Re: [ccp4bb] help needed with a rabbit-head-shaped blob

[James Gordon]

Or dumbo the elephant.
[Image]

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From: CCP4 bulletin board  on behalf of Frank Von Delft 

Sent: Saturday, November 3, 2018 7:41:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] help needed with a rabbit-head-shaped blob

It's more a Pacman ghost, innit??

Sent from Nine

From: Deborah Harrus 
Sent: Friday, 2 November 2018 21:25
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] help needed with a rabbit-head-shaped blob


Dear all,

I came across an unidentified rabbit-head-shaped blob and would need help for 
its identification. There are 2 molecules of protein per asymmetric unit but 
there are some differences between the two chains. The blob is located in 
between the two chains, and is surrounded by residues Asp, Pro, and Val.

The protein, a glycosyltransferase, was expressed in E. coli BL21(DE3) and 
purified on Ni-NTA followed by gel filtration. The purification buffer included 
Sodium phosphate and NaCl. The crystallization condition had Bis-Tris, ammonium 
acetate and PEG 2, and glycerol was used as cryoprotectant. From the size, 
bis-tris was the most probable, but I have tried to fit it and it is not 
convincing.

The structure is 1.6 angstrom resolution and this is the only thing left to be 
done, it's driving me crazy!

Pictures attached show the face, bottom and top of the head. 2Fo-Fc is at 1.1 
sigma, Fo-Fc at 3 sigmas.

Many thanks in advance for your suggestions!

Regards,
Deborah.



=
Deborah Harrus, PhD.

University of Oulu / Faculty of Biochemistry and Molecular Medicine
Aapistie 7 A, 90220 Oulu
Finland

office: F123B
email: deborah.har...@oulu.fi
phone: +358 50 3502387 / +358 44 2386351
http://www.oulu.fi/fbmm/node/20603
=



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Re: [ccp4bb] suggestions for cryoprotectant

[James Gordon]

This kit from molecular dimensions allows you to test a variety for cryo 
protectants
https://www.moleculardimensions.com/products/3732-CryoProtX/

Best

James
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From: CCP4 bulletin board  on behalf of Joseph Brock 

Sent: Sunday, October 21, 2018 2:08:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] suggestions for cryoprotectant

Dear Friduous,

This is also a good paper to look at once you know your crystals diffract at 
room temperature:
https://www.ncbi.nlm.nih.gov/pubmed/10944336

Many of the salts in your condition will be cryo-protective if you increase 
their concentration to saturated levels.

Cheers,
Joseph.


Joseph S. Brock | PhD
Researcher
Drew Lab
Department of Biochemistry and Biophysics
Stockholm University
From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Sunday, 21 October 2018 1:00:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 19 Oct 2018 to 20 Oct 2018 (#2018-267)

There are 5 messages totaling 760 lines in this issue.

Topics of the day:

  1. suggestions for cryoprotectant (4)
  2. RapiData at SSRL 2019



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--

Date:Fri, 19 Oct 2018 17:23:49 -0700
From:Christine Gee 
Subject: Re: suggestions for cryoprotectant

Dear Fridous

Have a look at https://www.ncbi.nlm.nih.gov/pubmed/14646118





Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown 
macromolecular crystals. - PubMed - NCBI


www.ncbi.nlm.nih.gov

Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2356-8. Epub 2003 Nov 
27.


Malonate as a cryoprotectant

Or https://www.ncbi.nlm.nih.gov/pubmed/20606259





Cryoprotection properties of salts of organic acids: a case study for a 
tetragonal crystal of HEW lysozyme. - PubMed - NCBI


www.ncbi.nlm.nih.gov

Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. doi: 
10.1107/S0907444910015416. Epub 2010 Jun 19.



Regards
Christine

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
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--

Date:Sat, 20 Oct 2018 02:21:41 +
From:"Smith, Clyde" 
Subject: RapiData at SSRL 2019

Hi everyone,

Applications are now open for the 2019 edition of RapiData at SSRL. RapiData is 
a practical course in macromolecular X-ray diffraction data collection, data 
processing and structure solution. The aim of the RapiData course is to educate 
and train young scientists in data collection and processing methods at 
synchrotron beamlines, using state-of-the-art software and instrumentation. The 
course will be held at the Stanford Synchrotron Radiation Lightsource (SSRL) 
located on the SLAC National Accelerator Laboratory campus in Menlo Park, CA, 
from May 5-10 2019. Course organizers for 2019 are Silvia Russi, Clyde Smith 
and Jeney Wierman.

The course will comprise hands-on experiments at the SSRL beamlines, software 
tutorials, and lectures on the following topics:
Specimen preparation, Data collection strategies, X-ray light sources, X-ray 
detectors, Data reduction, Structure solution by MAD, SAD and Molecular 
Replacement, Complementary methods (spectroscopy and small angle scattering)

Please visit http://smb.slac.stanford.edu/news/rapidata/rapidata-2019/ and use 
the links on the "registration" page to apply for the course. The application 
deadline is December 31 2018 Successful applicants will be notified early in 
January 2019