[ccp4bb] job offer - slightly off topic

[Karolina Michalska]

Argonne National Laboratory has the following job offer:  

Requisition Number: 405283 

Position Title: Postdoctoral Appointee  

Career Level: 700  

Division: ASD-Accelerator Systems Division  

Location: Lemont, IL  

Offsite Work Location: ANL  

Shift: 8:30 - 5:00  

Hours of Work: 40 

The Accelerator Systems Division (ASD) of the Advanced Photon Source
(APS) is offering a Postdoctoral position. You will work with a
multidisciplinary team of scientists and engineers to carry out magnetic
measurements and tuning of APS insertion devices (ID) and develop your
own program in the enhancement of magnetic measurement capabilities at
the APS. You will also have the opportunity to work in the areas of
novel ID design as well as participate in the development and
implementation of customized measurement techniques for future
generation light sources. 

We expect you to have: 

A strong background in experimental system design, build-up, and
commissioning. 

Good skills in running measurements, data collection and analysis. 

Familiarity with programming languages including Python, Matlab, C/C++,
and LabVIEW. 

Please, contact  Joseph Xu (x...@anl.gov) with informal inquires.



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

[ccp4bb] Beamline Scientist position at ANL

[Karolina Michalska]

Dear all, 

I'd like to draw your attention to the beamline scientist position
currently open at the Structural Biology Center, ANL. 

Job ID: 403448
Job Title: Beamline Scientist/Physicist
Job Details Link:
http://www.anl.gov/careers/apply-job/external-applicants?locale=en-us&cpUrl=https%3A%2F%2Fcareers.peopleclick.com%2Fcareerscp%2Fclient_argonnelab%2Fexternal%2Fen-us%2Fgateway.do%3FfunctionName%3DviewFromLink%26jobPostId%3D5827%26localeCode%3Den-us


For informal inquiries, please contact Andrzej Joachimiak,
andrz...@anl.gov 

Best, 

Karolina

Re: [ccp4bb] mystery feature near a PLP substrate

[Karolina Michalska]

Hi, 

The Lys-PLP adduct looks like a perfectly happy internal aldimine. I do
not see any indication of a covalent bond between PLP and the density in
question indicating any other intermediate. Which, to my knowledge,
would be rather hard to get accidentally. I do not know distances
between these fairly well resolved peaks in the 2Fo-Fc map but I am
guessing they would nicely accommodate solvent molecules. The peak
closer to PLP is weaker, maybe just another partly occupied solvent
molecule? Could the continuity of your difference map be coming from too
low contour level? 

Best, 

Karolina  

W dniu 2017-05-24 09:16, Eleanor Dodson napisał(a):

> Any ideas?

Re: [ccp4bb] cryoprotectant

[Karolina Michalska]

 

Hi Reza, 

Check the following reference: 

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

Bujacz G, Wrzesniewska B, Bujacz A. 

Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. 

Cheers, 

Karolina 

W dniu 2014-12-01 10:59, Reza Khayat napisał(a): 

> Hi,
> 
> Has anyone used citrate as the sole cryoprotectant? If so, 
> what concentration was needed?
> 
> Best wishes,
> Reza
> 
> Reza Khayat, PhD
> Assistant Professor
> The City College of New York
> Department of Chemistry, MR-1135
> 160 Convent Avenue
> New York, NY 10031
> Tel. (212) 650-6070
> www.khayatlab.org [1]

 

Links:
--
[1] http://www.khayatlab.org

Re: [ccp4bb] High Salt Cryo

[Karolina Michalska]

 

4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M
NaCl. 

Karolina 

W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a): 

> For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M AmmSO4, 
> we do in situ cryoprotection with sodium malonate. We add 2-4 uL of 1.9 M Na 
> malonate to the crystallization drop, wait 10 seconds and add 2-4 uL of 2.4 M 
> sodium malonate, repeat with 2.8 M and then 3.4 M. We do not bother 
> withdrawing aliquots to maintain a fixed volume. You may need to tweak the 
> volumes to optimize the resulting diffraction. You can also break the 
> additions at given concentration into smaller aliquots to reduce the osmotic 
> shock. This approach is much gentler than transferring the crystal directly 
> to 3 M sodium malonate. Do not leave the drop exposed to the air for more 
> than 3 minutes or so because salt crystals will start to grow. When there are 
> multiple crystals in a drop, often the unused crystals in the very high salt 
> solution will still diffract well up to a year later if the crystallization 
> chamber is resealed well; their diffraction might even improve with the 
> prolonged exposure
to high salt. 
> 
> Blaine Mooers
> Assistant Professor
> Department of Biochemistry and Molecular Biology
> University of Oklahoma Health Sciences Center
> S.L. Young Biomedical Research Center Rm. 466
> 
> Shipping address:
> 975 NE 10th Street, BRC 466
> Oklahoma City, OK 73104-5419
> 
> Letter address:
> P.O. Box 26901, BRC 466
> Oklahoma City, OK 73190
> 
> office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910
> e-mail: blaine-moo...@ouhsc.edu
> 
> Faculty webpage: 
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
>  [1]
> 
> X-ray lab webpage: 
> http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
>  [2]
> 
> Small Angle Scattering webpage: 
> http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
>  [3]
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katherine 
> Sippel [katherine.sip...@gmail.com]
> Sent: Tuesday, February 18, 2014 12:08 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] High Salt Cryo
> 
> Hi all,
> 
> I'm looking for a cryo condition for high NaCl (3+ M) crystallization 
> condition. I would do it the proper way, but our beam/cryostream is down.
> 
> I've tried a bunch of things at the moment. Ethylene glycol and PEG 400 nuke 
> the crystals immediately even at low concentrations. Prolonged exposure to 
> glycerol and sucrose starts to break them down so I'm thinking that the 
> diffraction will probably suffer. I can't find any reports of NaCl's 
> viability as a cryosalt. I've got Paratone/Paraffin oil/Mitegen's LV cryo oil 
> on tap but I was hoping to not put all my eggs in one basket.
> 
> I tried the ISRDB database through 
> archive.com  [4]> without any luck (no search function). I've gone to the PDB searching 
> for similar crystallization conditions and looked up the papers for their 
> cryos, but they are all glycerol. Google gives me the same.
> 
> I thought I'd see if anyone on the bb has an anecdotal "this worked for us" 
> story. I would love to hear it.
> 
> Thank you for your time,
> Katherine
> 
> --
> "Nil illegitimo carborundum" - Didactylos

 

Links:
--
[1]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
[2]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
[3]
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
[4]
https://urldefense.proofpoint.com/v1/url?u=http://archive.com&k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0A&r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e

[ccp4bb] which dimer?

[Karolina Michalska]

 

Hi all, 

I'm working with a protein that appears to be a dimer in solution, on
SEC in runs as 24 kDa, while the actual mass of a dimer is 30. And I am
trying to figure out which dimer is the biological one (it is a
regulatory protein but details are uknown). The crystal structure gives
me a few options (mol A and B in ASU plus P6122 symmetry), but none of
them is really convincing: for deltaGdiss PISA goes from -0.4 to 1.5
kcal/mol. 

Theoretically, I have two compact dimers (1 and 2) and one elongated
(3). 

In 1, I have two hydrophobic helices interacting and four hydrogen
bonds, 1550 A2 buried area (out of 14200 total). This interface applies
only to molecule A and its crystallographic mate, equivalent molecules B
are too far from each other. Moreover, even in the dimer made of mol A
there are channel at the interface. 

Interface 2 is purely hydrophobic, but at least it's consistent, i.e
there are comparable interactions for A-A and B-B pairs, 1850 A2 buried
area 

Interface 3 involves non-crystallographic copies, buried area is 1040
A2. The interacting elements are proline-rich, and there are four
main-chain - main chain hydrogen bonds plus two main-chain - side chain
ones. Formally, these fragments are not classified as beta-strands, but
the association does look like an intermolecular beta-sheet. This dimer
is not consistent with the SEC data though. I'm assuming that with an
elongated shape it would run as a bigger particle than it actually is,
not as a smaller one. 

So I think I can discard first option but I am still debating on 2 and
3. I'll appreciate your comments on this. 

Karolina 
 

[ccp4bb] small proteins that do not crystallize

[Karolina Michalska]

Hello everybody,

I'm looking for small proteins (up to 150 aa) that are known to express
reasonably in E. coli, are soluble and folded but failed to crystallize.
Can you provide some examples, especially of proteins that might be
interesting for a broad community?

Thanks,
Karolina

Re: [ccp4bb] The effect of His-tag location on crystallization

[Karolina Michalska]

You may want to have a look at 3ur7 and 3ur8. I have also another example
of a glycohydrolytic enzyme with an N-terminal His-tag sitting in the
active site of a symmetry-related molecule (not deposited yet).

Karolina



On 27/6/2012, "Brad Bennett"  wrote:

>I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase)
>that mediated crystal contacts with a symmetry related molecule. As I
>recall, this tag composed a B-strand that formed a nice interface with a
>"native" B-strand of the symmetry related molecule. Pretty cool...
>
>-Brad
>
>On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice  wrote:
>
>> With Flp recombinase - DNA complexes, a C-terminal His tag triggered a
>> different (but sadly not better) crystal form, and the His side chains
>> packed against the bases at the end of a neighboring DNA duplex.
>>
>> =
>> Phoebe A. Rice
>> Dept. of Biochemistry & Molecular Biology
>> The University of Chicago
>> phone 773 834 1723
>>
>> http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
>> http://www.rsc.org/shop/books/2008/9780854042722.asp
>>
>>
>>  Original message 
>> >Date: Wed, 27 Jun 2012 10:14:58 -0400
>> >From: CCP4 bulletin board  (on behalf of "R. M.
>> Garavito" )
>> >Subject: Re: [ccp4bb] The effect of His-tag location on crystallization
>> >To: CCP4BB@JISCMAIL.AC.UK
>> >
>> >   Most of the comments you will get will be anecdotal
>> >   in that people will report the successful results
>> >   and do not take the time or effort to characterize
>> >   the less successful results.  This often occurs
>> >   because the tagged portion of the protein is most
>> >   often disordered, even in the best crystals.  Thus,
>> >   other than saying "tagging on this end works, but
>> >   tagging on that end doesn't," there is little more
>> >   you can say.  Each case will be different, and it is
>> >   almost impossible to arrive at any generalized
>> >   conclusion.
>> >   We prefer C-terminal tagged proteins for a number of
>> >   reasons, but if an N-terminally tagged protein
>> >   crystallizes well, so be it.  Of the dozens of N-
>> >   and C-tagged protein structures we have solved in my
>> >   lab and with collaborators, I have only seen one
>> >   case of an ordered His-tag:  the His residues had
>> >   coordinated Cd ions, which proved essential for
>> >   getting good crystals.  However, beyond that there
>> >   was not much more to say.
>> >   For your protein and the resulting crystals, an
>> >   N-terminally tagged protein crystallized well.
>> >Whether you can draw any more conclusions from
>> >   these results depends on characterizing crystals of
>> >   both N- and C-tagged proteins.  Just assuming that
>> >   the C-tagged protein is trying to crystallize in the
>> >   same or related crystal form as the N-tagged protein
>> >   is an unwarranted assumption without experimental
>> >   evidence to back it up.  That is why most groups
>> >   just run with the winner.
>> >   Cheers,
>> >   Michael
>> >   
>> >   R. Michael Garavito, Ph.D.
>> >   Professor of Biochemistry & Molecular Biology
>> >   603 Wilson Rd., Rm. 513
>> >   Michigan State University
>> >   East Lansing, MI 48824-1319
>> >   Office:  (517) 355-9724 Lab:  (517) 353-9125
>> >   FAX:  (517) 353-9334
>> >Email:  rmgarav...@gmail.com
>> >   
>> >   On Jun 26, 2012, at 9:06 PM, weliu wrote:
>> >
>> > Dear all,
>> >
>> > We crystallized a protein and found that crystal
>> > quality greatly depended on the location of
>> > His-tag. When a His-tag was added at the
>> > C-terminus, only crystalline precipitate or
>> > spherical quasi crystals were grown. However, when
>> > the His-tag was moved to the N-terminus, single
>> > crystals were grown under a number of conditions,
>> > and the best one diffracted to 1.7 angstrom after
>> > optimization. I was wondering if there were
>> > published reports describing similar cases.
>> >
>> > Thank you in advance
>> >
>> > Wei Liu
>>

[ccp4bb] Buster & ions

[Karolina Michalska]

Hello,

Can someone explain how to deal with ion restraints in Buster? I have
zinc and chloride modeled and I get the following info: No ccp4 atom
type ZN+2UN so will use TNT bad contact term. The same for chloride.
Interestingly, the charges have been assigned correctly and Buster
reports that contact.dat dictionary defining metal distances is used.

Thanks,
Karolina

[ccp4bb] twinned refinement

[Karolina Michalska]

Hello everyone,

I'm having some troubles refining against twinned data. The space group
is I4, twin fraction 35%, 1.5 A resolution. I have selected Rfree set in
Phenix using lattice symmetry, so I believe twin operator was taken into
account.  Twinned refinement in Refmac without TLS gives R/Rfree
0.148/0.173, which seems OK, but I wanted to try TLS refinement and then
the program crashed with something like:

 ***TLS refinement cycle***5

   1
  6.3690789E-02  5.8194529E-02 -0.1404448 -1.0440781E-02
-4.1422732E-03
  1.5739001E-02
  9.1476954E-04 -5.5926095E-04  4.0188796E-04  1.7031586E-04 
2.0264904E-04
 -1.7866226E-04
  7.5498177E-04 -1.2186267E-03  1.7816454E-04  1.3809106E-03 
7.6890434E-03
  1.4953420E-03 -2.4529384E-03 -5.5452576E-04
 -0.1526335
 Problem
 xyz 2420   7.378197  -3.33  0.5405517
-2.3370266E-02
  0.3058620  0.3236508 -2.1583334E-02 -2.1171659E-02
-2.8666053E-03
  2.4862900E-02 -2.3370266E-02  0.2921907  0.3391090
-4.1098855E-03
 -0.8417150 -0.5399062
***
This is from Refmac_5.5.0109 but I have also tried Refmac_5.5.0110 and
different number of TLS groups always with the same result.

I have also tried phenix (ver.1.7-650) with and without TLS, but only in
the first macrocycle both R and Rfree go down; in the subsequent cycles
Rfree increases significantly.

I will appreciate your comments on that.

Karolina

[ccp4bb] ligand density

[Karolina Michalska]

Hi all,

I have a ligand-corresponding density which I cannot identify:

http://s1194.photobucket.com/albums/aa376/dziuba22/?action=view¤t=coot1.png

Here is the story. I collected a couple of datasets, three of them belong
to methylated protein and one to non-methylated. The protein is putative
chitinase.
Methylation reaction was carried out in a buffer composed of HEPES, NaCl,
BME, imidazole, glycerol. For the reaction, formaldehyde and
dimethylamine borane complex were added and reaction was quenched with
glycine. After that, buffer was exchanged to HEPES, NaCl and DTT. The
non-methylated protein was stored in the same buffer. Both proteins are
His-tagged.
The density that I'm showing here is present only in the crystals of
methylated protein, both were grown form PEG3350 and either NaI or NaSCN.
I have been trying to model something and histidine nicely fits on the
left side, but right fragment seems to be more complicated. In terms of
geometry, D-Thr-L-His would fit:

http://s1194.photobucket.com/albums/aa376/dziuba22/?action=view¤t=coot4.png

But the problem is that instead of CB from Thr I need a proton donor to
interact with a neighboring Asp (unless it is C-H...O interaction).
Moreover, if such a compound were grabbed from bacterial cells I would
expect it to be present also in non-methylated
crystals, which is not the case. This made me think of some sort of
intermediate of methylation reaction as dimethylamine borane complex
would fit in Thr position with nitrogen atom replacing CB, but with
borane I could not find an explanation for an equivalent of Thr amino
group. Moreover, I do not know what was the source of free histidine in
the methylated protein prep.

A separated big peak on right corresponds to an anion and it is also
conserved in methylated crystals. In case of crystals obtained from NaI
it is an iodide anion.

I will appreciate any suggestions,

Karolina

[ccp4bb] ADIT Validation server & Ramachandran outliers

[Karolina Michalska]

Hi all,

Can anyone tell me, why ADIT Validation server says that residues that
are in the generously allowed regions of Ramachandran plot are outside
the expected regions? Is there something new in the validation tools of
the server? Two days ago I validated the same pdb and in the validation
summary these residues were not listed as outliers.


Karolina 
-- 
***
Karolina Michalska, PhD Student
Dept. of Crystallography
Faculty of Chemistry
A. Mickiewicz University
ul. Grunwaldzka 6
60-780 Poznan, Poland
***

[ccp4bb] : density at crystallographic symmetry axis

[Karolina Michalska]

Dear all,

I have a 2A structure in C2 space group which is already refined quite
well (R=0.194, Rfree=0.229). It is MR solution of the same protein but
the crystal form is different. Everything seems to be OK apart from the
density on a symmetry axis. The density is not very well defined but it
seems to belong to the protein chain. Specifically, to the loop that is
ordered in the model structure and fits also into this density. I cannot
see any alternative conformation for this region. Does the protein chain
on a symmetry axis make a physical sense? Or maybe I have something
wrong here? 

Karolina


-- 
***
Karolina Michalska, PhD Student
Dept. of Crystallography
Faculty of Chemistry
A. Mickiewicz University
ul. Grunwaldzka 6
60-780 Poznan, Poland
***