmjEhodmXKcLzCHbPFV%2FjNO9nfQEkM%3D&reserved=0>
 :

The accuracy of AlphaFold structures at the time of wri

Re: [ccp4bb] problems with running PARROT

[Savvas Savvides]

  21.6

NCS operator:   4
 NCS masking: Mask volume as fraction of ASU: 0.89   Multiplicity: 9
  Contiguity score:  0.998   Self-overlap score:  0.179
 NXop refinement- correlation before:  0.423, after:  0.588
 NXop old:  100.4  178.5  -79.3-17.9   16.17.4-17.7   33.0   93.4
 NXop new:  144.0  179.8  -36.0-24.0   11.7   20.5-23.7   37.5   80.6


NCS operator statistics:
 Operator_number  Mask_volume/ASU  Correlation
11.0010.911
20.9980.912
30.9540.863
40.8880.854


$TABLE :Cycle 1 Electron density histograms:
$GRAPHS :Protein:N:1,4,5,6::Solvent:N:1,7,8::Simulation:N:1,3,4: $$
rho_min rho_max   Simulatn P_init P_trgt P_mod  S_init S_mod $$
$$
 -0.317  -0.282  0.000  0.000  0.017  0.008  0.000  0.000
 -0.282  -0.247  0.000  0.000  0.026  0.014  0.000  0.000
 -0.247  -0.212  0.002  0.002  0.036  0.024  0.000  0.000
 -0.212  -0.177  0.007  0.007  0.045  0.039  0.000  0.000
 -0.177  -0.143  0.022  0.019  0.057  0.056  0.003  0.000
 -0.143  -0.108  0.050  0.048  0.064  0.076  0.020  0.000
 -0.108  -0.073  0.093  0.094  0.071  0.081  0.069  0.000
 -0.073  -0.038  0.142  0.147  0.070  0.090  0.156  0.000
 -0.038  -0.003  0.172  0.175  0.072  0.091  0.234  0.000
 -0.003   0.032  0.172  0.173  0.068  0.085  0.237  1.000
  0.032   0.067  0.141  0.141  0.063  0.075  0.167  0.000
  0.067   0.101  0.099  0.094  0.057  0.071  0.080  0.000
  0.101   0.136  0.056  0.053  0.053  0.063  0.026  0.000
  0.136   0.171  0.029  0.026  0.049  0.053  0.006  0.000
  0.171   0.206  0.011  0.012  0.044  0.048  0.001  0.000
  0.206   0.241  0.004  0.005  0.038  0.038  0.000  0.000
  0.241   0.276  0.001  0.002  0.033  0.033  0.000  0.000
  0.276   0.311  0.000  0.001  0.030  0.025  0.000  0.000
  0.311   0.345  0.000  0.000  0.025  0.016  0.000  0.000
  0.345   0.380  0.000  0.000  0.021  0.014  0.000  0.000
$$


Gamma 0.24162

Log likelihood:  2.920853e+04  Log likelihood (free):  0.00e+00

$TABLE :Cycle 1 SigmaA statistics:
$GRAPHS :SigmaA statistics:N:1,2,3: $$
 1/resol^2  sigmaA(s)  sigmaA(w) $$
$$
0.012  0.682  0.675
0.025  0.629  0.635
0.035  0.659  0.664
0.043  0.666  0.670
0.051  0.619  0.638
0.059  0.515  0.593
0.066  0.452  0.656
$$

> On 25 Jun 2021, at 15:34, Eleanor Dodson  wrote:
> 
> The extract from the  log file looks OK - can you send the whole log.txt?
> Eleanor
> 
> On Fri, 25 Jun 2021 at 12:09, Savvas Savvides  <mailto:savvas.savvi...@ugent.be>> wrote:
> Dear colleagues,
> 
> I am trying to run Parrot via CCP4-7.1.014 and the CCP4i2 GUI on a MacBookPro 
> (OSX 10.15.7) and keep getting the following error report as the program is 
> in the process of outputting SigmaA statistics.
> 
> Below, I provide two pieces of information:
> (1) The Error message
> (2) The last page of the ouput file where the program ends up crashing with 
> the error message.
> 
> Thank you in advance for any insights/input on this issue.
> 
> Best wishes,
> Savvas
> 
> 
> 
> 
> 
> 
> Error Report for Job 41: Density modification - PARROT
> -ERROR- CTaskParrot:9 Error in wrapper parrot 0.0:: Failed starting external 
> process
> - this can be due to a number of things, but usually is due to the command 
> used by subprocess/QProcess not working for some reason.
> Missing input files, bad commands, non-functional programs etc. Check log 
> files and stdout.
> Process: cparrot
> 
>  -ERROR- CTaskParrot:47 Error in wrapper parrot 0.0:: Error in checking 
> external process after completion
> exit status and  code: -11 -11
> 
> 
> ———
> 
> -- Cycle: 1 
> 
> Suggested radius for solvent mask determination: 5.68666
> 
> NCS operator:   1
>  NCS masking: Mask volume as fraction of ASU: 1.00   Multiplicity: 10
>   Contiguity score:  1.000   Self-overlap score:  0.151
>  NXop refinement- correlation before:  0.539, after:  0.581
>  NXop old:  -57.7  179.7  122.8 -5.6   10.0   36.4 -5.5   39.0   64.4
>  NXop new:  105.8  180.0  -74.1 -7.4   11.2   40.1 -7.3   37.8   60.9
> 
> NCS operator:   2
>  NCS masking: Mask volume as fraction of ASU: 1.00   Multiplicity: 10
>   Contiguity score:  1.000   Self-overlap score:  0.154
>  NXop refinement- correlation before:  0.528, after:  0.577
>  NXop old:   57.2  179.7 -122.3 -5.5   39.0   64.4 -5.6   10.0   36.4
>  NXop new:  -94.6  179.8   85.4 -3.8   36.5   60.3 -3.8   12.5   40.6
> 
> NCS operator:   3
>  NCS masking: Mask volume as fractio

[ccp4bb] problems with running PARROT

[Savvas Savvides]

Dear colleagues,

I am trying to run Parrot via CCP4-7.1.014 and the CCP4i2 GUI on a MacBookPro 
(OSX 10.15.7) and keep getting the following error report as the program is in 
the process of outputting SigmaA statistics.

Below, I provide two pieces of information:
(1) The Error message
(2) The last page of the ouput file where the program ends up crashing with the 
error message.

Thank you in advance for any insights/input on this issue.

Best wishes,
Savvas






Error Report for Job 41: Density modification - PARROT
-ERROR- CTaskParrot:9 Error in wrapper parrot 0.0:: Failed starting external 
process
- this can be due to a number of things, but usually is due to the command used 
by subprocess/QProcess not working for some reason.
Missing input files, bad commands, non-functional programs etc. Check log files 
and stdout.
Process: cparrot

 -ERROR- CTaskParrot:47 Error in wrapper parrot 0.0:: Error in checking 
external process after completion
exit status and  code: -11 -11


———

-- Cycle: 1 

Suggested radius for solvent mask determination: 5.68666

NCS operator:   1
 NCS masking: Mask volume as fraction of ASU: 1.00   Multiplicity: 10
  Contiguity score:  1.000   Self-overlap score:  0.151
 NXop refinement- correlation before:  0.539, after:  0.581
 NXop old:  -57.7  179.7  122.8 -5.6   10.0   36.4 -5.5   39.0   64.4
 NXop new:  105.8  180.0  -74.1 -7.4   11.2   40.1 -7.3   37.8   60.9

NCS operator:   2
 NCS masking: Mask volume as fraction of ASU: 1.00   Multiplicity: 10
  Contiguity score:  1.000   Self-overlap score:  0.154
 NXop refinement- correlation before:  0.528, after:  0.577
 NXop old:   57.2  179.7 -122.3 -5.5   39.0   64.4 -5.6   10.0   36.4
 NXop new:  -94.6  179.8   85.4 -3.8   36.5   60.3 -3.8   12.5   40.6

NCS operator:   3
 NCS masking: Mask volume as fraction of ASU: 0.95   Multiplicity: 9
  Contiguity score:  0.999   Self-overlap score:  0.149
 NXop refinement- correlation before:  0.447, after:  0.606
 NXop old: -100.7  178.5   79.6-17.7   33.0   93.4-17.9   16.17.4
 NXop new: -140.4  179.8   39.7 -9.8   36.7   79.4 -9.9   12.4   21.6

NCS operator:   4
 NCS masking: Mask volume as fraction of ASU: 0.89   Multiplicity: 9
  Contiguity score:  0.998   Self-overlap score:  0.179
 NXop refinement- correlation before:  0.423, after:  0.588
 NXop old:  100.4  178.5  -79.3-17.9   16.17.4-17.7   33.0   93.4
 NXop new:  144.0  179.8  -36.0-24.0   11.7   20.5-23.7   37.5   80.6


NCS operator statistics:
 Operator_number  Mask_volume/ASU  Correlation
11.0010.911
20.9980.912
30.9540.863
40.8880.854


$TABLE :Cycle 1 Electron density histograms:
$GRAPHS :Protein:N:1,4,5,6::Solvent:N:1,7,8::Simulation:N:1,3,4: $$
rho_min rho_max   Simulatn P_init P_trgt P_mod  S_init S_mod $$
$$
 -0.317  -0.282  0.000  0.000  0.017  0.008  0.000  0.000
 -0.282  -0.247  0.000  0.000  0.026  0.014  0.000  0.000
 -0.247  -0.212  0.002  0.002  0.036  0.024  0.000  0.000
 -0.212  -0.177  0.007  0.007  0.045  0.039  0.000  0.000
 -0.177  -0.143  0.022  0.019  0.057  0.056  0.003  0.000
 -0.143  -0.108  0.050  0.048  0.064  0.076  0.020  0.000
 -0.108  -0.073  0.093  0.094  0.071  0.081  0.069  0.000
 -0.073  -0.038  0.142  0.147  0.070  0.090  0.156  0.000
 -0.038  -0.003  0.172  0.175  0.072  0.091  0.234  0.000
 -0.003   0.032  0.172  0.173  0.068  0.085  0.237  1.000
  0.032   0.067  0.141  0.141  0.063  0.075  0.167  0.000
  0.067   0.101  0.099  0.094  0.057  0.071  0.080  0.000
  0.101   0.136  0.056  0.053  0.053  0.063  0.026  0.000
  0.136   0.171  0.029  0.026  0.049  0.053  0.006  0.000
  0.171   0.206  0.011  0.012  0.044  0.048  0.001  0.000
  0.206   0.241  0.004  0.005  0.038  0.038  0.000  0.000
  0.241   0.276  0.001  0.002  0.033  0.033  0.000  0.000
  0.276   0.311  0.000  0.001  0.030  0.025  0.000  0.000
  0.311   0.345  0.000  0.000  0.025  0.016  0.000  0.000
  0.345   0.380  0.000  0.000  0.021  0.014  0.000  0.000
$$


Gamma 0.24162

Log likelihood:  2.920853e+04  Log likelihood (free):  0.00e+00

$TABLE :Cycle 1 SigmaA statistics:
$GRAPHS :SigmaA statistics:N:1,2,3: $$
 1/resol^2  sigmaA(s)  sigmaA(w) $$
$$
0.012  0.682  0.675
0.025  0.629  0.635
0.035  0.659  0.664
0.043  0.666  0.670
0.051  0.619  0.638
0.059  0.515  0.593
0.066  0.452  0.656
$$




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This message was issued to mem

[ccp4bb] Research fellow position at Ghent Un. and the VIB (Ghent, Belgium)

[Savvas Savvides]

Dear colleagues,

please find below a published vacancy for a post-doctoral research fellow in 
structural biology in our group at Ghent University and the VIB (Ghent, 
Belgium). For a summary of our research activities please visit:
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>

best wishes,
Savvas
---
Savvas Savvides
VIB Center for Inflammation Research
Ghent University, Dept. Biochemistry & Microbiology
Technologiepark-Zwijnaarde 71, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>




To apply please visit:
https://career012.successfactors.eu/career?company=C956575P&career_job_req_id=12740&career_ns=job_listing&navBarLevel=JOB_SEARCH


→   Aplication deadline 05/11/2019 23:59 (Brussels time)
→   Faculty of Sciences
→   Department WE10 - Biochemistry and microbiology
→   Reference number: 201909/WE10/DA/014
 
ABOUT GHENT UNIVERSITY
 
Ghent University is a world of its own. Employing more than 8,000 people, it is 
actively involved in education and research, management and administration, and 
technical and social services on a daily basis. It is one of the largest, most 
exciting employers in the area and offers great career opportunities. With each 
of its 11 faculties and more than 100 departments offering state-of-the-art 
study programmes that are grounded in research in a wide range of academic 
fields, Ghent University is a logical choice for its employees as well as its 
students.
 
YOUR TASKS
 
At least 70% of your assignment will be spent on academic research in 
structural biology within the Unit for Structural Biology embedded in the 
Department of Biochemistry and Microbiology (WE10) and you will provide 
technical support for data acquisition and analysis in single-particle electron 
microscopy. 
You will assist in teaching activities at the Bachelor’s or Master’s level.
You will counsel Master’s students in their exercises, work placement and 
Master’s dissertation.
 
WHAT WE ARE LOOKING FOR
 
You hold a doctoral degree in Biochemistry, Biotechnology, Biophysics, 
Structural Biology or a subject considered relevant by the selection committee. 
The degree requirements need to be fulfilled at the start of your appointment.  
  For diplomas awarded outside the European Union, a certificate of 
equivalence (NARIC) must be submitted. 
You have already conducted research and acquired technical skills in electron 
microscopy (EM) and in particular cryoEM towards the determination of the 
three-dimensional structure of proteins, protein-protein, and protein-ligand 
complexes. Such research experience needs to be supported by a publication 
track record in scientific journals that subscribe to the peer-review process. 
Additional research experience in one of the following complementary methods 
within structural biology could be an advantage:
- Macromolecular X-ray Crystallography
- Small-angle X-ray Scattering
You are familiar and have practical experience with biophysical methods for the 
study of protein-protein and protein-ligand interactions (e.g. Isothermal 
Titration Calorimetry, Biolayer interferometry, Multi-angle laser light 
scattering).
You have broad practical experience in molecular biology, protein biochemistry, 
and tissue culture for the production and purification of mammalian recombinant 
proteins and complexes thereof towards structural and biophysical studies.
You have distinguished yourself as a promising researcher during your doctorate.
You are interested in coaching students in the Bachelor’s, Master’s and/or 
Advanced Master’s programmes.
 
 
WHAT WE CAN OFFER YOU
 
We offer you a three-year appointment. The appointment may be renewed once for 
three years at the most, on condition that the previous term was given a 
positive evaluation. The term of renewal begins immediately after the first 
term has expired. Attention: If you have been previously appointed as a 
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Your appointment will start on 1 February 2020.
Your remuneration will be determined according to salary scale AAP5. More 
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INTERESTED?
 
Apply

Re: [ccp4bb] Optimization from needle shaped crystals

[Savvas Savvides]

Dear Prem,

before moving on with any optimization rounds it would be good to first confirm 
that these crystals are actually protein crystals, and in particular the 
protein of interest.

For example, a Silver-stained SDS-PAGE gel can be very informative.
You can run a couple of your crystalline clusters after 2 washing steps on 
SDS-PAGE. Make sure you also load the washing steps, the content of clear drops 
(from the same screen), and protein as purified,- as controls.
We have found that such a simple diagnostic early in the process can be 
crucially important.

best wishes
Savvas



> On 8 Sep 2019, at 06:39, Prem Prakash  wrote:
> 
> Dear all, 
> Sorry for a trivial query. I am trying to Co-crystallize my protein with its 
> substrate (peptide) using commercial screenings. In one condition of JCSG 
> plus (Molecular Dimension) that contains  0.2 M Magnesium chloride 
> hexahydrate,  0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like 
> crystals (picture attached). Does anyone have idea to optimize such needles 
> into better crystals. I would appreciate all your suggestions. 
> 
> Thank you 
> With kind regards,
> Prem Prakash  (Ph.D.) 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
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Re: [ccp4bb] different residues as alternate occupancy?

[Savvas Savvides]

The structure of milk proteins obtained from in vivo-grown crystals from a 
viviparous cockroach could also serve as an interesting case:

http://dx.doi.org/10.1107/S2052252516008903 


best wishes
Savvas


> On 24 Jun 2019, at 16:03, Holton, James M 
> <270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> A classic case of this is crambin.  Residue 25 of 3nir.
> 
> -James Holton
> MAD Scientist
> 
> On 6/24/2019 1:00 AM, Guenter Fritz wrote:
>> Dear all,
>> 
>> I am refining a multimer and mass spec data of the sample indicate 
>> that there is a mixture of two variants which differ in one amino acid 
>> residue. The density that we see is therefore most likely an average 
>> of both variants. I have created a pdb file with "alternate residues" 
>> each with 0.5 occupancy at this position to use it in refinement.  
>> However, the programs detect this as an error in the pdb file.
>> 
>> Has anyone  faced such a problem previously? Any suggestions are very 
>> much appreciated.
>> 
>> Thanks a lot and best regards, Guenter
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
> 
> 
> 
> 
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Re: [ccp4bb] A Max Perutz question

[Savvas Savvides]

Hey Zac
A good scan of page 228 in Georgina Ferry’s biography of Max Perutx (Max Perutz 
and the secret of life) will do the trick. It is a 18x13 cm photo. I have it in 
front of me!

best 
Savvas

> On 10 Apr 2019, at 16:29, Zachary A. Wood  wrote:
> 
> Hello Fellow Structural Enthusiasts,
> 
> My apologies for the slightly off-topic question. I am trying to track down a 
> higher resolution image of the jpg that I have attached. I use this photo of 
> Max Perutz when I am teaching about protein folding, and have always wanted a 
> better quality one. I believe it is credited to Nature, and I am trying to 
> find out what issue, but I am hoping that one of you may have more 
> information or perhaps even a better photo. Thanks for any help, and for 
> those of you who may never have seen this photo before, I hope you enjoy it. 
> I like to imagine that Perutz is considering the challenges associated with 
> folding that chain after he determined the crystal structure. If you have 
> never read the discussion in his famous Nature paper, I will leave you with a 
> relevant quote of him referring to the structural similarity between horse 
> hemoglobin and sperm whale myoglobin, in which he predicts the thermodynamic 
> hypothesis (Anfinsen’s dogma):
> 
> “How does this arise? It is scarcely conceivable that a three-dimensional 
> template forces the chain to take up this fold. More probably, the chain, 
> once synthesized and provided with a haem group around which it can coil, 
> takes up this configuration spontaneously, as the only one which satisfies 
> the stereochemical requirements of its amino acid sequence.” 
> 
> Thank you for any help you may be able to offer!
> 
>  Best regards,
> 
> Z
> 
> ***
> Zachary A. Wood, Ph.D.
> Associate Professor and Graduate Coordinator  
> Department of Biochemistry & Molecular Biology
> University of Georgia
> Life Sciences Building, Rm A426B
> 120 Green Street
> Athens, GA  30602-7229
> Office: 706-583-0304
> Lab:706-583-0303
> FAX: 706-542-1738
> ***
> 
> 
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Re: [ccp4bb] Purchasing slides with diffraction gratings for teaching diffraction

[Savvas Savvides]

Dear Pietro
the ones that I have just used in my lecture are from 
https://www.patonhawksley.com 

best wishes
Savvas

> On 5 Mar 2019, at 10:41, Roversi, Pietro (Dr.)  wrote:
> 
> Dear all,
> 
> I'd like to purchase slides with diffraction gratings for educational 
> purposes.
> 
> Ideally, to be used with a visible-light laser pointer.
> 
> And: with 1D and 2D patterns, of variable repeats - so as to be able to 
> illustrate the effect on the spacing in reciprocal space, as it were.
> 
> I am looking online but not having much joy.
> 
> Can anyone recommend a good provider to purchase from?
> 
> Thank you!
> 
> Pietro
> 
> 
> Pietro Roversi
> LISCB Wellcome Trust ISSF Fellow
> https://bit.ly/2I4Wm5Z 
> 
> Leicester Institute of Structural and Chemical Biology
> Department of Molecular and Cell Biology, University of Leicester
> Henry Wellcome Building
> Lancaster Road, Leicester, LE1 7HB
> England, United Kingdom
> 
> Tel. +44 (0)116 2297237
> 
> 
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[ccp4bb] upcoming VIB conference on Structural Biology

[Savvas Savvides]

Dear colleagues,

I am taking the liberty of bringing to your attention the VIB's bi-annual 
conference on Structural Biology, which is set to take place in the very 
accessible (and beautiful) Brussels, Belgium September 20-21st 2018.

We have put together a very exciting program covering many facets of structural 
biology.

Please take a look at:
https://vibconferences.be/event/structural-dynamics-in-cellular-communication-2nd-edition
 
<https://vibconferences.be/event/structural-dynamics-in-cellular-communication-2nd-edition>
 

The early-bird registration deadline is on August 10th.

On behalf of the organizers,
Savvas


---
Savvas Savvides
VIB Center for Inflammation Research
Dept. of Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Savvas Savvides]

Dear Partha
you do not specify which HEK293 cell line you have used, but if it so happens 
that it is the very handy HEK293S MGAT1-/- cell line (previously known as 
HEK293S GnTI-/- ) which produces N-linked Man5GlcNAc2 glycans you might want to 
consider using EndoH (e.g. see Verstraete et al. DOI: 10.1038/ncomms14937) or 
even Jack-bean alpha-mannosidase (e.g. see Bloch et al. 
https://doi.org/10.1016/j.immuni.2017.12.008 
<https://doi.org/10.1016/j.immuni.2017.12.008>).
Kifunensine, an inhibitor of N-glycosylation processing, tends to work quite 
well for protein expression in HEK293T and renders N-linked glycans digestible 
by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix et al. 
http://dx.doi.org/10.1016/j.str.2015.06.019 
<http://dx.doi.org/10.1016/j.str.2015.06.019> ; Elegheert et al. 
doi:10.1038/nsmb.2367).

If resources and protein material allow, you might also want to consider the 
permutation exercise of subjecting the complex to deglycosylation, or the 
individual components followed by complex formation/purification, or just one 
of the two components followed by complex formation/purification, or even one 
of the two components followed by further deglycosylation of the complex. We 
are becoming more and more apprehensive of the possible role of glycans in 
complex formation.
And then there is of course the option to apply mutagenesis, e.g. via N—>Q, to 
eliminate certain N-linked glycans either as a standalone approach or in 
combination with enzymatic glycan digestions as described above.

Best wishes
Savvas


---
Savvas Savvides
VIB Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>

> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar  
> wrote:
> 
> Dear All, 
> 
> I am in a situation, almost for the first time within my limited experience, 
> that deglycosylation might be necessary to obtain crystal. So, I thought of 
> tapping to vast experience of CCP4BBers, while I am searching literature. 
> 
> I have protein that has been expressed in HEK293 cells, secreted into media, 
> purified over IMAC and SEC columns. Crystallization-screens with its binding 
> partners (they form good complexes based on analytical SEC) have not produced 
> any useful hits (whereas complexes with related proteins worked well). So, I 
> plan to re-try complex formation and \crystallization screen after 
> deglysosylation. 
> 
> My question is: In practice, Does a kit (for example here: 
> https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en®ion=US 
> <https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en®ion=US>)
>  containing Endo F1, F2, F3 be sufficient or should this be tried in 
> combination with PNGase (which requires
> desaturating conditions)?!! 
> 
> Many Thanks in advance for your suggestions, and reference. 
> 
> Best Wishes,
> Partha 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] His-6 versus His-10 tag

[Savvas Savvides]

Hi Herman
the survey article by Carson in Acta D DOI: 10.1107/S0907444906052024 
 from a decade ago may provide 
relevant information/insights.
best wishes
Savvas




> On 19 Sep 2017, at 12:10, herman.schreu...@sanofi.com wrote:
> 
> Dear BB,
>  
> We are planning the production of a protein for crystallization. From 
> literature, we know that the construct with a 6-histidine tag crystallizes. 
> However, for other biophysical measurements, we would prefer to have a 
> 10-histidine tag.
>  
> Does anyone has experience with His-6 versus His-10 tags in terms of 
> crystallization success?
>  
> Thanks for your help!
> Herman

Re: [ccp4bb] CC(1/2) reference

[Savvas Savvides]

Dear Nicola
the most thorough discussion I have seen regarding recommendations on cutoffs 
(or at least ways to think about them) for such crystallographic data 
indicators can be found in:

Assessing and maximizing data quality in macromolecular crystallography
P Andrew Karplus and Kay Diederichs
Current Opinion in Structural Biology 2015, 34:60–68
http://dx.doi.org/10.1016/j.sbi.2015.07.003 

best wishes
Savvas



Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>



> On 29 Aug 2017, at 16:05, Nicola Evans  wrote:
> 
> Hello all, I have heard at several CCP4 meetings and also at Diamond training 
> that a good "cut off" for CC(1/2) is around 0.3 and I/sig(I) is 0.2, but I am 
> struggling to find any journal references to say this (other than 
> demonstrating the merits of CC(1/2) over Rmeas). 
> 
> Can anyone point me in the right direction? Thanks all!
> 
> Nicola

Re: [ccp4bb] A question about Cys and fluoro benzene ring

[Savvas Savvides]

Dear Cheng,

it is possible that the sulfhydryl of the cysteine might have reacted with the 
methyl fluoro benzyl group via nucleophilic aromatic substitution. Fluorine is 
not the best leaving group among the halides but it would certainly allow this 
especially if the ligand-protein incubation time was sufficient and if the the 
pH of the reaction is above 7. This would get the Cys-SH closer to the 
cysteinyl moiety (S:-). The pKa of the Cys-SH group is just under 8.5 and in 
your case it might even be lower depending on local structure environment, 
resulting in an even better nucleophile.

Your electron density indeed suggests that the sulfur from the Cys-SH is 
covalently bonded to the benzyl ring and this would happen at the carbon that 
originally carried the fluorine. You will have to remodel your ligand, keeping 
in mind that the fluorine will no longer be part of your ligand model. The 
methyl group will of course be retained.

It might also be useful to try to confirm the reactivity of the ligand of 
interest with the protein (reminiscent of a suicide or covalent inhibitor 
against cysteine proteases?) via some kind of an orthogonal method (e.g. mass 
spectrometry). This would even tell you if the particular cysteine in your 
structure is truly the relevant covalent target. In this way you can even 
compare washed and dissolved crystals of your protein-ligand complex with the 
complex in solution.

best wishes
Savvas


---
Savvas Savvides
VIB-UGent Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>




> On 23 Aug 2017, at 17:01, Cheng Zhang  wrote:
> 
> Hi everyone,
> 
> We recently got a structure of a receptor bound to a ligand. The ligand has a 
> fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
> receptor. The density for the ligand and the Cys seems to suggest a covalent 
> bond. However, I don't know if a covalent bond is chemically possible. Also, 
> I believe Cys is rarely involved in cation-pi interactions? Any suggestions 
> for placing the Cys and the fluoro methyl benzene ring?
> 
> Thanks! 
> 
> Cheng
> 
> 
> 
> -- 
> -
> Cheng Zhang

Re: [ccp4bb] Glycoprotein expression question

[Savvas Savvides]

Dear Bernhard
Our campaigns over the years aiming to produce mammalian cytokines and the 
ectodomains of cytokine receptors via eukaryotic expression systems (mainly in 
several HEK293 flavors) for structural biology, have taught us that the 
N-linked glycosylation issue remains a very empirical exercise. Our protein 
targets are typically 15-70 kDa and have 2-6 predicted N-linked glycosylation 
sites. For instance, we have seen that elimination of even one such site by 
mutagenesis can abrogate protein secretion. So for those cases one may even 
make an argument for a glycosylation ‘hotspot'.  Also, eliminating all possible 
N-linked glycosylation sites at once in the couple of cases tried has been 
synonymous with zero protein secretion. Our consensus of the ‘magic combo’ in 
terms of expression levels and stability is a reduction of N-linked 
glycosylation sites by up to 1/3. Such reduced levels of glycosylation also 
appear to be amenable for enzymatic glycan trimming in subsequent stages of 
sample preparation with good results.  
The article http://dx.doi.org/10.1016/j.sbi.2013.04.003 by Aricescu and Owens 
may provide some additional perspectives.
 
best wishes
Savvas


> On 11 Apr 2017, at 22:34, Bernhard Rupp  wrote:
> 
> Hi Fellows,
>  
> a humble question for our glyco-expressionists: 
>  
> I have mutated out the Asns of the N-glycoslation consensus sites for Asp 
> (Asp simply because the PNGaseF treated protein stays stable so I thought 
> that might be a good guess) 
> and indeed the unglycosilated mutant expresses well and gets secreted as 
> planned. 
>  
> But rumor has it that glycoproteins that are mutated to non-glyc often are 
> not processed correctly and 
> that we had just dumb luck. 
>  
> May I poll the educated opinion of the erudite here?
>  
> Cheers, BR
>  
> --
> Bernhard Rupp
> Crystallographiae Vindicis Militum Ordo
> http://www.hofkristallamt.org/
> b...@hofkristallamt.org
> +1 925 209 7429
> +43 767 571 0536
> --
> :(){ :|: & };:
> --

[ccp4bb] post-doctoral position at VIB-Plant Systems Biology, Belgium.

[Savvas Savvides]

Within the framework of an ERC Consolidator project, an initial 2-year 
postdoctoral position is available in the Advanced Live Cell Imaging group of 
Daniel Van Damme at The VIB/UGent Department of Plant Systems Biology to 
investigate the ultrastructure of the plant endocytic TPLATE complex (TPC).

The TPC has its evolutionary origins between the emergence of COPI and the 
functional specification of the AP complexes. While the TPC remained essential 
for plants, it was evolutionarily lost in yeasts and animal cells, indicating 
that endocytosis operates differently in plants (Gadeyne et al., Cell 2014: 
Hirst et al., Elife 2014). The project aims to generate structural and 
mechanistic insights into membrane recruitment and cargo identification of the 
TPC as well as its interplay with the evolutionarily conserved AP-2 complex. 
The project will run in close collaboration with structural biology groups 
within the VIB institute (Savvas Savvides, VIB/UGent and Rouslan Efremov, 
VIB/VUB).

The successful candidate will spearhead the structural elucidation of an 
enigmatic endocytic complex and will become part of a lively research group 
with a long track record on unravelling the role of this key endocytic complex 
in plants (Van Damme et al., Plant Journal 2004; Van Damme et al., Plant Cell 
2006; Van Damme et al PNAS 2011; Gadeyne et al., Cell 2014).

Motivated candidates with a PhD and a proven track record in structural biology 
and/or molecular biology (e.g. protein/complex purification, recombinant 
protein biochemistry, X-ray crystallography, single-particle EM etc) are 
requested to send their CV (including academic record, publications and proof 
of relevant experience), a statement of interest focusing on the project and 
the contact details of two referees (name, organization, phone, e-mail address) 
to:

daniel.vanda...@psb.vib-ugent.be <mailto:daniel.vanda...@psb.vib-ugent.be> 

Prior experience in the field of endocytosis is an asset. The succesful 
candidate can start immediately and the call will remain open until a suitable 
candidate is found. Selected candidates will be invited for an interview or 
skype meeting.



 

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Savvas Savvides]

Dear Pascal,

the EM2DAM program from the ATSAS package for SAXS 
(https://www.embl-hamburg.de/biosaxs/manuals/em2dam.html 
<https://www.embl-hamburg.de/biosaxs/manuals/em2dam.html>) can convert any EM 
envelope to a dummy-atom model in pdb format.
Also, the recent SUPALM algorithm might provide additional options as well.
http://journals.iucr.org/j/issues/2016/03/00/ap5002/ap5002.pdf 
<http://journals.iucr.org/j/issues/2016/03/00/ap5002/ap5002.pdf>


best wishes
Savvas


Savvas Savvides
Ghent University, L-ProBE
VIB Inflammation Research Center (IRC)
Technology Park 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 
<http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx>



> On 25 Oct 2016, at 10:00, Randy Read  wrote:
> 
> Dear Pascal,
> 
> I'm assuming that you're talking about using the negative stain image as an 
> MR model.  I don't recall hearing of this having ever worked (though I would 
> be very interested of course if anyone has managed to do this!), but my 
> intuition is that it's not going to work.  Negative stain just gives you an 
> external shape, whereas a cryo-EM reconstruction has internal features as 
> well.
> 
> Presumably you don't have atomic models of the individual components of the 
> complex?  If you did, using those directly for MR would be my first choice, 
> but you could also consider making a pseudo-atomic model by docking them into 
> the shape of the negative stain image.  Such a model would add substantial 
> higher-resolution information.
> 
> Best wishes and good luck,
> 
> Randy Read
> 
>> On 25 Oct 2016, at 02:49, Pascal Egea > <mailto:pas...@msg.ucsf.edu>> wrote:
>> 
>> Dear All,
>> 
>> I would like to know if it is possible to use a low resolution EM 
>> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
>> help molecular replacement in a 4.5A resolution X-ray diffraction data set 
>> of the same complex
>> I am aware of the possibility of using low resolution cryoEM maps for MR as 
>> described in the review from Jackson et al in Nature Protocols but I was 
>> wondering if there is an intrinsically impossibility for negative stain 
>> reconstructions.
>> 
>> Any thoughts or advice will be greatly appreciated.
>> 
>> Best,
>> 
>> -- 
>> Pascal F. Egea, PhD
>> Assistant Professor
>> UCLA, David Geffen School of Medicine
>> Department of Biological Chemistry
>> Boyer Hall room 356
>> 611 Charles E Young Drive East
>> Los Angeles CA 90095
>> office (310)-983-3515
>> lab  (310)-983-3516
>> email pegea at mednet.ucla.edu <http://mednet.ucla.edu/>
> 
> --
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research  Tel: + 44 1223 336500
> Wellcome Trust/MRC Building   Fax: + 44 1223 336827
> Hills RoadE-mail: rj...@cam.ac.uk 
> <mailto:rj...@cam.ac.uk>
> Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk 
> <http://www-structmed.cimr.cam.ac.uk/>

Re: [ccp4bb] additional density on cysteine residue

[Savvas Savvides]

Dear Sreetama,

I would consider the possibility that this active site cysteine is involved in 
a mixed-disulfide with beta-mercaptoethanol, which is present at a considerable 
concentration in your protein buffer. 
The fact that the residual density in both the Fo-Fc and 2Fo-Fc maps actually 
increased beyond the modeled S-OH group after refinement and the features 
thereof, provide evidence for the likelihood of a mixed-disulfide with betaME.

best wishes
Savvas
 

Savvas Savvides
Unit for Structural Biology, L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html 
<http://www.lprobe.ugent.be/xray.html>




> On 17 Jan 2015, at 19:22, sreetama das  wrote:
> 
> Dear Users,
> 
> I am solving a structure from x-ray diffraction data (1.62A resolution).
> 
> The protein has a single cysteine residue (which is also the catalytic 
> residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). 
> The positive density is retained upto 11.5 sigma level.
> 
> Modelling with water retains the positive density (fig 2; R/Rfree = 
> 16.85/19.94) upto 5.2 sigma level.
> 
> Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) 
> produces partial positive and negative densities, which are retained upto 5 
> sigma. Moreover, after real-space refinement in coot followed by refinement 
> in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor 
> is its C-terminus bonded to the succedding residue.
> 
> All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map).
> The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and 
> beta-mercaptoethanol (2mM), while the crystallization condition contained 
> citric acid (pH 3.5) and ammonium sulfate.
> 
> Please suggest how to interpret the data.
> 
> thanking in advance,
> sreetama
> 

Re: [ccp4bb] Space group problem?

[Savvas Savvides]

Dear Chen,

how sure are you that your crystals contain the protein of interest? Repeatedly 
washing harversted crystals in crystal stabilization solution followed by 
SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver 
staining) or western blotting can give a pretty conclusive answer.
In our group, SDS-PAGE followed by Silver-staining is done routinely and in 
most cases leads to conclusive results. Here is a summary of the protocol:


- select a drop containing substantial crystalline material. The crystals can 
be many and small (crystal shower) or few and large.
- prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of 
motherliquor containing a 10% higher concentration of precipitant).
- transfer all the crystalline material from the drop into the PCR-tube using a 
pipet (You can use stabilization buffer from the PCR tube to collect all 
crystals). One can also use a cryo-loop to harvest the crystals if they are 
large enough to allow efficient harvesting.
- centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals 
under the microscope to make sure they are at the bottom of the PCR-tube.
- remove as much of the supernatant as you can using a pipet making sure not to 
remove the crystals. Then add crystal stabilization buffer to wash the 
crystals, and centrifuge again.
- repeat this washing procedure a few times (typicaly 3-4 times).
- after the final washing step, centrifugation and removal of the supernatant, 
add Laemmli-buffer to the crystals and use this sample for loading onto the 
SDS-PAGE gel.
- include a positive control (e.g. solubilize another drop directly in 
Laemmli-buffer) and a negative control (final washing buffer). Including a 
pre-crystallization sample of your protein as a control is also recommended, to 
control for the integrity of the protein under crystallization conditions.
- use silver staining to visualize the protein.
---


best regards
Savvas



On 31 Mar 2014, at 23:48, Chen Zhao  wrote:

> BTW, I forgot to mention that phenix.autosol also gave similar result.
> 
> 
> On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao  wrote:
> Dear all,
> 
> I am now trying to phase a structure in C2 using anomalous scattering at 5-6 
> A. It is hard to improve the derivative resolution at the moment. Shelxd is 
> able to locate 6 sites with a distinct CC and FOM. After density modification 
> in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 
> 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and 
> MAD are similar, and the solvent boundary is quite clear. However, the 
> problem is that the electron density blob passes through the 2-fold rotation 
> axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small 
> for the molecule. I am afraid that the space group assignment is wrong, but I 
> am a beginner so I nearly have no clue. I did reprocess the data in P1 and 
> looked at the self-rotation function with a radius at 200 A. From the list it 
> seems that there is only one 2-fold rotation axis. I am quite confused. Could 
> anybody give me some hint of this problem?
> 
> Thanks a lot in advance!
> 
> Sincerely,
> Chen
> 

[ccp4bb] university-funded research position in structural biology at Ghent University, Belgium

[Savvas Savvides]

Dear colleagues,

I have taken the liberty of drawing your attention to a university-funded 
post-doctoral position in structural biology that is currently available at the 
Unit for Structural Biology, Department of Biochemistry and Microbiology, Ghent 
University (Belgium).

The succesful candidate will be appointed for an initial period of 3 years and 
is expected to start by September 1st, 2014 the latest.  The appointment can be 
renewed for an additional term of 3 years upon a positive evaluation.

Candidate profile
·  Doctoral degree in Biochemistry, Biophysics, or related disciplines 
(this academic credential should be attained by the start of the appointment).
·  Research experience in one or several of the following methodological 
approaches in structural biology in the context of interdisciplinary protein 
research:
o   Macromolecular X-ray Crystallography
o   Small-angle X-ray Scattering
o   Electron Microscopy
·  Experience in biophysical methods for the study of protein-protein and 
protein-ligand interactions.
·  Broad practical experience in molecular biology, protein biochemistry, 
and tissue culture for the production and purificationof recombinant 
glycosylated proteins and complexes thereof towards structural and biophysical 
studies.
·  Publication record in international journals that subscribe to the 
peer-review process.
·  Experience and strong interest in the supervision of BA- and MA-level 
students.

Function
·  Interdisciplinary research (70% of the time) in structural biology.
·  Supervision of students carrying out MA-thesis work.
·  Part-time teaching activities in biochemistry-related courses at the BA- 
and MA-level.

Application instructions
Deadline 30/04/2014. 
Reference number: 2014-03-54
Applications should include a cover letter (addressed to the Chairman of the 
Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, 
Belgium), CV and copy of the required academic transcripts, and should be 
submitted as a single PDF by e-mail to recruitment...@ugent.be with cc to 
savvas.savvi...@ugent.be .
The candidate will receive an e-mail confirming receipt of the application.

[ccp4bb]

[Savvas Savvides]

Dear Yarrow
your toroidal structure suggests that the protein may actually have the 
propensity to assemble as such in solution, hinting a connection to a 
biologically relevant state.  Do you have any experimental information that it 
does so? e.g. via SAXS, MALS, native PAGE etc.? 

best regards
Savvas


Savvas Savvides
Unit for Structural Biology, L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html



On 19 Mar 2014, at 16:58, Yarrow Madrona  wrote:

> Thank you to everyone for their input. I am posting a picture to some of the 
> symmetry related molecules shortly. There are six dimers related by symmetry 
> (60 degrees) with a "donut" hole in the middle. This was troubling to me as I 
> have solved mostly tighter packing structures (monoclinic or orthorhombic) in 
> the past. If expanded further there are a bunch of tightly packed donut holes 
> (though I didn't show these).
> 
> I want to know if this is really a viable solution. The crystals are huge 
> (300microns X 300microns) and this would maybe explain why they are only 
> diffracting to 3.2 angstroms. Thank you!
> 
> https://www.dropbox.com/s/r01u37owbkz9pon/donut.png
> 
> -Yarrow
>  
> 
> 
> On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona  wrote:
> Yes in the first couple of rounds of refinement it refines very well for a 
> 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs 
> contiguously except for a "donut" hole in between six dimers that are related 
> by symmetry. Trying to put a molecule there disrupts the symmetry and leads 
> to clashes. I have a synchrotron trip next week, hopefully this should help 
> clear things up a bit.
> 
> 
> On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson  
> wrote:
> I think you have solved it! That is an excellent LLG and if you can't see 
> anything else in the map, then there s prob. not another molecule. 
> Does it refine? If you look at the maps following refinement any missing 
> features should become more obvious.
> Solvent content of 65% is not uncommon.
> Eleanor 
> 
> 
> On 19 Mar 2014, at 03:46, Yarrow Madrona wrote:
> 
>> Hello CCP4 Users,
>> 
>> I recently collected data in-house on an Raxis IV and am trying to solve a 
>> 3.2 angstrom structure.
>> I have obtained only "partial solutions" using Phaser and would like some 
>> help. I believe I only have two molecules in the ASU instead of three as 
>> suggested by the mathew's calculation. I believe I have two molecules in the 
>> ASU with a space group of P312 despite a high solvent content. I have 
>> outlined by line of reasoning below.
>> 
>> 1. Indexes as primitive hexagonal
>> 
>> 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm 
>> assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis 
>> in the P312 space group. See this link, 
>> https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf
>> 
>> 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. 
>> Phaser gives solutions for only 4 molecules.
>> 
>> 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. 
>> Phaser gives solutions for only 2 molecules.
>> Mathews calculation for data scaled in P312:
>> 
>> For estimated molecular weight   44000.
>> 
>> Nmol/asym  Matthews Coeff  %solvent   P(3.20) P(tot)
>> 
>> 
>> 
>>   1 6.8482.03 0.00 0.00
>> 
>>   2 3.4264.07 0.18 0.13
>> 
>>   3 2.2846.10 0.81 0.86
>> 
>>   4 1.7128.13 0.01 0.01
>> 
>>   5 1.3710.17 0.00 0.00
>> 
>> 
>> 
>> Phaser Stats:
>> 
>> 
>> Partial Solution for data scaled in P312:
>> 
>> RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 
>> TFZ==30.3
>> 
>> 6. No peaks in patterson map (No translational symmetry).
>> 
>> 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 
>> bromo-phenyl Immidazole) despite not including them in the search model.
>> 6. There is only one "black hole" where it would be possible place another 
>> subunit but there is not much interpretable density and the symmetry of the 
>> space group would be bro

Re: [ccp4bb] N-linked glycans mediate inter-molecular protein-protein interactions

[Savvas Savvides]

Dear Tianyu,
I have found the recent review by:
Nagae and Yamaguchi in Int. J. Mol. Sci. 2012, 13, 8398-8429; 
doi:10.3390/ijms13078398 quite a good starting point.

best
Savvas




On 05 Feb 2014, at 19:53, Gmail  wrote:

> Dear colleagues, I am looking for structures of protein complexes in which 
> N-linked glycans mediate inter-molecular protein-protein interactions. Can 
> anyone point me in the right direction? Thanks!
>  
> Tianyu
>  
> Gmail via foxmail

Re: [ccp4bb] Puzzling Structure

[Savvas Savvides]

i meant to say P21212

On 12 Apr 2013, at 21:47, Savvas Savvides  wrote:

> The CRYST1 in the pdb header is problematic.
> CRYST1   95.520   47.815   88.570  90.00  90.00  90.00 P 21 21 218 
> it looks like the number '1' was paired to the space group rather than the 
> space grop number (i.e. nr. 18 for P21212)
> Table 1 in the paper specifies P212121 as the space group.
> 
> 
> 
> 
> On 12 Apr 2013, at 21:14, Michel Fodje  wrote:
> 
>> Has anyone else noticed a problem with the structure  of the N-terminal 
>> capsid domain of HIV-2  PDB 2wlv.
>> Load it up to in coot and navigate to residue B118.
>>  
>> /Michel.
> 

Re: [ccp4bb] Puzzling Structure

[Savvas Savvides]

The CRYST1 in the pdb header is problematic.
CRYST1   95.520   47.815   88.570  90.00  90.00  90.00 P 21 21 218 
it looks like the number '1' was paired to the space group rather than the 
space grop number (i.e. nr. 18 for P21212)
Table 1 in the paper specifies P212121 as the space group.




On 12 Apr 2013, at 21:14, Michel Fodje  wrote:

> Has anyone else noticed a problem with the structure  of the N-terminal 
> capsid domain of HIV-2  PDB 2wlv.
> Load it up to in coot and navigate to residue B118.
>  
> /Michel.

Re: [ccp4bb] refinement protein structure

[Savvas Savvides]

Dear Tom
in addition to the very valuable input you have received already, I would like 
to point out that based on the fact that the  of your data is above 
3 in the highest resolution shell, you will likely be able to push the 
resolution limits of your analysis to go beyond 1.4 angs resolution.  I would 
definitely enoucrage you to do so even if Rmeas values end up being higher than 
what common practice and rules of thumb may prescribe. 
Recent work by Karplus and Diederichs (Science 336, 1030 (2012)) and an 
accompanying editorial by Phil Evans (Science 336, 986 (2012)) will provide the 
necessary food for thought to proceed.

Very best wishes
Savvas


Savvas Savvides
Unit for Structural Biology, L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype
http://www.LProBE.ugent.be/xray.html



On 27 Mar 2013, at 17:22, Tom Van den Bergh 
 wrote:

> Dear members of ccp4bb,
> 
> I need some help with the refinement of my structure of a variant of mRFP 
> (monomer red fluorescent protein, sequence in attachment). I have done 
> molecular replacement with phaser with model 2VAD of protein database. Then i 
> have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
> flags and log file attached) When i refine with phenix.refine my structure i 
> get a R-value of 0,42 which is still way too high. (redfluorescent 
> protein.pdb, .mtz and logfile attached) When i look at the structure in coot 
> i find many unmodelled blobs and many outliers in density analysis and 
> rotamer analysis. The problem is that there are so many problems with my 
> structure, that i dont know where to begin. Could you try some refinement for 
> me, because this is first structure that i need to solve as a student and i 
> dont have too many experience with it.
> 
> Greetings,
> 
> Tom
> 
> 

Re: [ccp4bb] FW: [ccp4bb] [ccp4bb] disulfide engineering

[Savvas Savvides]

We have had some good luck with hydrogen peroxide [for technical details plus 
validation via a crystal structure see Van der Meeren et al JBC  
288(2):1214-1225, (2013)].  
best of luck
Savvas


On 28 Feb 2013, at 12:14, "McEwan, Paul"  wrote:

> Rather than looking at “anti-DTT” it is more important to set up an 
> appropriate redox system. This can be a combination of reduced and oxidised 
> glutathione  or cysteine. If you check some of the commercial protein 
> refolding screens this should give you an idea about relative concentrations.
>  
> Best regards,
> Paul..
>  
> -
> Dr. Paul A. McEwan
> Senior Scientist (Structural Biology)
> Evotec (UK) Ltd.
> 114 Innovation Drive | Milton Park | Abingdon | Oxfordshire | OX14 4SA
>  
> email: paul.mce...@evotec.com
> Tel: +44 (0)1235 861561
> Fax:+44 (0)1235 863139
> direct line: +44 (0)1235 838802
> www.evotec.com
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob 
> Keller
> Sent: 28 February 2013 11:09
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] disulfide engineering
>  
> Along these lines, what reagents do people use to promote disuflide bonds, 
> i.e., the "anti-DTT?"
>  
> JPK
> 
> On Thu, Feb 28, 2013 at 2:06 AM, David Briggs  
> wrote:
> You might want to try "Disulfide by design"
> 
> http://cptweb.cpt.wayne.edu/DbD2/
> 
> Cheers
> 
> Dave
> 
> On Feb 28, 2013 6:55 AM, "Careina Edgooms"  wrote:
> Dear CCP4 members
>  
> I wish to engineer a disulfide bond at the dimer interface of a protein I am 
> working with. Does anyone know of any available software to assist with this?
>  
> Best
> Careina
> 
> 
>  
> -- 
> ***
> Jacob Pearson Keller, PhD
> Postdoctoral Associate
> HHMI Janelia Farms Research Campus
> email: j-kell...@northwestern.edu
> ***
> Evotec (UK) Ltd is a limited company registered in England and Wales.  
> Registration number:2674265.  Registered Office:  114 Milton Park, Abingdon, 
> Oxfordshire, OX14 4SA, United Kingdom
> 

Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!

[Savvas Savvides]

Hi Jacob,

check out Figure 1 in

Natively inhibited Trypanosoma brucei cathepsin B structure determined by using 
an X-ray laser.
Redecke L, et al.
Science. 2013 Jan 11;339(6116):227-30.

and 

In vivo protein crystallization opens new routes in structural biology.
Koopmann R. Nature Methods. 2012 Jan 29;9(3):259-62.

best
Savvas 




On 15 Feb 2013, at 20:44, Jacob Keller wrote:

> Dear Crystallographers,
> 
> I was looking at some live, control HEK cells expressing just eGFP, and to my 
> great surprise, saw littered across the dish what appeared to be small 
> fluorescent needles (see attached--sorry about the size, but it's only ~1MB 
> total.) Can these possibly be fortuitous protein crystals? They were too 
> small to mount I think, and for what it's worth, parallel-transfected HeLa 
> cells did not have these things. But, some needles could be seen in the DIC 
> images as well, and the needles were only fluorescent with GFP filter sets, 
> and not CFP, YFP, or texas red filters. I thought of whale myoglobin 
> crystallizing on the decks of ships, but never thought I would see this
> 
> Jacob
> 
> -- 
> ***
> Jacob Pearson Keller, PhD
> Postdoctoral Associate
> HHMI Janelia Farms Research Campus
> email: j-kell...@northwestern.edu
> ***
> 

Re: [ccp4bb] S-nitrosylation protein

[Savvas Savvides]

Dear Uma,

A very complehensive survey of the state-of-the-art in this topic can be found 
in the following literature resources:

(1) 
Garman EF.
Radiation damage in macromolecular crystallography: what is it and why should 
we care?
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):339-51.

(2) 
The January 2013 issue of the Journal of Synchrotron Radiation featuring a 
special section with papers presented at last year's Intl workshop on X-ray 
damage of biological crystalline samples.
The annotated editorial by Garman and Weik in that issue is also very helpful.

Best regards
Savvas


On 13 Feb 2013, at 23:38, Uma Ratu wrote:

> Dear All:
>  
> I plan to use X-ray crystallography method to study the S-nitrosylated 
> protein structure.
>  
> The native protein crystals diffracted to 2A with synchrontron. I now have 
> the crystals of S-ntrosylated protein.
>  
> Since S-NO moiety appears to be unstable to synchrotron radiation, could you 
> advice /  comments on the stratage on the data collection of S-nitrosylated 
> protein crystals?
>  
> The protein crystals did not diffract well with in house X-ray.
>  
> Thank you for your comments.
>  
> Uma
> 

Re: [ccp4bb] a challenge

[Savvas Savvides]

Dear James

> I actually chose 3dko because it is a kinase (with a ligand), and
> therefore an interesting candidate for a molecular replacement
> "score".  I have not set this up yet, but I think if you look for PDB
> entries that contain the word "kinase" and try to molecular-replace
> all of them into the 3dko dataset, what fraction of them will "work"?
> I think that fraction would make a good "score" for a given molecular
> replacement pipeline.

At the recent CCP4 SW in Nottingham Giovanna Scapin from Merck gave a talk on 
MR during which she reflected upon their attempts from some time ago to 
troubleshoot a recalcitrant MR case of a kinase by searching with hunderds of 
models derived from all kinase structures known at that time. However, I am not 
quite sure if they published these results anywhere (at least I could not fish 
out a relevant reference).

Along these lines, 'Wide Search MR' (Stokes-Rees and Sliz (2010) PNAS 107: 
21476-21481) and (www.sbgrid.org) may also provide some options to  establish 
such benchmarking or MR 'scores'.

Best regards
Savvas

> 
> 
> On Mon, Jan 14, 2013 at 2:31 PM, Nat Echols  
> wrote:
>> On Mon, Jan 14, 2013 at 11:18 AM, Tim Gruene  
>> wrote:
>>> I admit not having read all contributions to this thread. I understand
>>> the "John Henry Challenge" as whether there is an 'automated way of
>>> producing a model from impossible.mtz'. From looking at it and without
>>> having gone all the way to a PDB-file my feeling is one could without
>>> too much effort from the baton mode in e.g. coot.
>> 
>> This should be even more possible if one also uses existing knowledge
>> about the expected structure of the protein: a kinase domain is quite
>> distinctive.  So, James, how much external information from homologous
>> structures are we allowed to use?  Running Phaser would certainly be
>> cheating, but if I take (for instance) a 25% identical kinase
>> structure, manually align it to the map and/or a partial model, and
>> use that as a guide to manually rebuild the target model, does that
>> meet the terms of the challenge?
>> 
>> -Nat

Re: [ccp4bb] estimate of effective concentration

[Savvas Savvides]

Dear Filip,

I believe that you may find interesting methods, insights and principles to get 
you going in the following paper:

Wu et al.
Transforming binding affinities from three dimensions to two with application 
to cadherin clustering.
Nature. 2011 Jul 27;475(7357):510-3
doi: 10.1038/nature10183

best regards
Savvas


Savvas Savvides
Unit for Structural Biology, L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype
http://www.LProBE.ugent.be/xray.html








On 21 Jun 2012, at 01:08, Filip Van Petegem wrote:

> Dear crystallographers,
> 
> I have a question concerning effective concentration. Say you have a crystal 
> structure whereby two loops, each part of a different domain but within the 
> same molecule happen to be juxtaposed and can form an interaction.  The loops 
> have some degree of flexibility, but are ordered when interacting. The 
> domains on which they are attached have a rigid configuration due to the 
> remainder of the structure. The interaction is potentially very weak and 
> mainly driven by the fact that the effective concentration is extremely high. 
> 
> The question: how can one obtain a rough estimate of the effective 
> concentration of these two juxtaposed loops?   The simple straightforward 
> answer would be to just divide number (1 each) by volume (some box drawn 
> around the loops), and convert this to molar. That's easy. However, this is 
> over-simplified and really an underestimate of 'effective' concentration, 
> because these loops cannot rotate freely when attached to the domains.  
> Hence, there are constraints that allow them to interact more readily 
> compared to the isolated loops within the same box. So I'm looking for a 
> model that also takes limited conformational freedom into account.
> 
> If anybody has any pointers to some reference text or paper that has 
> performed such an analysis, I would be very interested.
> 
> Regards,
> 
> Filip
> 
> -- 
> Filip Van Petegem, PhD
> Associate Professor
> The University of British Columbia
> Dept. of Biochemistry and Molecular Biology
> 2350 Health Sciences Mall - Rm 2.356
> Vancouver, V6T 1Z3
> 
> phone: +1 604 827 4267
> email: filip.vanpete...@gmail.com
> http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] help regarding structure solution

[Savvas Savvides]

Dear Sonali
have you run any diagnostics on your dataset e.g. via xtriage in PHENIX, or the 
ccp4 programs to detect issues such as twinning and pseudotranslation. 
You also did not provide any information regarding the data quality. Processing 
your dataset via Xia2 from the ccp4 suite could also provide an important 
(additional) dataset variant to work with. 

Best regards
Savvas

On 20 Jun 2012, at 20:13, sonali dhindwal  wrote:

> Dear All,
> 
> I am working on a protein for last so many years and for which i have got 
> crystal now in a tray which i kept 1 years ago. It diffracts well and 
> resolution is 2.2A, which is good. 
> 
> I indexed in HKL2000, mosflm and automar and it shows P21 space group in all 
> data reduction packages. But when I tried using molrep or phaser then I do 
> not get any solution. The sequence of my protein is having  46% identity with 
> other available crystal structure.
> Also when I tried to get matthews coffecient, it calculates its molecular 
> mass less ( about 35 kDa) than which should be (original 54kDa) with solvent 
> content 47%.
> 
> I have also run the silver staining gel of the protein which contained 
> crystal that shows about 45 kD protein band which is 10 less than the 
> original.  Also I tried to run gel on crystal but it did not give anything as 
> it was a small crystal. 
> 
> I have tried all combinations of the search model and tried to break 
> available pdb many ways to make different search models but have not got any 
> good solution. Molrep gives contrast even 10 or more but no good electron 
> density map yet. Free R and figure of merit becomes 52% and 42% respectively 
> in Refmac with all the solutions.  
> 
> I will highly appreciate all the suggestions for this kind of problem.
> 
> Thanks and regards
> 
> -- 
> Sonali

Re: [ccp4bb] Do my SAXS data agree with the crystal structure?

[Savvas Savvides]

It might also be useful to go through the  recent paper by Jacques et al 
Acta cryst D68:620-6 (2012) towards the standardization of saxs data analysis, 
diagnostics and reporting. 

Best regards,
Savvas

On 17-jun.-2012, at 13:01, David Briggs  wrote:

> Dear Xun,
> 
> Regarding your monomer vs dimer, theoretical vs observed crysol plots
> - yes - they are significantly different.
> 
> If you focus at the very lowest q part of the curve - the deviation
> there in your monomer plots indicate that there is a significant size
> difference between your PX monomer and your SAXS data - the PX dimer
> is a much better fit at low q.
> 
> This should be enough to demonstrate to a reviewer that the dimer you
> see in PX is also present in solution.
> 
> Other experiments that could support this are SEC-MALLS or perhaps AUC.
> 
> HTH,
> 
> Dave
> 
> David C. Briggs PhD
> Father, Structural Biologist and Sceptic
> 
> University of Manchester E-mail:
> david.c.bri...@manchester.ac.uk
> 
> Webs : http://flavors.me/xtaldave
> Twitter: @xtaldave
> Skype: DocDCB
> 
> 
> 
> On 17 June 2012 06:11, Xun Lu  wrote:
>> Drs.Caldwell, Briggs, and Gupta,
>> 
>>   Thank you very much for the advices.   I regret that I didn't show any
>> figure in the earlier post.  Here I've attached a figure showing the data
>> quality and some fittings.
>>   Data look OK, right? This question may sound silly, but I just want to
>> make sure.
>>   As I said in the earlier post, I tried Crysol.  I used the crystal
>> structure (dimer+DNA) as the model, and the fitting was OK, right?  In fact,
>> I also tried monomer+DNA as the model (I simply deleted one monomer from the
>> PDB file).  This kind of comparison may be meaningless, but I was just
>> curious.  I am wondering how people judge whether the fit is good or not.
>> 
>> 
>>Another question, I tried to generate an envelope from SAXS data using
>> Gasbor and Dammin (people say Dammin is better at protein-DNA complex,
>> although it still uses the same bead for both DNA and protein?).  The
>> generated envelope was nothing like my crystal structure.  As people have
>> pointed out, protein and DNA scatter differently.  SANS is the way to go.
>> So I should give up on modeling SAXS data?  I've almost given up, because
>> anyways I have the crystal structure, and SAXS is only a small part of this
>> paper.
>> 
>> 
>> 
>> Thanks,
>> 
>> Xun
>> 
>> 
>> 
>> On Sat, Jun 16, 2012 at 6:36 PM, Kushol Gupta 
>> wrote:
>>> 
>>> Two cents -
>>> 
>>> 
>>> 
>>> A good deal of caution must be exercised when working with composite
>>> particles such as a protein-DNA complex in SAXS because of the contrast
>>> problem.  Simply, protein and DNA scatter differently in x-rays, with a bias
>>> towards the DNA component.  As a result, experimental Rgs could be slightly
>>> deflated versus what their true values would be at infinite contrast.  Mass
>>> estimation by I(0) analysis with a protein standard of known mass and
>>> concentration is not really valid because the contrast terms are different.
>>> Because the particle is heterogeneous in composition and distribution, shape
>>> reconstruction from SAXS alone, which assumes homogeneity, can also be
>>> misleading (although in practice it is still reasonably instructive).  It is
>>> for these reasons that SANS and the contrast variation approach can be
>>> extremely useful.
>>> 
>>> 
>>> 
>>> With those caveats, the strategy you describe - comparison of experimental
>>> and theoretical profiles from an experimental structure using CRYSOL or FoxS
>>> is definitely the best way to go in the case of a protein-DNA complex with
>>> SAXS alone.  Showing comparisons of the experimental with the calculated
>>> should make the point.  Test other possible models inferred from lattice
>>> packing to further your point (if applicable).
>>> 
>>> 
>>> 
>>> Regarding populations of monomer and dimer -
>>> 
>>> 
>>> 
>>> · it is generally good to constrain your interpretation of
>>> scattering data with other orthogonal solution measures which demonstrates
>>> the homogeneity of your complex in comparable experimental conditions, such
>>> as sedimentation velocity or gel filtration.
>>> 
>>> 
>>> 
>>> · Have some determination of affinity of the complex in the same
>>> solution conditions (including temperature!).  This will allow you to argue
>>> that your sample concentrations are well in excess of any monomer-dimer
>>> association behavior (eg, mixtures!).  Scattering of mixtures can undermine
>>> your ability to accurately assess the structural properties of your complex.
>>> 
>>> 
>>> 
>>> · Collect a concentration series and extrapolate to infinite
>>> dilution, if possible, to ensure elimination of the S(q) term from your
>>> data.  Interparticle interactions can be an issue with complexes containing
>>> DNA if the buffers aren’t qui

Re: [ccp4bb] CSX

[Savvas Savvides]

Dear Uma
Your modeling of a Cysteine sulfenic group (SOH) is reasonable given the 
observed difference density but do keep in mind that sulfenic groups are very 
susceptible to further oxidation to sulfinic and sulfonic acids. 
Stabilization of sulfenic and sulfinic groups in enzyme active sites is 
possible as shown crystallographically by Becker et al Nat Struct Biol 
5:267-271 (1998). 
Also make sure that you flip the Histidine in the active site that faces the 
SOH group to account for a possible hydrogen bond. 

Best regards
Savvas



On 04 May 2012, at 20:24, Uma Ratu  wrote:

> Dear All:
>  
> Thank you very much for your advices and comments.
>  
> Following your instructions, I am able to change the CYS to CSX.
>  
> Both "Get Monomer" and "Replace Residue" work well.
>  
> I have the refined conformation CSX (by "real space refinment" ) attached 
> (named as M-CSX-1).
>  
> The Fo-Fc map is shown with sigma @2.0.
>  
> Is this conformation reasonable? Why there are two bond conformation there?
>  
> Thank you for comments.
>  
> Uma
> 
> On Fri, May 4, 2012 at 12:10 PM, Hugo Correia  
> wrote:
> Dear Uma,
> 
> You can do this using coot. Go to Extensions > Modelling > Replace Residue... 
> and enter the three letter code.
> 
> Cheers
> 
> Hugo Correia
> 
> 
> 2012/5/4 Uma Ratu 
> Dear All:
>  
> My protein has a key cysteine residue involved in catalytic activity.
>  
> The template structure used for the modeling has the same key cysteine. In 
> the template structure, this key cysteine residue is assigned as CSX based on 
> the observation from its electronic density.
>  
> I compared the electron density from the template as well as my model. I 
> can't tell if the cysteine in my model is oxidized or not. The ones from the 
> template also looks different from each other, although both assigned as CSX.
>  
> I have the snapshots of these cysteines attached. The ones from my model 
> named as "M-", and the ones from the template named as "T-".
>  
> Plus, how to change the residue label from Cys to CSX if the cystein is 
> oxidized? In coot, I could not find such function.
>  
> Thank you very much for your advice
>  
> Uma
> 
> 
> 

Re: [ccp4bb] Suggestions for solving a structure with 8-10 copies per asymmetric unit

[Savvas Savvides]

Dear Jiyuan
have you run any data diagnostics on your dataset? Ccp4 offers quite some 
options (truncate, detwin etc), and xtriage from PHENIX is also very powerful. 
And how sure are you of your laue group symmetry and space group (pointless via 
ccp4 and xtriage via PHENIX can be very helpful)? Also, is your data strongly 
anisotropic?
Furthermore, the presence of pseudotranslation may not be an unlikely scenario 
given your large unit cell and large number of copies in the asu. MolRep in 
ccp4 can deal well with that, and is worth trying anyway regardless of 
pseudotranslation issues.  Also I could not help notice that your c axis is 
b+b/2.

best regards
Savvas
 
On 30 Apr 2012, at 17:41, Ke, Jiyuan wrote:

> Dear All,
>  
> I have a question regarding solving a crystal structure by molecular 
> replacement. It is a single protein with a molecular weight of 25.5 kDa. The 
> cell dimension is rather big from the diffraction data ( 90.9 Å, 143.9 Å, 
> 216.3Å, 90°, 90°,  90°). The possible space group is P212121. With such a big 
> unit cell, we predicted that there are 8-10 molecules per asymmetric unit. We 
> have a decent model with sequence similarity of 49%. I tried several times 
> with Phaser search with the current model and had difficulty to find any 
> clear solution. Has anyone seen such cases and any suggestions to solve the 
> structure? Thanks!
>  
> Jiyuan Ke, Ph.D.
> Research Scientist
> Van Andel Research Institute
> 333 Bostwick Ave NE
> Grand Rapids, MI 49503
>  

Re: [ccp4bb] Aggregated protein for crystallization

[Savvas Savvides]

Dear Raji
Running a blue-native gel with lanes in the presence and absence of a reducing 
agent could prove quite informative. DLS could also return a quick result on 
the particle distribution in your sample. In that case I would measure samples 
as fractionated from the superdex200 and compare the measurements after 
centrifuging the same samples at 100k x g for one hour. 

Best regards
Savvas

On 22 Feb 2012, at 00:21, Raji Edayathumangalam  wrote:

> Hi Folks,
> 
> As crazy as it sounds, if you have crystallized and managed to solve the 
> structure of a protein from aggregated protein, please could you share your 
> experience.
> 
> After many constructs, many many expression schemes and after the usual 
> rigmarole of optimization that is also often discussed on ccp4bb (buffers, 
> glycerol, salt concentrations, pH, detergent, additives etc.), I now have a 
> decently expressing truncated construct for my protein (80 kDa) that is pure 
> but aggregated (elutes in the void volume from a Superdex200 column). I am 
> tempted to make a boatload of aggregated protein and set up some crystal 
> trays (after perhaps testing by CD). So I'd like to hear from folks who have 
> been successful in solving structures from aggregates when many many known 
> and tested optimization methods still leave one with aggregated protein.
> 
> Thanks.
> Raji
> 
> -- 
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
> 
> 

Re: [ccp4bb] mutagenesis based on ligand-bound crystal structures

[Savvas Savvides]

Dear Chris
A whole series of studies from the early to late 90's did his for the growth 
hormone-GHR interaction. 
Papers by Pearce, Cunningham, Clackson and Wells come to mind. 
Also a recent study on the IL13-IL13R interaction by Lupardus et al has covered 
most of the interaction epitope. 

Best regards
Savvas

On 07 Feb 2012, at 10:16, Chris Ulens  wrote:

> Dear colleagues,
> I am looking for a few example studies in which a complete mutagenesis of all 
> residue-ligand interactions has been conducted to evaluate the energetic 
> contributions of each individual interaction.
> Thank you.
> Best regards.
> -Chris
> 
> ---
> Chris Ulens, Ph.D.
> Lab of Structural Neurobiology
> Department of Cellular and Molecular Medicine
> Campus Gasthuisberg, ON1
> Herestraat 49, PB 601
> B-3000 Leuven
> Belgium
> e   chris.ul...@med.kuleuven.be
> t+32 16 330689
> f+32 16 345699
> w   http://www.xtal.be

Re: [ccp4bb] Freezing crystal

[Savvas Savvides]

Dear Theresa
Cryo-cooling the crystals straight out of their drops or after brief 
incubations in crystal stabilization solutions containing >2M ammonium sulfate 
may be the way to go.
Here is a copy/paste piece from Kyndt et al (2007) Biochemistry 46, 95-105.
All the best
Savvas

Aliquots (0.5 í L) of the seed suspension (diluted 1:100 in stabilization 
buffer) were
introduced into a series of fresh hanging drops (containing
4 í L of protein sample and 4 í L of reservoir solution) that
had been equilibrated for 24 h over reservoirs containing 3
M ammonium sulfate and 20 mM sodium phosphate, pH
5.4- 6.2. Single crystals with bipyramidal morphology grew
after 1 week to a final size of 0.150 mm   0.150 mm
 0.100 mm.
To prepare crystals for data collection under cryogenic
conditions (100 K), crystals were flash-cooled by plunging
them directly from their native drops into liquid nitrogen. A
series of cryocooling conditions using a variety of cryoprotecting
reagents such as glycerol, sucrose, PEG 400, and
paratone indicated that only crystals flash-cooled by plunging
them directly from their native drops into liquid nitrogen
produced diffraction of acceptable quality.



On 05 Feb 2012, at 23:49, Theresa H. Hsu wrote:

> Hi all
> 
> Is there a list of conditions to be tried *first* for cryoprotectant? My 
> crystals diffract at room temperature capillary but no in 30% PEG 400. 
> Crystals are from 2 M ammonium sulfate.
> 
> Thank you.
> 
> Theresa

Re: [ccp4bb] model building at 3.2 A

[Savvas Savvides]

Dear Kumar
Could you please provide some information on your refinement protocol/progress 
and quality of your partial model thus far. Also, what is the quality and 
completeness of your data, in particular in the lowest resolution to 10 angs 
range.   In this way one might be able to provide more concrete feedback. 
Experience from several structures around such resolution tells me that it is 
very much worth spending time optimizing what you can reliably fit and refine. 
This approach will pay off handsomely in terms of map quality and might lead to 
modeling what now may seem uninterpretable. 
The latest Phenix and Refmac versions are great, and their B-sharpening 
protocols can reveal a lot. In addition Buster with its handling of missing 
domains in Fc can also help a lot. 
Did you try any phase improvement by density modification? No. of molecules in 
asu? Also getting some experimental phase info (even at low resolution) might 
also help. 

Best regards
Savvas



On 08 Dec 2011, at 07:47, atul kumar  wrote:

> Dear all
> 
> I have crytals which diffract up to 3.2 A at synchrotron, I am solving this 
> by molecular replacement. I have built 70% of the model successfully,but the 
> problem is it have a very poor density for some 30-40 residues at N terminal. 
> I can't build anything in this region,this could be because of disordered 
> structure or because of low resolution.what are the things which I can try to 
> improve this?
> 
> suggestion are requested!
> 
> thanks
> regards'
> Atul Kumar
> 

Re: [ccp4bb] Movements of domains

[Savvas Savvides]

Dear Filip
'Annoying' MR problems for which the answer often lies in relatively small 
differences between the search model and 'RB-shifted' domains and/or subdomains 
in the actual structure, are I think a good experimental indication of the 
significance of such issues.

To extrapolate from this, RB refinement of whole domains that are initially 
misplaced by 0.5-2 angstroms will show that X-ray data to 4-8 angstrom 
resolution are often sufficient to refine the model to a position that 
decisively agrees better with data as judged by crystallographic refinement 
R-factors and electron density. So, my reaction every time I see such behavior 
is that the observed domain shift must be significant and that I should do my 
crystallographic best to model and cross-validate it as well as the data 
quality and resolution will allow.

However, the biological interpretation and impact of such significant 
displacements are of course quite specific to the system under study.

Best regards,

Savvas


On 21 Nov 2011, at 23:04, Filip Van Petegem wrote:

> Dear crystallographers,
> 
> I have a general question concerning the comparison of different  structures. 
>  Suppose you have a crystal structure containing a few domains.  You also 
> have another structure of the same, but in a different condition (with a 
> bound ligand, a mutation, or simply a different crystallization 
> condition,...).  After careful superpositions, you notice that one of the 
> domains has shifted over a particular distance compared to the other domains, 
> say  1-1.5 Angstrom.   This is a shift of the entire domain.  Now how can you 
> know that this is a 'significant' change?  Say the overall resolution of the 
> structures is lower than the observed distance (2.5A for example).
> 
> Now saying that a 1.5 Angstrom movement of an entire domain is not relevant 
> at this resolution would seem wrong: we're not talking about some electron 
> density protruding a bit more in one structure versus another, but all of the 
> density has moved in a concerted fashion.  So this would seem 'real', and not 
> due to noise.   I'm not talking about the fact that this movement was 
> artificially caused by crystal packing or something similar. Just for 
> whatever the reason (whether packing, pH, ligand binding, ...), you simply 
> observe the movement.   
> 
> So the question is: how you can state that a particular movement was 
> 'significantly large' compared to the resolution limit?  In particular, what 
> is the theoretical framework that allows you to state that some movement is 
> signifcant? This type of question of course also applies to other methods 
> such as cryo-EM.  Is a 7A movement of an entire domain 'significant' in a 10A 
> map? If it is, how do we quantify the significance?
> 
> If anybody has a great reference or just an individual opinion, I'd like to 
> hear about it.
> 
> Regards,
> 
> Filip Van Petegem
> 
> -- 
> Filip Van Petegem, PhD
> Assistant Professor
> The University of British Columbia
> Dept. of Biochemistry and Molecular Biology
> 2350 Health Sciences Mall - Rm 2.356
> Vancouver, V6T 1Z3
> 
> phone: +1 604 827 4267
> email: filip.vanpete...@gmail.com
> http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] effect of Izit dye within the crystal structure

[Savvas Savvides]

Dear Adam and colleagues

The enzyme human glutathione reductase (hGR) binds dyes such as safranin and 
xanthene, which are analogous to methylene blue (aka IZIT) with respect to 
their heterocyclic ring structures, in a cavity deep at the dimer interface. 
Safranin and xanthene are also inhibitors of hGR and crystal structures of 
complexes of GR with safranin and xanthene have been reported:
Karplus et al. 1989. Eur J Biochem. 178(3):693-703.
Savvides & Karplus 1996. Journal of Biological Chemistry 271(14):8101-7.

Given the general resemblance of these compounds to methylene blue and just to 
satisfy my curiosity at the time, I soaked methylene-blue into crystals of GR 
and collected good data to 2 angs. I was basically able to confirm that 
methylene blue bound specifically in the cavity at the dimer interface just 
like the other two compounds. However, this work has remained unreported.

best regards
Savvas


On 11 Nov 2011, at 05:34, adam andres wrote:

> 
> 
> Hi crystallographers
> 
> Has anyone actually collected data on a crystal that
> has been treated with Izit dye? If so did you
> see any structural effects or know any structure that present this dye? 
> 
> Cheers
> 
> Adam Campos
> 

Re: [ccp4bb] Parrot NCS averaging

[Savvas Savvides]

Dear Herman
In some cases density modification starting with MR phases can indeed prove 
very powerful. 
We recently used Parrot to bootstrap a difficult case at 4.3 angs and were able 
to obtain very good maps for entire missing domains. 
See Supplementary Fig 1 in:Verstraete et al 2011 BLOOD 118:60-68
and more technical info and fig5 in:
Verstraete et al 2011 Acta F67:325-331. 

The "Ncs mask radius" card in parrot proved to be an important one to 
experiment with, and is probably more important to do so at medium-low 
resolution. We provide details of our observations/implementation on page 330 
of the actaF paper.
We also found that post-MR HL coefficients written out by Phaser were also key. 
So in subsequent runs, after having built and refined partial
models, we would do a phase calculation run in Phaser to obtain HL by 'fixing' 
the partial model via a template .sol file with 0 values for 
rotation/translation across the board. 

Best wishes
Savvas


On 05 Oct 2011, at 17:13, herman.schreu...@sanofi-aventis.com wrote:

> Dear CCP4'ers,
>  
> I do have a similar case with 8 molecules in the asymmetric unit, related by 
> improper symmetry and the structure is solved by molecular replacement. I do 
> have 2 questions:
>  
> 1) Does it make sense to average to improve phases? The molecular replacement 
> phases perfectly obey the non-crystallographic symmetry?
> 2) For refinement I do not need average maps since Buster and other 
> refinement programs are very capable to handle ncs. However, I am not a 
> computer and for manual rebuilding and display purposes it would be very good 
> to use averaged maps. Which program could I best use for this: Parrot, DM, 
> Solomon, other? None of them seem to be really made for molecular replacement 
> phases. 
>  
> Also my many thanks for any help, advice etc.
> Herman Schreuder
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter 
> Canning
> Sent: Wednesday, October 05, 2011 4:42 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Parrot NCS averaging
> 
> Dear CCP4’ers,
> 
>  
> 
> I am working on refining the structure of a protein complex consisting of two 
> different chains. Data were collected to 2.3A in space group C2 and phased by 
> molecular replacement without any problem, but the ASU contains 6 complexes 
> (so 12 chains in total). In the ASU, the 6 complexes form 3 dimers. Dimer 1 
> is related to dimer 2 by a rotation operation and to dimer 3 by translation – 
> I hope that makes sense!
> 
>  
> 
> When I run Parrot to do density improvement and NCS averaging, Parrot works 
> beautifully (final FOM is 0.87) but NCS averaging causes the average NCS 
> correlation coefficient to drop (from 0.94 to 0.64) and the average mask 
> volume to increase from 0.31 to 0.7 (minimum volume increases from 0.05 to 
> 0.07 and maximum volume goes from 1.2 to 2), which seems a bit high.
> 
>  
> 
> Unless I have misunderstood something, I thought the NCS correlation 
> coefficient should increase during averaging and the average mask volume 
> should decrease. If this is the case, has anyone any idea what I’m doing 
> wrong?
> 
>  
> 
> Also, at the risk of asking a dumb question, is there a particular way my 
> Parrot modified file should be input into Refmac to get the best results? The 
> FOM from my refmac runs never seems to be as good as that output by Parrot.
> 
>  
> 
> Thanks in advance, all advice gratefully received, etc etc…
> 
>  
> 
> Peter Canning
> 
> SGC
> 
>  
> 
>  
> 

Re: [ccp4bb] crystals sticks to the plate surface

[Savvas Savvides]

Der Atul
this topic was previously summarized on the ccp4wiki

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Sticky_crystals

best
Savvas

On 02 Sep 2011, at 08:00, atul kumar wrote:

> hi all
> 
> I have crystals which have tendency to stick with the plate and while i try 
> to pick them for mounting on the loop they get broken.I have also tried to 
> tools to detach the crystals from the plate but couldnt get success. before 
> picking the best crystals I would like to know if someone has experience of 
> tackling this kind of problem. suggestions are requested..
> 
> thanks
> regards
> Atul Kumar

Re: [ccp4bb] Methods for dehydrating crystals

[Savvas Savvides]

Dear Andrea
check out:
Post-crystallization treatments for improving diffraction quality of protein 
crystals.
Heras B, Martin JL.
Acta Crystallogr D Biol Crystallogr. 2005 Sep;61(Pt 9):1173-80.

All the best
Savvas

On 26 Aug 2011, at 22:53, Andrea L Edwards wrote:

> Hi all,
> 
> What are the most successful methods you know of for dehydrating a crystal 
> prior to freezing it? I am trying to push the resolution of my crystals.
> 
> Thanks,
> Andrea

Re: [ccp4bb] spherulites and PEG3350

[Savvas Savvides]

Dear Jan
I would recommend running the following protocol on your spherulites. Just 
pretend that they are crystals :)
This was posted some time ago on the ccp4bb.
best regards
Savvas

> On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete <
> kenneth.verstra...@ugent.be> wrote:
> 
> > Hi Ivan,
> >
> > there are several tests (e.g. Izit dye, crush test) you can do discern
> > protein from salt crystals but what was always very informative to me (and
> > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the
> > crystals using the following protocol:
> >
> > - select a drop which contains some substantial crystalline material. The
> > crystals can be many and small (crystal shower) or few and large.
> > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother
> > liquor containing a 10% higher concentration of precipitant)
> > - transfer all the crystalline material from the drop into the PCR-tube
> > using a pipet (use stabilizing buffer from the PCR tube to collect all
> > crystals)
> > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the
> > crystals under the microscope. They should be at the bottom of the PCR-tube.
> > - Remove as much as supernatant as you can (make sure not to remove your
> > crystals), add stabilizing buffer to wash the crystals, and centrifuge again
> > - repeat this washing protocol a few times
> > - after the final washing step, add Laemli-buffer to the crystals and use
> > this sample to load the SDS-PAGE gel
> > - include a positive (eg. solubilize another drop directly in
> > Laemli-buffer) and a negative (final washing buffer) control
> > - use silver staining to visualize the protein
> >
> > This always works for me. If you don't see a band at this point I would be
> > worried that it is salt. You could then choose to do a Western blot instead
> > of silver staining to increase the sensitivity. Make sure to include control
> > samples then.
> >
> > Kind regards,
> >
> > Kenneth Verstraete
> > L-PROBE
> > Ghent University
> > Belgium

On 24 Aug 2011, at 20:05, Jan van Agthoven wrote:

> Dear all,
> 
> I recently obtained some spherulites while trying to crystallize my protein. 
> The spherulites are manually reproducible, but changing pH, protein 
> concentration, and salt concentration does not result in crystal formation. 
> Microseeding with crushed spherulites isn't a solution either as it only 
> yields new spherulites. Next stepp is the use of an optimization kit but I 
> have a limited amount of material, and I start doubting that these are 
> protein spherulites, as the spherulites are not particularly soft. The 
> condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 
> forms easily spherulites around that concentration?
> 
> 
> Thanks, 

Re: [ccp4bb] FW: [ccp4bb] highly glycosylated protein

[Savvas Savvides]

The HEK293S/ I-/- strain (Reeves et al PNAS 2002) does not lack glycosylation 
capacity. It is deficient in a key glucosyl transferase responsible for adding 
glycans beyond the Man5GlcNac2. This produces N-linked glycosylated proteins 
with homogeneous glycan trees. 

We recently established stable cell lines for inducible expression of a 
generously glycosylated human protein using the HEK293S/ I-/- strain. This 
turned out to be a very effective approach for dealing both with low yield 
transient expressions and the glycosylation issue. 
Verstraete et al Acta Cryst F67 pp. 325-31 (2011)
and
Verstraete et al  Blood. 2011
PMID: 21389326

Best regards
Savvas



On 17 May 2011, at 19:09, "Bernhard Rupp (Hofkristallrat a.D.)" 
 wrote:

> I have limited cautionary experiences with Lec1 CHO expression, although with 
> a highly and complex glycosilated protein. Subsequent glycan analysis showed 
> that the glycans are indeed more homogeneous and less branched, but not 
> exclusively the Man5GlcNac2 type that are reported/expected. There is still 
> some fraction of higher glycans left, and the crystals are correspondingly 
> ratty in the first round. On the other hand, successful crystallization has 
> been reported:
> 
>  
> 
> Chen, L., Gorman, J.J., McKimm-Breschkin, J., Lawrence, L.J., 
> Tulloch, P.A., Smith, B.J., Colman, P.M., and Lawrence, M.C. 2001. The 
> structure of the fusion glycoprotein of Newcastle disease virus suggests a 
> novel paradigm for the molecular mechanism of membrane fusion. Structure 
> 9:255-266.
> 
>  
> 
> I’d be interested to hear about the completely glycosylation deficient cell 
> lines mentioned below – surprised the cells live at all.
> 
>  
> 
> BR
> 
>  
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wei Li
> Sent: Tuesday, May 17, 2011 9:36 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] FW: [ccp4bb] highly glycosylated protein
> 
>  
> 
>  
> 
> Dear Pascal and Matthias,
> 
> I am sorry for the delay of reply, thanks very much for your suggestions on 
> the glycosylation protein. Now I am  trying to do a stable cell line with CHO 
> lec 3.8.2.1 cells, this cell line could express protein with shorter glycans. 
> I hope several weeks later I could get some better result. I will also try to 
> use the Glycosylation deficient cell lines.
> 
>  
> 
> I am still working on it, thanks again for your valuable advice.
> 
>  
> 
>  
> 
> Best regards,
> 
>  
> 
> Wei
> 
>  
> 
>  
> 
>  
> 
>  
> 
>  
> 
>  
> 
>  
> 
>  
> 
> From: Matthias Zebisch [mailto:matthias.zebi...@bbz.uni-leipzig.de] 
> Sent: 2011年5月13日 18:15
> To: Wei Li
> Subject: Re: [ccp4bb] highly glycosylated protein
> 
>  
> 
> Try to get hold of GntI deficient HEK293S cells (not commercially available). 
> Expression takes two weeks but you can achieve comparable yields to HEK293T.
> These cells yield very homogenous bands on SDS PAGE. However, check also for 
> O glycosylation prediction.
> As you appear to be from Braunschweig, ask Prof. Sträter in Leipzig. He can 
> send you these cells.
> 
> Good luck,
> 
> Matthias
> 
> From: Pascal Egea [mailto:pas...@msg.ucsf.edu] 
> Sent: 2011年5月13日 18:01
> To: Wei Li
> Cc: CCP4BB@jiscmail.ac.uk
> Subject: Re: [ccp4bb] highly glycosylated protein
> 
>  
> 
> Hi Wei,
> 
>  
> 
> Glycosylation usually stabilize proteins although it is a source of 
> structural heterogeneity for us crystallographers.Since you are expressing in 
> HEK293 cells, there is a strain of cells that is deficient for glycosylation 
> (it was designed by Gobind Khorana at the MIT I believe). You may want to try 
> this. This is particularly useful when you express membrane proteins, it 
> avoids hyperglycosylation. You may want to try a lightly glycosylated version 
> of your protein and see if it behaves correctly,
> 
> The other extreme solution is to identify all occupied sequons in your 
> protein and eventually inactivate them by mutagenesis to have a completely 
> deglycosylated protein. This solution is probably not the best since 
> glycosylation usually stabilize proteins and may be essential to their 
> biological function and activity. So it is to be considered with a lot of 
> caution.
> 
>  
> 
> Hope this helps.
> 
>  
> 
> 
> -- 
> Pascal F. Egea, PhD
> Assistant Professor
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> 356 Boyer Hall
> office (310)-983-3515
> lab  (310)-983-3516
> email   pe...@mednet.ucla.edu
> 
>  
> 
> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
> Braunschweig | www.helmholtz-hzi.de
> 
> Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
> Bundesministerium für Bildung und Forschung
> Stellvertreter: MinDirig Heiko Gevers, Niedersächsisches Ministerium für 
> Wissenschaft und Kultur
> Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
> Gesellschaft mit beschränkter Haftung (GmbH)
> Sitz der Gesellschaft: Braunschweig
> Handelsregister: 

Re: [ccp4bb] off topic: problematic protein

[Savvas Savvides]

I would like to thank all of you  who promptly replied to my posting with so 
many ideas and suggestions (18 answers so far). I will post a summary soon.

best wishes to all
Savvas


>> 
>> 
>> On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote:
>> 
>>> Dear colleagues
>>> 
>>> We are working on a large bacterial protein (featuring a large number of 
>>> repeats) that appears to copurify with a lot of other proteins after 
>>> Ni-affinity chromatography and gel-filtration. We have tried adjusting the 
>>> ionic strength of these runs and have gone to as high as 5M NaCl but only 
>>> saw marginal improvements.  It appears that the protein likes to stick to a 
>>> lot of stuff, and in fact the number of repeats in a given construct 
>>> appears to correlate with the extent of contaminants in our purification 
>>> steps. We have admittedly never seen anything like this among the so many 
>>> different, and often challenging, proteins, we have worked on in our group 
>>> over the last few years.
>>> 
>>> We are now thinking of trying detergents in the buffers (at non-micellar 
>>> concentrations), in conjunction with playing a bit with the pH to see if 
>>> such an approach provides a 'stripping' effect. Interestingly, the protein 
>>> has a calculated pI of 3.5 !
>>> 
>>> As the options for handling this protein are indeed quite numerous, we 
>>> would be grateful for any additional input and possible tips/tricks.
>>> 
>>> I will prompty post a summary of the thread.
>>> 
>>> Best regards
>>> Savvas et al.
>>> 
>>> 
>>> 
>>> Savvas Savvides
>>> Unit for Structural Biology @ L-ProBE
>>> Ghent University
>>> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
>>> Tel/SMS/texting +32  (0)472 928 519
>>> Skype: savvas.savvides_skype
>>> http://www.LProBE.ugent.be/xray.html
>>> 

[ccp4bb] off topic: problematic protein

[Savvas Savvides]

Dear colleagues

We are working on a large bacterial protein (featuring a large number of 
repeats) that appears to copurify with a lot of other proteins after 
Ni-affinity chromatography and gel-filtration. We have tried adjusting the 
ionic strength of these runs and have gone to as high as 5M NaCl but only saw 
marginal improvements.  It appears that the protein likes to stick to a lot of 
stuff, and in fact the number of repeats in a given construct appears to 
correlate with the extent of contaminants in our purification steps. We have 
admittedly never seen anything like this among the so many different, and often 
challenging, proteins, we have worked on in our group over the last few years.

We are now thinking of trying detergents in the buffers (at non-micellar 
concentrations), in conjunction with playing a bit with the pH to see if such 
an approach provides a 'stripping' effect. Interestingly, the protein has a 
calculated pI of 3.5 !

As the options for handling this protein are indeed quite numerous, we would be 
grateful for any additional input and possible tips/tricks.

I will prompty post a summary of the thread.

Best regards
Savvas et al.


----
Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype
http://www.LProBE.ugent.be/xray.html

Re: [ccp4bb] Off topic: How to identify a unknown ligand

[Savvas Savvides]

Hi Xiaopeng
To add to Artem's comments:
Does the presumed gsh make a mixed disulfide in the active site?i.e. is it 
covalently bonded to the active site via a s-s bond?
If yes then MS on your purified sample should easily give you the answer.
If a mixed s-s is indeed the scenario then purifying the enzyme in the presence 
of reducing agents and playing with the pH a bit should yield a gsh free 
preparation.  

Best regards
Savvas

On 16 Mar 2011, at 02:19, Artem Evdokimov  wrote:

> Tightly bound ligands commonly survive purification :) Several unexpected 
> discoveries have been made this way!
>  
> If you think your stuff is GSH, soak some 'real' GSH or co-crystallize with 
> it, and see if density shape changes from what you had before. This is not 
> guaranteed to work because sometimes the ligand may be bound so 
> tightly/exchange slowly that the original ligand just won't budge. This was a 
> significant issue with some of the kinase inhibitors and nuclear hormone 
> receptor antagonists that I've had a chance to work with - the binding was 
> almost 'one way' in real-time situation, and (partial) denaturation was 
> required to get the ligands out.
> If your ligand is indeed GSH, in equimolar amount with the protein, then you 
> could also try MS as detection technique. The ligand should come off when 
> protein is subjected to the normal MS environment (typically 0.1% TFA or 
> formic acid, in a mixture of water and acetonitrile, etc.). To detect it, 
> don't forget to expand the mass window to get the mass, and set the 
> ionization mode to positive - you should see a clear 308.3 Da peak, with a 
> lovely isotope splitting pattern (assuming you have access to MS). In 
> negative mode the mass will be 1/2 of the already low m.w. of 307, since GSH 
> has two negative charges. Notably, GSH should also accept e.g. an 
> iodoacetamide group on the -SH, meaning that you should be able to treat 
> crystals with iodoacetamide and observe the addition of -CH2CONH2 to the 
> sulfur. Naturally, the S atom should be pretty prominent in the density 
> anyway. Ditto mercurials, but they may wreck the crystal. Since your enzyme 
> may be a GST (assumption on my part here) it also may present GSH to be 
> reactive with whatever substrates that yor GST is targeting, so you may be 
> able to identify conjugation products.
>  
> Artem
> 2011/3/14 Xiaopeng Hu 
> Dear all,
> 
> Sorry for this off topic question.
> 
> We are working on protein/inhibitor complex structure although we can not get 
> our inhibitors in. However, we did find a strange density at the active site, 
> it looks really like GSH, the natural co-enzyme of thiis protein.We tried to 
> use very simple solution to get crystal then exclude the possiblity of buffer 
> moleculors,but that density is aways there.
> 
> I am wondering how this ligand (if it is GSH) can survive all purification 
> steps and want to indentify it. Are there any methodes to do this work? Let's 
> say to pick up a crystal and do some analysis?
> 
> Many many thanks!!!
> 
> 
> Xiaopeng
> 

Re: [ccp4bb] Crosslinking of Protein-Protein complex for crystallisation

[Savvas Savvides]

Dear Catrine
can you comment on the pH range of your crystallization hits compared to the pH 
of the sample you use for your crystallization trials? The pH can be a major 
issue depending on the nature of the interaction interface.

best regards
Savvas







On 14 Mar 2011, at 13:21, Catrine Berthold Siöberg wrote:

> Dear CCP4bb,
> I am trying to solve the structure of a protein-protein complex. The two 
> proteins are co-expressed and co-purified and I get a homogeneous 1:3 
> complex, which is the predicted composition. I get several hits using 
> commercial crystallisation screens (including the MD ProPlex screen), but all 
> crystals only contain the trimeric, more stable protein of the two.
> 
> I am now considering to crosslink them, but my problem is that I cannot 
> modify them separately to get a specific crosslink, since one of the proteins 
> precipitates by itself.
> I would appreciate all protocols for crosslinking of proteins aimed for 
> crystallisation, or references to structures solved of crosslinked proteins.
> 
> Thank You,
> Catrine
> 
> -- 
> Catrine Berthold Siöberg, PhD
> Stockholm Center for Biomembrane Research
> Department of Biochemistry and Biophysics
> Stockholm University
> S-106 91 Stockholm
> Sweden
> e-mail: catrine.siob...@dbb.su.se
> phone: +468-162451

Re: [ccp4bb] How to crystallize a helicase

[Savvas Savvides]

I would certainly try cocrystallizing with adp, amppnp, or ATPgS
Many helicases have been crystallized in this way.   Papers by Sawaya et al and 
Singleton et al come readily to mind. 

Best of luck
Savvas

On 09 Mar 2011, at 15:52, Ed Pozharski  wrote:

> On Wed, 2011-03-09 at 10:52 +, Art wrote:
>> No protein crystal was found after some typical conditions (kit I, kit
>> II, and Index) were repeatly screened for more than three times.
> 
> See Einstein's definition of insanity :)
> 
>> Protein concentration gradient was considered, but it can not work.
> 
> Not sure what you mean by this, but you can also try diluting reservoir
> solutions.  Also, given your protein buffer, you may be more or less
> locking yourself into a narrow pH range - try 10mM MES instead.
> 
> -- 
> "I'd jump in myself, if I weren't so good at whistling."
>   Julian, King of Lemurs

Re: [ccp4bb] optimization of dumbbell like crystal

[Savvas Savvides]

Dear Harvey
Based on the experimental history you provided I would try two quick things in 
order to obtain additional dimensions for your condition. 
(1) set up your best consensus condition against an additive screen (e.g. From 
Hampton). 
(2) I would repeat your last pH vs  precipitant grid against different starting 
protein concentrations. You can conveniently play with different drop ratios to 
screen for that. 

Best regards
Savvas

On 21 Feb 2011, at 03:29, Harvey Rodriguez  wrote:

> Dear CCP4BBers,
>  
> Recently I got a crystal which appeared in the condition 2.1M DL-Malic Acid, 
> pH 7.0. The crystal looks like a dumbbell but was composed of a cluster of 
> very thin needles as shown in the picture. The crystal can be repeated but 
> the diffraction is poor. I have tried the grid optimization by pH and salt 
> concentration gradients, however there was no improvement in either the shape 
> or the quality of the crystal. Does anyone have some experiences in 
> optimizing this kind of crystals? Any suggestion is appreciated!
>  
> Harvey
> 

Re: [ccp4bb] Detaching crystals from glass cover slides

[Savvas Savvides]

Dear Wataru
check out the following entry on the CCP4-wiki on 'sticky crystals' which we 
compiled based on feeback from many crystallographers:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Sticky_crystals


Best regards
Savvas

----
Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype
http://www.LProBE.ugent.be/xray.html






On 09 Feb 2011, at 16:11, Colbert, Christopher wrote:

> Hi Wataru,
> 
> I second Bill's suggestion.  Additionally, you might want to migrate to a
> sitting drop setup and use the vacuum grease with that.  It worked for me.
> 
> Happy Fishing,
> 
> Chris
> 
> --
> Christopher L. Colbert, Ph.D.
> Assistant Professor
> Department of Chemistry and Biochemistry
> North Dakota State University
> P.O. Box 6050 Dept. 2710
> Fargo, ND 58108-6050
> PH: (701) 231-7946
> FAX: (701) 231-8324
> 
> 
> 
> 
> 
> On 2/8/11 8:19 PM, "William G. Scott"  wrote:
> 
>> Hi Wataru:
>> 
>> I hope all is well.  For the ones you already have grown, try very gently
>> prying them off with a wedge-shaped needle.
>> 
>> If you can grow more, try using a very thin smooth layer of vacuum
>> grease, and apply the drop to that.  I managed to get RNA crystals to
>> grow that way that otherwise irreversibly adhered to the surface.
>> 
>> All the best,
>> 
>> Bill
>> 
>> 
>> On Feb 8, 2011, at 6:06 PM, Wataru Kagawa wrote:
>> 
>>> Hi all,
>>> 
>>> I have crystals growing by the hanging-drop method, using 24-well VDX
>>> plates and Hampton Research siliconized glass cover slides. Most
>>> crystals are attached to the cover slide, and I am having difficulties
>>> detaching the crystals (using a cryoloop) without breaking them. There
>>> are few smaller crystals floating in the drop, and they diffract X-rays
>>> pretty well (clean spots, ~3.5A resolution using RAXIS). However, I
>>> would like to try the bigger ones, because they may diffract to a higher
>>> resolution.
>>> 
>>> Any suggestions for detaching crystals from cover slides will be
>>> greatly appreciated.
>>> 
>>> Wataru
>> 
> 

Re: [ccp4bb] Strange density on Serine oxygen.

[Savvas Savvides]

Hi Vinson
is O-glycosylation possible at all given the origin of the protein and the 
expression system used? 
best 
Savvas




On 24 Nov 2010, at 14:42, Vinson LIANG wrote:

> Dear Savas and all,
>  
> Thank you very much for all your quick suggestions.
>  
> I have tried Tyr and it turns out to fit the density very well. I will have 
> the protein sequenced again to see if it is wrong sequece or O-linked 
> glycosylation.
>  
> I'll let you know if it turns out to be O-linked glycosylation.
>  
> All the best,
>  
> Vinson 
> 发件人： Savvas Savvides 
> 收件人： Vinson LIANG 
> 抄 送： CCP4BB@JISCMAIL.AC.UK
> 发送日期： 2010/11/24 (周三) 9:27:31 下午
> 主 题： Re: [ccp4bb] Strange density on Serine oxygen.
> 
> Hi Vinson
> Beyond the possibility for another type of residue as already suggested by 
> Phil and Mark, there is also the possibility of O-linked glycosylation of the 
> serine and threonine, if your protein undergoes such post-translational 
> modification and it has been expressed via an expression system that 
> processes the protein in that way.
> Ser/Thr tandems are well known targets for O-glycosylation 
> (http://www.cbs.dtu.dk/databases/OGLYCBASE/).
> 
> best regards
> Savvas
> 
> 
> Savvas Savvides
> Unit for Structural Biology @ L-ProBE
> Ghent University
> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
> Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html
> 
> 
> 
> On 24 Nov 2010, at 13:10, Vinson LIANG wrote:
> 
>> Dear all,
>>  
>> I'm refining a structure and find some strange triangle density on the 
>> oxygen of Ser and Thr at the C terminus. One picture of the strange density 
>> is attached here. Could anyone please give me some suggestions on what this 
>> could be?
>>  
>> The buffer used during purification is PBS, Tris and NaCl. And 
>> crystallization condition contains PEG3,350 and Mg(NO3)2.
>>  
>> Thank you all in advance for any suggestion.
>>  
>> Best,
>>  
>> Vinson Liang
>>  
>>  
>> 
>>  
> 
> 
>  
> 
>  

Re: [ccp4bb] Strange density on Serine oxygen.

[Savvas Savvides]

Hi Vinson
Beyond the possibility for another type of residue as already suggested by Phil 
and Mark, there is also the possibility of O-linked glycosylation of the serine 
and threonine, if your protein undergoes such post-translational modification 
and it has been expressed via an expression system that processes the protein 
in that way.
Ser/Thr tandems are well known targets for O-glycosylation 
(http://www.cbs.dtu.dk/databases/OGLYCBASE/).

best regards
Savvas


Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 24 Nov 2010, at 13:10, Vinson LIANG wrote:

> Dear all,
>  
> I'm refining a structure and find some strange triangle density on the oxygen 
> of Ser and Thr at the C terminus. One picture of the strange density is 
> attached here. Could anyone please give me some suggestions on what this 
> could be?
>  
> The buffer used during purification is PBS, Tris and NaCl. And 
> crystallization condition contains PEG3,350 and Mg(NO3)2.
>  
> Thank you all in advance for any suggestion.
>  
> Best,
>  
> Vinson Liang
>  
>  
> 
>  

Re: [ccp4bb] High Rmerge with thin frames

[Savvas Savvides]

Dear Sergei
how much do the refined unit cell parameters (given a fixed detector distance) 
vary as a function of frame number? We have been using such initial diagnostic 
approach to trace radiation damage issues (among other problems) for a number 
of crystal forms that maximally diffracted in the 3.8-4.5 angstron range. 

I copy/paste an excerpt from the XDS wiki that reinforces this approach:

REFINE(INTEGRATE)
The defaults (REFINE(INTEGRATE)=DISTANCE BEAM ORIENTATION CELL) could be 
modified by omitting DISTANCE, because one should assume that the distance is 
constant. This is particularly recommended if SPACE_GROUP_NUMBER=0 or 1. 
Furthermore, by fixing the distance one can better see from the results of the 
refinement whether the cell parameters are stable, or whether they change due 
to radiation damage. There are situations when one wants to reduce the number 
of parameters to be refined even more, see Optimization.

Best regards
Savvas


Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 05 Nov 2010, at 09:40, Sergei Strelkov wrote:

> Dear All,
> 
> I am processing a dataset collected (not by me) with 0.1 degree oscillations.
> The diffraction is quite weak even though there is a clean diffraction 
> pattern to about 3A.
> 
> Either Mosflm or XDS processes the data readily with +/- default settings
> but both yield a high overall Rmerge of about 0.23 in the expected symmetry.
> Processing in P1 yields an overall Rmerge of ~0.18, but what is especially 
> disappointing
> is that Rmerge is as high as 0.15 at ~5A resolution already.
> 
> The question is, how can we process the data so that the merging statistics
> becomes more reasonable?
> 
> Apparent mosaicity turns out to be ~0.5A. My naive way of thinking is
> to try treating each five consecutive frames as a single 0.5 degree frame.
> Does anyone have experience with this?
> 
> Many thanks in advance,
> Sergei

Re: [ccp4bb] offtopic: effect of compound impurities on ITC?

[Savvas Savvides]

Hi Francis
I guess it depends on how much residual high-affinity binder you have in the 
mixture and what the difference in affinity is between Y and deriv-Y. Another 
issue is of course whether Y and derY compete for the same binding site and 
have the same stoichiometry. A well designed displacement ITC experiment and 
comparisons thereof with ITC data for your high-affinity binder should lead to 
some good answers.  Knowing the ratio of Y vs deriv-Y in your starting compound 
solution will be an advantage.

A very useful reference in thinking about and carrying out displacement ITC in 
our group has been the one by Velazquez-Campoy and Freire. This article was 
specifically written to address the application of displacement titrations in 
ITC. We have applied this approach to address several types of questions 
concerning interactions in the uM-pM range.
 
Velazquez-Campoy A, Freire E.
Isothermal titration calorimetry to determine association constants for 
high-affinity ligands
Nat Protoc. 2006;1(1):186-91. 

Best regards
Savvas


Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html



On 24 Aug 2010, at 17:11, Francis E Reyes wrote:

> Hi All
> 
> I'm curious the effect of small impurities in commercially synthesized 
> compounds on ITC and its analysis. Say if compound Y is the high affinity 
> binder, but you make a derivative that differs from a single functional group 
> from Y (you used Y to make this new compound) and you never are able to 
> completely get rid of Y. How does this affect the analysis of determining the 
> derivative's affinity by ITC?
> 
> References or personal experience is appreciated!
> 
> F
> 
> -
> Francis E. Reyes M.Sc.
> 215 UCB
> University of Colorado at Boulder
> 
> gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D
> 
> 8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] DLS

[Savvas Savvides]

Hi Wojtek
I can easily second Dave's comment!
Best 
Savvas


Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519
http://www.LProBE.ugent.be/xray.html


On 04/02/10 14:54, "David Briggs"  wrote:

> Hiya.
> 
> My two penneth on the Malvern Zetasizer.
> 
> Simple, idiot-proof operation. Software is easy and intuative. Reports
> are customisable to give you what information you want.
> Problems easy to trouble shoot IMHO.
> 
> Does DLS, SLS, melting point determination.
> 
> After-care support is good from Malvern.
> 
> Not used the Wyatt instrument.
> 
> HTH,
> 
> Dave
> 
> 
> David C. Briggs PhD
> Father, Structural Biologist and Sceptic
> 
> University of Manchester E-mail:
> david.c.bri...@manchester.ac.uk
> 
> Internetz: http://xtaldave.posterous.com/
> Twitter: @xtaldave
> Skype: DocDCB
> 
> 
> 
> 
> On 4 February 2010 13:36, Wojciech Rypniewski  wrote:
>> Dear Fellow Crystallographers
>> 
>> We are going to buy a DLS instrument to help us assess crystallizability
>> of our proteins. I would be grateful for any opinions - either
>> on this BB or in my mail box (in which case I'll keep them to myself,
>> naturally).
>> 
>> We have looked more closely at two instruments:
>> 
>> DynaPro NanoStar from Wyatt and
>> Zetasizer Micro V from Malvern
>> 
>> and both seem to be rather similar in terms of parameters and price.
>> 
>> I would appreciate user opinions as to the reliability,
>> ease of use or any features that make a given instrument
>> particularly suited for assessing biological samples.
>> 
>> Many thanks,
>> 
>> Wojtek
>> 
>> --
>> --
>> Prof. dr hab. Wojciech Rypniewski      tel: +48-61-8528503
>> Institute of Bioorganic Chemistry      fax: +48-61-8520532
>> Polish Academy of Sciences             e-mail: wojt...@ibch.poznan.pl
>> Noskowskiego 12/14                     www: www.man.poznan.pl/~wojtekr/
>> 61-704 Poznan, Poland
>> --
>> 

Re: [ccp4bb] Solubilization buffer

[Savvas Savvides]

Hi Meg
I would highly recommend giving 6M guanidinium chloride a go in parallel, to
compare the solubilization efficiency of the two approaches. We have had a
couple of cases in the lab where homologous proteins showed strong
preference either to urea OR guanidinium chloride when it came to
solubilization of their inclusion bodies for refolding protocols.
best wishes
Savvas

 
Savvas Savvides 
http://www.lprobe.ugent.be/xray.html




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joerg
Standfuss
Sent: Thursday, December 03, 2009 8:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Solubilization buffer

Dear Meg,

There is a database of refolding conditions from which you could get some
inspiration.

http://refold.med.monash.edu.au/search.php

The Urea conditions I have seen there mostly use 37C. for
solubilization/refolding and nearly all contain a reducing agent. Have a
look. There are also a great number of other additives people use to
improve things.

Joerg


> Hi all,
>
> We use 8M urea solubilization buffer for our protein in inclusion bodies
> and
> recommended temperature is 10-15º C. but in 8M conc the urea does not
> dissolve and is in crystalline form only, will it have any effect on
> solubilzation efficiency. Our solubilization time is 1 Hr and after that
> we
> centrifuge and use the supernatant for refolding via dialysis. however the
> pellet after centrifugation of solubilzation show presence of our protein
> on
> sds page analysis. what should we do so that the process of solubilization
> is complete and our protein is not lost in pellet.
>
> thanks in anticipation.
>
> meg
>





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Re: [ccp4bb] Phase separation to crystals

[Savvas Savvides]

Hi James,

just out of curiosity. Did IZIT convincingly color the crystals? We have
recently seen that crystals grown in high concentrations of MPD do not color
with IZIT. This phenomenon was documented in Eckert et al. Acta Cryst.
(2003). D59, 139-141.

 

Best regards

Savvas

 

 

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
james09 pruza
Sent: Saturday, October 17, 2009 7:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Phase separation to crystals

 

Dear CCP4bbers,

Surprisingly, only At pH 3.6  the needles are appearing after 3 days but
they are very thin and long rods. I have checked it running SDS-PAGE as well
as using IZIT dye . Since the proteins has already 50mM of NaCl so I added
100mM of NaCl in the crystallization mix. The crystal appeared at 70% of
MPD. Bunch of florets appears without NaCl at the same condition. Will the
Additives like Ethanol or 2-propanol help in this condition? Should  I not
use NaCl in the mix? Changing temperature from 4degree to 25 degree will
help?
Please advice.

Thanks a lot.
James





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Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Savvas Savvides]

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great succes by
cryo-cooling xtals grown at 3.2M AS straight out of their crystallization
drops. You can consult the "M&M" section in Kyndt J. et al Biochemistry 2007
Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer





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Re: [ccp4bb] cyseteine modification

[Savvas Savvides]

Dear Debajyoti

There is also the sulfenic acid species (-S-OH) which is actually the first
oxidized form of sulfhydryls on the way to sulfonic acid.  However sulfenic
acids are very susceptible to further oxidation to sulfinic and sulphonic
acids, and therefore need a protective chemical environment to remain
stable. 

See for example some previous work of ours on sulfenic and sulfinic forms of
active-site cysteines in glutathione reductase (Nature Structure Biology vol
5, 267-271, 1998) and the corrresponding pdb entries 1dnc and 1gsn.

Best regards

Savvas

 

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Debajyoti Dutta
Sent: Wednesday, August 19, 2009 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cyseteine modification

 

Dear Sir,

Is there any other oxidation states of cysteine other than cysteine
sulphinic acid and cysteine sulphonic acid. In my protein, the cysteine
molecule is definitely overoxidized but the electron density is not
corresponding to the sulphinic or the sulphonic acid. The positive density
looks as if it can accomodate only one oxygen atom and not more.

Thank you for reply in advance. 

Sincerely
Debajyoti Dutta


 
<http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/sign
atureline@middle?> 

 





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Re: [ccp4bb] heavy atom derivative choice

[Savvas Savvides]

Hi Sebastiano,
Given that your crystallization condition contains KSCN, I would certainly
give Hg(SCN)2 a go among other Hg options for a protein containing free
cysteines. Hg(SCN)2 might end up being more compatible with your crystal
form, and might thus minimize non-isomorphism issues. 

Best of luck
Savvas




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David
Briggs
Sent: Wednesday, July 15, 2009 3:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] heavy atom derivative choice

Hi Sebastiano,

Free Cys' are crying out for Mercury derivatives. Try 2-3 with varying
sizes of additional groups - HgCl2, K2HgI4, PCMB,  from the "Magic
seven" would be a good place to start.

Watch out for problems with your low pH, though. Some salts will form
insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay)

Bart Hazes used to have an excellent set of notes online about heavy
atom derivatisation - I can't seem to find the URL right now...

HTH,

David

2009/7/15 Sebastiano Pasqualato :
> Hi all,
> I've got crystals of a protein of ca 200 residues, with 2 free cysteines,
5
> histidines, 2 methionines.
> We have nice diffraction for the native crystals, that grow in 150 mM
KSCN,
> 17% PEG 3350, bis tris propane pH 8.8.
> We are crystallising the SeMet derivative, but I'm not completely sure I
> will be able to have nice crystals by saturday, when we have tunable time
at
> the ESRF.
> I was thinking of trying with some heavy atom soaks, but only have like 30
> crystals, so limited trials allowed!
> Which compound would you advice as more likely to work, and thus worth
> testing?
> Thanks in advance for the suggestions,
> ciao
> s
>
>
>
> --
> Sebastiano Pasqualato, PhD
> IFOM-IEO Campus
> Dipartimento di Oncologia Sperimentale
> Istituto Europeo di Oncologia
> via Adamello, 16
> 20139 - Milano
> Italy
>
> tel +39 02 9437 5094
>



-- 

David C. Briggs PhD
Father & Crystallographer
http://drdavidcbriggs.googlepages.com/home 
Skype: DocDCB






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Re: [ccp4bb] unknown density

[Savvas Savvides]

Dear Bert,
Your density reminded me a little bit of the paper by Pearson et al.
Biochemistry 39:8575-8584(2000) which describes the water structure in the
catalytic center of urease. I just went back to it and it looks like Figure
3 in that paper may hint some similarities to your case, in that you may
indeed have a mixture of water models.

You did not say if your data comes from cryo-cooled crystals that were
manipulated in similar or different ways, or whether one of the two is a
room temperature data set. One of the largely overlooked issues related to
crystal cryo-cooling concerns the structure attained by the solvent.
Structures of urease at 100K and 298K (pdb entries 1ejw and 1ejx) show
marked differences in the water structure in the active site and elsewhere.
This phenomenon has been observed in other structures as well, and not just
with respect to water structure but also for loops and side-chains. Even two
cryo-cooled crystals that are manipulated in similar ways may exhibit
differences.
Bart Hazes addressed some of these issues in an interesting paper in Acta
Cryst. (2005). D61, 80-87.

Nonetheless, to maximize the chances for the best interpretation possible I
would first improve phase accuracy by completing the solvent model as much
as possible (except from the contested density), and then use
post-refinement residual Fo-Fc density to evaluate possible water models
(and other possible interpretations) for the density in question. 

Best wishes
Savvas

-
Savvas Savvides
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Van
Den Berg, Bert
Sent: Tuesday, July 07, 2009 10:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unknown density

Dear all,


I have attached a jpeg of some (2fo-fc) density in the active site of a
protein I'm refining (1.9 A data, Rfree 24%, still need to pick waters etc;
oxygen atom placed in the approximate center for reference). It looks like
there are 3 non-hydrogen atoms sandwiched between an Asp, His and Arg. The
molecule seems hydrogen bonded to all these three residues.
Has anybody any idea what this can be? In a map of another crystal of the
same crystal form at lower res (2.3 A), there is a clear, single water
molecule between the Asp and His. Are they waters at partial occupancies
(there is not enough room for 3 separate waters)? The only other thing I can
come up with is formate, but that wasn't used in the crystallization.

Any hints appreciated!

Thanks, Bert


Bert van den Berg
UMass Medical School
Worcester, MA 01605







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Re: [ccp4bb] multi-domain protein with identical tertiary structure

[Savvas Savvides]

Hi Shankar

gamma-crystallin is likely a good candidate [Blundell et al. Nature
289:771-777, 1981], and it in fact goes a step further. It combines two
nearly identical  greek-key motifs in each domain, which are then connected
via a loop to make a dimer.  This is all in one chain.

Best wishes

Savvas

 

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Shankar Prasad Kanaujia
Sent: Thursday, July 02, 2009 7:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] multi-domain protein with identical tertiary structure

 

Dear CCP4 users,
Is there any multi-domain protein (with at least two domains) which has
identical tertiary structure of each domain ?

Thanking you.

-regards
shankar






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Re: [ccp4bb] Halide soaking

[Savvas Savvides]

Hi everyone,
the in situ iodination reaction described in the following classic paper by
the late Paul Sigler works quite well.

Iodination of a single tyrosine in crystals of alpha-chymotrypsin.
Sigler PB.
Biochemistry. 1970 Sep 1;9(18):3609-17.

The primary purpose of my experiment (which took place 11 years ago
according to my notebook) was indeed to iodinate tyrosines, but difference
fourier analysis using calculated phases from the final refined MIR
structure to reveal the complete iodination model (out of curiosity), showed
that in addition to iodination of two tyrosines, two histidines had also
been iodinated! In retrospect, I had actually run across those peaks in my
cross-difference fourier maps but thought that they were too 'secondary' to
be included in the heavy atom model.

Best wishes
Savas

---- 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Alessandro Vannini
Sent: Tuesday, March 31, 2009 9:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Halide soaking

Hi,
it worked very nice for me in 1 out of 1 case where I tried it :-).  
Very well diffracting crystals (1.8  Ang), rather small protein 20  
kDa, 50 %solvent content, 1 mol/ASU. 20-30 s soak in 0.5 M NaBr  
resulted in 6 nice ordered sites.
It was crucial for us  to collect a 3 wavelength MAD data set. A SAD  
data set (using just the peak, even if with high redundancy ) was not  
enough to obtain traceable electron density map, even-though
one could distinguish clearly protein boundaries and solvent channels.

Good luck

Ale

On 31 Mar 2009, at 18:19, tat cheung cheng wrote:

> Hi all
>
> I am now trying to do bromide soaking, but i am not really sure does  
> the bromide atom enter my crystal. So is there any signs that  
> indicate the entry of bromide atom? e.g. does the space group, cell  
> dimension change? or just nothing change, and the bromide atom just  
> get in?
> Thanks very much.
>
> T.C. Cheng
>
>
>  Yahoo!香港提供�W上安全攻略，教你如何防��黑客! ��前往 http://hk.pro 
> mo.yahoo.com/security/ 了解更多!





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Re: [ccp4bb] Se oxidation

[Savvas Savvides]

I think that SeMet oxidation has been a problem in the past in at least one
case that I know, that of TolC by Koronakis et al.  The same group addressed
these problems in more detail in a second paper (see below):

 

Crystal structure of the bacterial membrane protein TolC central to
multidrug efflux and protein export.

Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C.

Nature. 2000 Jun 22;405(6789):914-9.

 

Oxidation of selenomethionine: some MADness in the method!

Sharff AJ, Koronakis E, Luisi B, Koronakis V.

Acta Crystallogr D Biol Crystallogr. 2000 Jun;56(Pt 6):785-8.

 

Best wishes

Savvas

 

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of aka
akaka
Sent: Monday, February 09, 2009 7:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Se oxidation

 

Dear All

 

I would like to know whether oxidation of Se entails any problem for SAD or
MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents
in my protein (extracellular and disulphide bonds are important).

Thanks

 

Dr. R.Depetris

Weill Cornell Medical College

 

 

 

  _  

Get 5 GB of storage with Windows Live Hotmail. Sign up today.
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[ccp4bb] 'Sticky Crystals' on ccp4-wiki

[Savvas Savvides]

Dear colleagues,
I have just deposited an entry on the ccp4-wiki that summarizes input/ideas
on dealing with 'sticky crystals'. Please feel free to edit it further as
necessary.
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Crystals#Crystal
_handling 

best wishes
Savvas

---- 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html








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[ccp4bb] SUMMARY: Sticky crystals

[Savvas Savvides]

Dear colleagues,

I would like to thank all of you who responded  so generously (either
privately or publically on the ccp4bb) to my question on how to deal with
'sticky crystals'.  I provide below a consensus of all the many answers
grouped according to the proposed solution.

Best wishes

Savvas

 

OPERATE ON THE PLASTIC RATHER THAN ON THE CRYSTAL!

---

-I dig the microtool into the plastic as close as I can get to the crystal
without touching the crystal. Usually a small deformation of the plastic
causes the crystal to pop off intact.

-I have had similar problems with crystals sticking to the plastic of the
crystallisation plates (96-well Grenier plates). I use an acupuncture needle
and dig into the plastic right next to the crystal, directing the needle
below the crystal. By digging and twisting, you can generate little
movements across the plastic which can be enough to free the crystal. If you
slip, you end up playing golf with your crystals, but with a steady hand, it
has worked well for me, though I guess it might be quite dependant on the
strength of the plastic of the plate. Worth trying though if you have
crystals.

-Take a sturdy needle (like one of the microneedles from a Hampton kit or a
very thin syringe needle) and (while observing the whole thing under a
scope) stick the needle into the plastic a bit away from the crystal. Push
hard. If you're using polarizers, you may be abe to visualize the stress
forces in the plastic by the shifting of the colors. The basic idea here is
to stress the plastic under the crystal w/o touching the crystal in any way.
In my case the bloody things just popped off. Sometimes you have to push
quite hard, and to wiggle the needle a bit - and beware, they can slip and
ruin your drop. But this really worked quite well!

 

COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE


-

-Additionally, once I realized this was going to be a long term problem, I
started coating the sitting drop depressions with a thin layer of vacuum
grease.  The crystals just slid right off the grease and I never saw any
changes in the diffraction data to suggest the grease was giving me issues.

-You only need a very thin layer of the grease (i.e. keep wiping until its
almost completely gone) and it usually has no affect on the crystallization.

-Another approach is to grease the wells of the plate.  If you then gently
warm and melt the grease (use Vaseline or other petroleum jelly rather than
silicone grease) it will set almost clear.  This is sometimes used with
microbatch.

 

COAT THE CRYSTALLIZATION SURFACE WITH SILICON



You can try various siliconizing fluids.  Hampton used to sell one called
Aquasil which you mix up in water.  This will not melt the plastic as eg.
Repelcote will.

 

CHANGE CRYSTALLIZATION PLATE

-

You can also try plates made from COC (cyclic olefins), such as the "UVP"
plates made by SwissCi (sold by lots of companies including us). They are
less sticky than polystyrene plates.

COC is halfway between polystyrene and polypropylene.  Polypropylene is even
less sticky than COC but is not rigid, therefore harder to dispense to
automatically.  You can get plates made of "clarified polypropylene" from
Emerald, and you can also get polypropylene "bridges" that you place in
Linbro wells.  I think Hampton still sells them.

 

CRYSTAL BOWLING

---

If you have good and bad crystals in the same drop, I've had success pushing
a crummy crystal into a good crystal and having it release that way.

 

CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE

--

Crystals form inside the very soft gel and they are hold in place by this
meshwork.

So, they are mechanically protected and do not fall down onto the bottom of
the sitting-drop well. A final concentration of 0.1-0.2 % (w/v) agarose is
sufficient. When you harvest a crystal cut generously around it with a
microtool, pick it up (e.g. using a nylon loop) and do not mind if some
agarose comes with it.

Reference:

Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C.

Crystallization of biological macromolecules using agarose gel.

Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9

PMID: 12351881

 

FLOATING DROP CRYSTALLIZATION METHOD 

--

References

1.  Application of a two-liquid system to sitting-drop vapour-diffusion
protein crystallization. Adachi, H. et al, Acta Cryst. (2003) D59, 194-196

2.  Promotion of large protein crystal growth with stirring solution.
Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027

3.  Two-liquid hanging-drop vapour-diffusion technique of protein
crystallization. Hiroaki Ada

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

[Savvas Savvides]

Hi Fred,
I am not sure if this has been brought up already. Another possibility might
be to generously pipet your Ni- or Co-matrix of choice directly to buffered
solubilized inclusion bodies, incubate for some time (even overnight if you
feel its necessary), and then use the slurry to pack a column for further
chromatographic manipulations. To quickly check if this approach might be
promising, you can do this on a small scale in epps.  IN fact working on a
small scale like this might allow you to test a number of buffer choices and
incubation protocols. You can spin down your beads followed by several
washing steps with your column buffer. You can then go two ways: (1) either
pellet with a hard spin and just add your SDS-PAGE sample buffer to the
pellet to visualize binding on gel (make sure you boil your sample) OR (2)
'elute' your protein with your imidazole-containing buffer followed by
analysis of the supernatant via SDS-PAGE. We have found that this approach
works quite well for difficult metal-affinity chromatography cases. 


Best wishes
Savvas




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred
Sent: Wednesday, January 28, 2009 9:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

Just to let you know. No way, Talon also don't work. I am gonna try the 
GE His-trap column.

Fred wrote:
 > Hi everyone,
 > Thanks for answer my question. Just to add some more notes regarding 
to my expression system. The insert-vector (pET28) has been sequenced 
and the his-tag is N-terminal. The anti his-tag WB is positive and the 
binding buffer's pH is 8.2 (double-checked).
 > I had already experienced the same problem before, which I solved 
just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no 
binding at all.
 > I'm currently running a SDS-PAGE with samples eluted from Talon and 
let you know the results.
 > All the Best,
 > Fred
 >>
 >>
 >> --- Fred // schrieb am *Di, 27.1.2009:
 >> *
 >>
 >> *Von: Fred 
 >> Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
 >> An: CCP4BB@JISCMAIL.AC.UK
 >> Datum: Dienstag, 27. Januar 2009, 22:00
 >>
 >> *
 >>
 >> *Hi ccp4 list,
 >> I am trying to purify a his-tag protein by metal affinity 
chromatography. The
 >> protein was expressed in inclusion bodies and its his-tag 
doesn't bind the
 >> Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). 
Playing with
 >> NaCl and detergents didn't help much.
 >> Any help is appreciated.
 >> Fred   *
 >>
 >>





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[ccp4bb] sticky crystals

[Savvas Savvides]

Dear colleagues,

we have been growing crystals of a protein complex  in sitting-drop geometry
that stick to the bottom of the drop remarkably well. It's as if they are
glued onto the plastic. This makes crystal handling next to impossible
without destroying the crystals. We have tried whiskers, loops, all kinds of
micro-tools, and pipetting techniques to no avail.  I can say at the outset
that we have been unsuccessful in growing these crystals in hanging-drops or
at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we
are only able to get crystals from homogeneously glycosylated protein
produced in HEK293S/I- cells. 

 

 In the meantime we are playing with the idea of  siliconizing the
sitting-drop depressions to alter the crystal/plate interface. But then
again, nucleation events on the plastic  may be the reason we are getting
crystals in the first place. We have also thought of trying microseeding to
have more control on nucleation issues. Our protein production is quite
limiting and forces us to be very selective with our experimentation.

 

Nonetheless,  while we are waiting for fresh material  to explore some of
these ideas we would like to make the most out of the crystals we have grown
thus far. We would therefore very much appreciate any input/ideas on
manipulating these crystals for data collection.

 

Best wishes

Savvas

 

 

---- 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Katarina Moravcevic
Sent: Tuesday, January 27, 2009 10:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo translation

 

Hi all,

here is a question from a beginner. I have a home source data set  that
indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783,
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing
to get a MR solution with Phaser I ran the phenix.xtriage which showed that
I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo
translational symmetry. I was wondering if there is anything I could do with
this data to get around this problem. Given that I don't have a lot of
experience any suggestion/explanation would be fantastic. 

Thanks in advance

K





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Re: [ccp4bb] Crystals grown from high ammonium sulphate

[Savvas Savvides]

Dear Sabine,
I recently dealt with a very similar situation as follows:

-I ended up growing the crystals in 4+4 uL drops. Skin formation tends to be
less of a problem in larger drops. This kind of experimentation is of course
only possible if protein production is not a limiting factor.
-For crystal manipulation, I used to add 10-20 uL of the reservoir solution
directly to the drop. In this way I could easily manipulate the 5-6 crystals
that grew per drop within 5 minutes without any noticeable effects on the
crystals.
-I found out that cryo-cooling the crystals by plunging them into liquid
nitrogen straight out the drop was the only way to effectively cryo-cool
such crystals. In fact the crystal condition was very similar to yours (3.2
M AS, 20 mM potassium phosphate pH 6.0).

I cite the most relevant paragraph from our paper (Kyndt et al.
Biochemistry. 2007 46(1):95-105.):

"To prepare crystals for data collection under cryogenic
conditions (100 K), crystals were flash-cooled by plunging
them directly from their native drops into liquid nitrogen. A
series of cryocooling conditions using a variety of cryoprotecting
reagents such as glycerol, sucrose, PEG 400, and
paratone indicated that only crystals flash-cooled by plunging
them directly from their native drops into liquid nitrogen
produced diffraction of acceptable quality."

Best wishes
Savvas


---- 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: [EMAIL PROTECTED] 
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html 

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sabine
Schneider
Sent: Tuesday, November 04, 2008 7:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystals grown from high ammonium sulphate

Hi everyone,

We got crystals that grew in ~3.2M ammonium sulphate and some 
tris-buffer at 18dgC. Unfortunately the crystals take a while to grow 
(~4-5 weeks) and so far we only have 4-5 xtals.
I tried to freeze the crystals, but as soon as I broke though the skin 
of the drop the ammonium sulphate started to crystallise. I got the 
crystals out, froze them using sort of an artifical mother liquor with 
sodium malonate as cryo and tested the diffraction. The freezing seems 
to be OK and it is definitely a protein crystal. The crystal suffered 
when the ammonium sulphate in the drop started to crystallise, but 
didn't seem to deteriorate anymore in the cryo. Well the corners had 
already more or less disappeared by the time I got them out of the drop...
Since we only have a few xtals at the moment and I can't try out a lot 
of things, I was wondering if anyone has experienced and solved a 
similar problem? My freezing attempt so far was in an airconditioned 
room with 18dgC. I thought about higher humidity and temperature in the 
room, and/or adding the cryo directly to the drop 
Any ideas are very much appreciated!

Sabine

--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry and Pharmacy
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77846
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/





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Re: [ccp4bb] birefringent spacegroups

[Savvas Savvides]

Hi Jacob,
Chek out Section 2 in the following paper:

Echalier et al. (2004) Assessing crystallization droplets uding
birefringence.
Acta Cryst D60, 696-702.

It offers a very effective summary of the physical basis of crystal
birefringence and reiterates the classification of crystal optics based
on isotropic, uniaxial and biaxial systems. My understanding is that a
favorable crystal orientation with respect to the direction of view is
important for being able to observe appreciable birefringence from
uniaxial and biaxial crystals. This can be especially tricky to get from
biaxial crystals, i.e. crystals with orthorhombic or monoclinic or
triclinic point-group symmetry, projecting their optically anisotropic
axis in the plane of the view. Crystals with cubic symmetry are
optically isotropic and are therefore not birefringent.

Best wishes
Savvas




-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Jacob Keller
Sent: Thursday, June 12, 2008 2:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] birefringent spacegroups


Dear Crystallographers,

is there a list somewhere of spacegroups which can and cannot be 
birefringent? Upon what feature of the spacegroup does this depend?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
*** 

Re: [ccp4bb] phenix.refine and refmac

[Savvas Savvides]

Hi Yang
how many reflections do you have in your test-set for calculating
R-free? Too few reflections, typically less than 500, may not
constitute a statistically robust cross-validation data set, and thus may
lead to fluctuations in R-free plus a tendency for R-free to increase  
as a function of refinement cycle. Some of the early

publications from Axel Brunger on crystallographic cross-validation
address the need for enough reflections (>500) in the test-set. In  
addition, the presence of even a handful of strong but inaccurately  
measured/integrated low-resolution reflections in a limited test-set  
can aggravate abnormal behavior in R-free.


Best wishes
Savvas


toQuoting yang li <[EMAIL PROTECTED]>:


Dear All,
  I have post a similar question about CNS and refmac before, now in
another structure I met a similar problem. I have an almost finished
structure, the Rfree of which
is about 0.28 by refmac. Then I used phenix to refine it, below is the
result:
REMARK  REFINEMENT SUMMARY: QUICK FACTS
***
REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213
REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374
REMARK

Since the map from phenix couldnot be opened by coot directly--or I
donnot know how to--I used refmac to get a mtz map file. But I found that at
the first several cycle of
refmac the Rfree decreased, then both the R and Rfree values  continued
increasing and FOM decreasing.
 The best R/Rfree/FOM during the refinement is

-
Overall R factor = 0.1932
Free R factor= 0.2513
Overall figure of merit  = 0.8168
-
 And after 40 cycles the final result is:
-
Overall R factor = 0.2008
Free R factor= 0.2772
Overall figure of merit  = 0.7902
-
The values looks like keep going up if increase the cycles. Then which
value should I take as the final result? The phenix or the best Refmac
result or  I have to take  a converged
value from refmac?

Re: [ccp4bb] wich program to test an alternative phaser solution

[Savvas Savvides]

Hi Fred,
in future PHASER runs you can also ask the program to write out an X
number of the top solutions as separate coordinate files via the keyword
TOPFILES (e.g 'TOPFILES 10' for the top ten solutions). In the PHASER
GUI you can enter that request in the 'Define Data' block.

best wishes,
Savvas


Savvas N. Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Vellieux
Sent: Tuesday, January 15, 2008 10:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] wich program to test an alternative phaser solution


Dear all,

We have a phaser output with 2 alternative solutions. Not differing much

by the statistics. Phaser provides the solution with the highest LLG. 
However I am not convinced by that solution.

What program should be used with the input PDB and the Euler angles and 
translation parameters (found in the .sol file) to generate the 
alternative solution?

Thank you in advance,

Fred.

Re: [ccp4bb] His tag on membrane protein

[Savvas Savvides]

Hi Deliang,

My own experience with two membrane proteins suggests that His-tag  
issues pertaining to soluble proteins also seem to apply to membrane  
proteins.


In one case, for example, cleaving the N-terminal His-tag improved the  
solubility of the protein dramatically. Interestingly, expression  
levels of the protein without the His-tag were much lower than with it!


In the other case, we have a construct with a His-tag at the  
N-terminus and another at the C-term. Both behave well when it comes  
to solubility and stability but we thus far only have diffracting  
crystals from the construct with the C-term His-tag. However, we have  
not yet tried to remove the tags from those two constructs to see  
whether that improves crystallizability.


My feeling is that if you do not have solubility/stability/aggregation  
problems with your His-tagged membrane protein, then there is no  
reason to expect lower success rates in crystallization trials. Screen  
with it and hope for the best!


By the way, there is a recent paper on the effects of His-tags on  
crystal structures of soluble proteins. It's worth a look.


Carson M et al. His-tag impact on structure.
Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):295-301.


Best wishes
Savvas


Savvas N. Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html






Quoting deliang <[EMAIL PROTECTED]>:


Hi there,

I purified a membrane protein with traditional His-tag on the C   
terminal. Before crystallization, I wonder how this tag may affect   
the result. Does anyone have  experience that the removel of this   
tag may improve the result or not? or can provide some references   
which may give some statistic, like how many membrane proteins have   
been crystallized with or without His-tag?


Thanks so much.

Deliang

Re: [ccp4bb] Flexible proline?

[Savvas Savvides]

Hi Tiancen,
provided that you know for sure that your Gly->Pro mutation has been  
incorporated (see Artem's comment) I would spend some more time  
modeling your Pro.


Here are some things to consider:

(1)  If the view of the model you provided has not fooled me, I  
believe that the main-chain model for your Proline is quite atypical  
for prolines. I estimate something like phi=-90 and psi=-10. That  
would of course be just fine for a GLY. Did you model the Proline  
according to evidence in difference electron density maps, or did you  
just mutate your Glycine using a graphics program and just let REFMAC  
push it in the 'right' place. Of course at 3.3 angs resolution you  
cannot expect much on that front. What does your Ramachandran plot  
look like for the mutant and the wild-type structures at this position?


(2) Given that this is a rather drastic mutation, it may just be that  
the PRO side-chain is behaving suboptimally and is simply  
flip-flopping between the preferred chi-1 angles of +/- 30 deg. This  
could explain the absence of density around the C-gamma position.


(3) I agree with Bill Scott's suggestion to model the cis-Pro as well.  
There is a well known variant of Ribonuclease A, the P114G (Schultz et  
al 2005), in which a cis-Pro is mutated to a Gly which in turn adopts  
the trans configuration. Configurations aside, this the opposite  
mutation to yours, but I could not refrain hallucinating about a  
possible reversal of scenarios...:), i.e transGLY->cisPRO.


Best wishes
Savvas


Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html





Quoting HTC <[EMAIL PROTECTED]>:


Dear all,

Recently, I crystallized a mutant protein in which a Glycine was changed
to Proline. The resolution of the crystal is 3.35A. I performed molecular
replacement and refinement with CCP4. Surprisingly, no electronic density
can be detected for the side chain of Proline. Since Proline is always
considered rigid, I wonder if it is possible that this Proline is so
flexible that its map becomes undetectable? The density map is attached.

Thanks in advance!

Tiancen Hu
Shanghai Institute of Materia Medica
Rm. 2107, #555, ZuChongzhi Rd.
Shanghai 201203
P.R. China

Re: [ccp4bb] Solvent content of membrane protein crystals

[Savvas Savvides]

I would like to thank Michael Caravito and Gert Van den Berg for  
taking the time to share their knowledge and insights on  
protein-detergent/micelle complexes.


A series of experiments I carried out using DLS some time ago showed
that the protein-detergent/micelle complex (with LDAO as detergent)  
for the membrane protein I was studying had a hydrodynamic radius that  
was 20 angs larger than the empty-micelles.  The experiments were done  
in the protein storage buffer: 20 mM Tris 8.0, 250 mM NaCl, 0.15 %

LDAO, which is far from a crystallization condition!

As Michael and Gert pointed out, it is a risky business to
draw quantitative conclusions from such measurements due to the
effects of so many factors. Nonetheless, it was quite
informative to me at the time to see a marked difference between the
protein-detergent complex and the empty-micelles. Aside form bringing  
home the concept that the interaction of the protein with  
detergent/micelle had a true physical meaning that could be translated  
to particle augmentation, it also helped me benchmark my  
gel-filtration runs.


Best regards
Savvas


Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html

Quoting "R.M. Garavito" <[EMAIL PROTECTED]>:


Saavas and Tommi,

The questions of what is the detergent content of a membrane protein
crystal and how to explicitly determine the amount of detergent in a
crystal are extremely difficult to address.  Moreover, is it
worthwhile to even attempt to correct the Matthews coefficient?  I
personally don't for a number of reasons.  However, one point I would
like to make in this discussion is that ANYTHING concerning micellar
structure or behavior cannot be naively extrapolated to a protein-
detergent complex without firm experimental data.  Moreover, when the
protein-detergent complex is in a crystal, it gets even worse.  Very
little quantitative work has been done on what is the detergent
structure and behavior in a protein-detergent complex.  Peter Timmins
has done the most using neutron diffraction with me and Wolfram Welte
on crystalline systems, as well as in solution (one paper is below).

Pebay-Peyuola, E., Garavito, R.M., Rosenbusch, J.P., Zulauf, M., and
Timmins, P.A.  (1995)  "Detergent structure in tetragonal crystals of
porin from the outer membrane of E. coli." Structure 3, 1051-1059.

One immediate take home message is that a membrane protein IS NOT in  a
micelle, even by definition from surfactant chemistry, nor does a
membrane protein insert into a micelle. In many of the experiments on
detergent binding in surfactant chemistry using styrene beads,
detergent adsorbs onto a hydrophobic surface from single monomer
accretion, and perhaps by micelle fusion.  Hence, one should forget
about micelles when talking about a protein-detergent complex.  My
rule of thumb from experience is that an "average" membrane protein  of
about 50 KD binds about a micelle's worth of detergent, but it  would
be a mistake to assume it has all the characteristics of a free  and
pure detergent micelle.

Getting back to the amount of detergent in a crystal and the Matthews
coefficient, the detergent layer of protein-detergent complex can
behave like a hard sphere in a crystal or it can fuse with its
neighbors, depending on the detergent used.  Changing the detergent
concentration around the crystal, as we do when manipulating a  crystal
for many experiments, will change the detergent concentration  in the
crystal and can impact the detergent layer of protein- detergent
complex.  Thus, efforts to get accurate, detergent- corrected Matthews
coefficients for membrane proteins, may not be  worth worrying about.

Regards,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  [EMAIL PROTECTED]



On Sep 24, 2007, at 2:29 AM, Tommi Kajander wrote:


Quoting Edward Berry <[EMAIL PROTECTED]>:


Savvas Savvides wrote:

Indeed, but wouldn't consideration of micelle size affect our
estimation of the number of molecules in the asu, in some cases
significantly?

Good point- I think now that is taken into account by just saying
"membrane proteins tend to have a high solvent content" and taking
that into consideration when you guess the number of molecules.
But it would be nice to account for the detergent explicitly.
Say by analyzing detergent content of the crystals, or in some
ideal cases neutron diffr

Re: [ccp4bb] Solvent content of membrane protein crystals

[Savvas Savvides]

Indeed, but wouldn't consideration of micelle size affect our  
estimation of the number of molecules in the asu, in some cases  
significantly?
The crystal packing of some membrane proteins shows that they tend to  
pack as "potatoes in space" with relatively few protein-protein  
contacts and with detergent micelles presumably providing the rest of  
the crystal packing interactions. That also explains the often  
significant diffraction anisotropy observed in such crystals. One  
classic example is the prototypical potassium channel structure (KCSA)  
(PDB entry 1bl8).


Savvas


Quoting Edward Berry <[EMAIL PROTECTED]>:


I would use a very general definition for "solvent",
including disordered detergent and lipids.
As you know in many cases ordered detergents and lipids
have been modeled in the coordinates, so they are part of
the model not the solvent. In some cases I think waters
should be included in the model not solvent- say for
structural waters buried in the protein at least.

Ed

Savvas Savvides wrote:


Dear colleagues,

in estimating the solvent content of membrane protein crystals it
would only seem reasonable that micelle size should also be taken   
into account. Depending on the aggregation number and MW of a given  
  detergent, the concentation of detergent used, and the buffer
conditions, one may have micelles on the order of 15-25 kDa or even  
  35-50 kDa for detergents with alkyl chains of more than 10 carbons.


However, when I took a look in a handful of papers reporting   
Matthews' numbers for membrane protein crystals, it became apparent  
 that only  the protein MW is used in such estimates. I am  
beginning  to wonder if  one should even bother reporting a  
Matthews number  for a membrane  protein crystal given the  
uncertainties surrounding  size and role of  micelles in crystal  
packing.


Any thoughts on this?

best wishes
Savvas

[ccp4bb] Solvent content of membrane protein crystals

[Savvas Savvides]

Dear colleagues,

in estimating the solvent content of membrane protein crystals it  
would only seem reasonable that micelle size should also be taken into  
account. Depending on the aggregation number and MW of a given  
detergent, the concentation of detergent used, and the buffer  
conditions, one may have micelles on the order of 15-25 kDa or even  
35-50 kDa for detergents with alkyl chains of more than 10 carbons.


However, when I took a look in a handful of papers reporting Matthews'  
numbers for membrane protein crystals, it became apparent that only  
the protein MW is used in such estimates. I am beginning to wonder if  
one should even bother reporting a Matthews number for a membrane  
protein crystal given the uncertainties surrounding size and role of  
micelles in crystal packing.


Any thoughts on this?

best wishes
Savvas

Re: [ccp4bb] the worst molecular replacment probes

[Savvas Savvides]

The paper below can put things in perspective a little bit.


Bernstein and Hol (1997) Acta Crystallogr D Biol Crystallogr.  53(Pt  
6):756-64.
Probing the limits of the molecular replacement method: the case of  
Trypanosoma brucei phosphoglycerate kinase.


best wishes
Savvas

[ccp4bb] pseudo-translation vectors in molrep vs other programs

[Savvas Savvides]

Dear colleagues,
I would like to thank J. Murray, J. Wright, K. Futterer, E. Dodson, A. Forster,
and F. Long for responding to my posting of two days ago on pseudo-translation
vectors in molrep vs other programs (see original posting at the end of this
message).
I should have said at the outset that we are dealing with a limiting data set
(see stats below), but since this is the only data we were ever able to collect
on this membrane protein, we have no option but to milk it as much as we can.

P21 with 104.82  151.28  109.49   90.00  118.13   90.00
Resolution: 30-4.2 angs (4.3-4.2)
Rmeas=0.15 (0.380)
I/sigma: 7.2 (1.9)
Completeness=93% (75%)
Redundancy= 2.3 (2.1)
Mosaicity= 1.1 deg
High data anisotropy, primarily along the K reciprocal axis.

The comments from Eleanor Dodson and Klaus Futterer prompted me to take another
look at the data frame per frame. I concluded that in several frames there were
a few reflections in the 40-30 angs range that obviously did not fit my
spot-integration strategy very well.  After failing repeatedly to get them to
integrate acceptably without compromising the rest of the data too much, I
decided to exclude all reflections between 40 and 30 angs res.

This has resulted in three important improvements:

(1) Better data integration and scaling statistics across the board.
(1) The spurious peaks clustering around the origin in the native patterson are
fewer, and those that do remain have a peak-height around 10-12% of the origin.
(2) The new data set has yielded unambiguous peaks in the self-rotation function
consistent with a 2-fold NCS axis.

I have now used this SRF peak in MolRep and came up with a reasonable MR
solution. I will soon try to implement this SRF info in PHASER as well via the
"Rotate around" option.

Best regards
Savvas


Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html



> > Dear colleagues,
> >
> > For a particular MR problem I am dealing with, 'analyse_mr' suggests
> > that there maybe a pseudo-translation vector as evidenced by the very
> > significant non-origin peaks in the native patterson: e.g
> >
> > GRID  80 112  80
> > CELL  104.8290  151.2840  109.4910   90.  118.1310   90.
> > ATOM1   Ano   0.  0.  0.  181.08  0.0 BFAC  20.0
> > ATOM2   Ano   0.9483  0.  0.0106   46.89  0.0 BFAC  20.0
> > ATOM3   Ano   0.0517  0.  0.9875   46.89  0.0 BFAC  20.0
> > ATOM4   Ano   0.9494  0.9911  0.0090   40.66  0.0 BFAC  20.0
> > ATOM5   Ano   0.0506  0.9911  0.9875   40.66  0.0 BFAC  20.0
> > ATOM6   Ano   0.0572  0.9911  0.   37.26  0.0 BFAC  20.0
> >
> > BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007,
> > i.e. very similar to the output from 'analyse_mr'.
> >
> > Yet, Molrep fails to recognize this possibility (in "auto' mode for
> > the PST) claiming that the 0.125 limit for the peak height compared to
> > the origin has not been reached. When I look at the output from
> > 'analyse_mr' it is quite clear the peak is at 0.25 of the origin peak.
> >
> > Why is there such a discrepancy in the interpretation of the native
> > patterson map?
> >
> > Best regards
> > Savvas

[ccp4bb] pseudo-translation vector in molrep

[Savvas Savvides]

Dear colleagues,

For a particular MR problem I am dealing with, 'analyse_mr' suggests
that there maybe a pseudo-translation vector as evidenced by the very
significant non-origin peaks in the native patterson: e.g

GRID  80 112  80
CELL  104.8290  151.2840  109.4910   90.  118.1310   90.
ATOM1   Ano   0.  0.  0.  181.08  0.0 BFAC  20.0
ATOM2   Ano   0.9483  0.  0.0106   46.89  0.0 BFAC  20.0
ATOM3   Ano   0.0517  0.  0.9875   46.89  0.0 BFAC  20.0
ATOM4   Ano   0.9494  0.9911  0.0090   40.66  0.0 BFAC  20.0
ATOM5   Ano   0.0506  0.9911  0.9875   40.66  0.0 BFAC  20.0
ATOM6   Ano   0.0572  0.9911  0.   37.26  0.0 BFAC  20.0

BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007,
i.e. very similar to the output from 'analyse_mr'.

Yet, Molrep fails to recognize this possibility (in "auto' mode for the
PST) claiming that the 0.125 limit for the peak height compared to the
origin has not been reached. When I look at the output from 'analyse_mr'
it is quite clear the peak is at 0.25 of the origin peak.

Why is there such a discrepancy in the interpretation of the native
patterson map?

Best regards
Savvas



Savvas N. Savvides
[EMAIL PROTECTED] for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html

Re: [ccp4bb] /usr/lib/libGL.so.1: no version information available

[Savvas Savvides]

Dear Paul,
thanks for confirming that this is a benign remark from Coot. In the 
meantime, I

got replies from other users who are seeing the same thing on their systems.

best regards
Savvas


Quoting Paul Emsley <[EMAIL PROTECTED]>:


On Fri, 2007-06-01 at 14:35 +0200, Savvas Savvides wrote:

Dear Coot users and developers,
upon starting up Coot-0.3.1 as distributed in the CCP4_v6.0.2 
binaries for Red

Hat 8 we get the following:

current_exe_dir is /usr/local/CCP4_v6.0.2/Coot-0.3.1/bin
COOT_PREFIX is /usr/local/CCP4_v6.0.2/Coot-0.3.1
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: 
/usr/lib/libGL.so.1: no version

information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real)
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: 
/usr/lib/libGL.so.1: no version

information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/lib/libgtkgl.so.5)
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: 
/usr/lib/libGL.so.1: no version

information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/lib/libglut.so.3)

We are running RHEL4 with FX3500 or FX1400 NVIDIA cards and the 9746 nvidia
drivers. I am quite intrigued by these messages (which we also get for our
installation of Coot-0.1.1) while Coot appears to woek fine. Nonetheless, I
cannot help but wonder about any possible loss of functionality.


Dear Savvas Savvides,

These occur as a result of executing:

  glXGetClientString(GDK_DISPLAY(), GLX_VENDOR)
  glXGetClientString(GDK_DISPLAY(), GLX_VERSION)
  glXGetClientString(GDK_DISPLAY(), GLX_EXTENSIONS)

If your libGL does not have that info you get those message.  On a
machine here I get

VENDOR : NVIDIA Corporation
VERSION: 1.3
EXTENSIONS : GLX_ARB_get_proc_address GLX_ARB_multisample
GLX_EXT_visual_info GLX_EXT_visual_rating GLX_EXT_import_context
GLX_SGI_video_sync GLX_NV_swap_group GLX_NV_video_out GLX_SGIX_fbconfig
GLX_SGIX_pbuffer GLX_SGI_swap_control GLX_NV_float_buffer
GLX_ARB_fbconfig_float

It helps me debug what is going on sometimes.  Those messages are
harmless, there is no loss of functionality (I am surprised that you
don't get a sensible reply though).

Paul.

[ccp4bb] /usr/lib/libGL.so.1: in Coot

[Savvas Savvides]

Dear CCP4 users and developers,
upon starting up Coot-0.3.1 as distributed in the CCP4_v6.0.2 binaries for Red
Hat 8 we get the following:

current_exe_dir is /usr/local/CCP4_v6.0.2/Coot-0.3.1/bin
COOT_PREFIX is /usr/local/CCP4_v6.0.2/Coot-0.3.1
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: /usr/lib/libGL.so.1: no version
information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real)
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: /usr/lib/libGL.so.1: no version
information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/lib/libgtkgl.so.5)
/usr/local/CCP4_v6.0.2/Coot-0.3.1/bin/coot-real: /usr/lib/libGL.so.1: no version
information available (required by
/usr/local/CCP4_v6.0.2/Coot-0.3.1/lib/libglut.so.3)

We are running RHEL4 with FX3500 or FX1400 NVIDIA cards and the 9746 nvidia
drivers. I am quite intrigued by these messages (which we also get for our
installation of Coot-0.1.1) while Coot appears to work fine. Nonetheless, I
cannot help but wonder about any possible loss of functionality.

Best wishes
Savvas


Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html

Re: [ccp4bb] Crystallisation of a extremly soluble protein

[Savvas Savvides]

Hi Sabine,
I vaguely recall that an old colleague had used a mini detergent screen (as
additive) to solve a problem such as yours. It may be worth setting up a plate
or two to screen a few conditions before you embark into more molbio. You can
use the 96 well plates with multiple wells (Corning sells plates with 5
depressions in sitting drop geomtery). In that way you could screen up to five
detergents per condition.

best of luck
Savvas

PS. A broad additive screen may also be worth a shot for that matter.

Quoting Schneider Sabine <[EMAIL PROTECTED]>:

> Hi everyone,
>
>
>
> I am trying to crystallise an extremely soluble and charged protein. It
> is ~30kDa and has an estimated PI of 5.2 and theoretical charge over pH
> range 4-10 from + 24 to -29. It is still happy at a concentration of
> 190mg/ml and fully reconstituted with its ligand.
>
>
>
> I have tried high throughput crystallisation with 10 different screens
> from Nextal with concentrations of  60, 100 and 150mg/ml with no NaCl
> and NaCl concentrations of 100mM, 300mM and 1M in either Hepes pH 8 or
> Tris-HCl pH 7.5.
>
>
>
> The distribution of heavy precipitation, light crystalline precipitation
> and clear drops through out the screens locks like I am in the right
> concentration range around the 100mg/ml, but I am not getting any real
> hit. There are some drops with extreme phase separation. I also tried
> changing the temperature from 20C to 4C.
>
>
>
> I chased up a few conditions with this strong phase separation (or where
> I imagined little objects...) by manual screening and also adding
> additives like 3% Succrose, 50-200mM LiCl, 100mM EDTA, varying the PEGs
> (1500, 3350, 4000, 6000, 8000) as well as adding NaCl  to the reservoir
> solution in sitting as well as hanging drop screens. But I am just
> getting nowhere - either just precipitation or the drop stays clear with
> the strong phase separation.
>
>
>
> I also re-cloned it with chopping of a few more residues on the N-term
> where according to a secondary structure prediction a helix starts and
> it is still very happy at high concentrations, but again nothing in the
> high-throughput screens.
>
>
>
> Has anyone any suggestions what else I could try?
>
>
>
> Thanks!
>
>
>
> Sabine
>
>
> This message has been checked for viruses but the contents of an attachment
> may still contain software viruses, which could damage your computer system:
> you are advised to perform your own checks. Email communications with the
> University of Nottingham may be monitored as permitted by UK legislation.
>
>


--

Re: [ccp4bb] Fancy crystal, poor diffraction

[Savvas Savvides]

Dear Tiancen,
for a survey of post-crystallization options, I highly recommend the following
review article:

Heras B, Martin JL. 2005
Post-crystallization treatments for improving diffraction quality of protein
crystals.
Acta Crystallogr D Biol Crystallogr. 61, 1173-80.

best of luck
Savvas




Quoting Tiancen Hu <[EMAIL PROTECTED]>:

> Dear all,
>
> Sorry for the non-CCP4 question. I think this is an old story but our
> knowledge to deal with it is very limited. So any suggestions will be greatly
> appreciated.
>
> We have crystallized a 21KD protein with 2 disulfide bonds grown for one
> month in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)2SO4 and 1M Li2SO4. The
> crystals look big (~0.4mm x 0.4mm x 0.3mm) and pretty (sharp edge, clean
> surface) but diffracted to only 4A in-house. The spots are quite strong and
> isotropic at low resolution but decay sharply beyond 5-6A. The crystal
> belongs to P4 pointgroup (P422 is also possible) with cell parameters of
> 127.6, 127.6, 162.5, 90, 90, 90. The solutions we can think of to elevate its
> diffraction ability are as follows:
>
> 1)Try synchrotron radiation
> 2)Try a lot of similar crystals and hope one of them diffracts better than
> others
> 3)Let the crystals grow for a longer time and hope it could pack more
> ¡°orderly¡±
> 4)Additive screen based on the original condition
> 5)Check the original plates for other crystallizing conditions
> (unfortunately until now this is the only one out of ~300)
> 6)Screen with other forms of the protein, i.e., N/C-terminus truncated 
> ones,
> complexed with its ligands etc.
>
> I believe many protein crystallographers have encountered similar problems,
> are there any successful stories from these fancy poor crystals? Any
> suggestions or references will be highly appreciated.
>
> Thanks in advance!
>
> Tiancen Hu
> Shanghai Institute of Materia Medica
> Rm. 2107, #555, ZuChongzhi Rd.
> Shanghai 201203
> P.R. China
> Tel: +86-21-50806600 ext 2107
> Email: [EMAIL PROTECTED]
>
>


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