[ccp4bb] PhD and postdoc positions at Heidelberg University Biochemistry Center

[Klemens Wild]

*PhD and Postdoctoral Fellowship**s**- Structural biology**in 
***co-translational processes and **protein *homeostasis

*

Heidelberg University Biochemistry Center, University of Heidelberg, Germany

The group of Prof. Irmgard Sinningat the Heidelberg University 
Biochemistry Center(BZH) located in the picturesque Heidelberg of 
southwest Germany offers PhD and postdoctoral fellow/associate positions 
tohighly motivated scientists interested in tackling challenging 
questions in co-translational processes and protein homeostasis. We use 
an integrated approach of structural biology applying X-ray 
crystallography, single particle cryo-EM and complementary biophysical 
and biochemical techniques, and have constant access to the ESRF and 
state-of-the-art cryo-EM setup (Glacios and KRIOS/K3). We are part of an 
agile scientific community and have a long standing interestin 
unraveling detailed mechanismsof macromolecular machines from large 
protein/RNA complexes to membrane proteins. For details visit:


www.bzh.uni-heidelberg.de/sinning/ 



We are looking forward to your application including a CV, (degree) 
certificates, a list of publications, a letter of motivation and two 
letters of reference. Please send your application as single pdf 
document or ask for more information by email to: 
irmi.sinn...@bzh.uni-heidelberg.de 





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Re: [ccp4bb] Flexible C terminus

[Klemens Wild]

On 12.03.20 08:53, chitra latka wrote:

Dear All,

I am working on a protein that has flexible C terminus. None of the 
available structures even in homologs have density for C term region 
(around 20 odd residues). All the available pdb entries have missing 
density for these 20 residues at C terminus.


I am going to try my luck crystallising the entire protein in hope of 
getting density for C term residues as well (Fingers crossed).


Has anyone faced a similar problem where they have managed to get 
density for a flexible terminus successfully?


Any suggestions would be appreciated.

Cheers !

Chitra Latka




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Dear Chitra

I would nevertheless try. Sometimes flexible termini fold back either in 
cis or in trans (crystal packing, a case I just had Yesterday) and you 
might learn sth important for biological regulation if you are lucky. At 
the same time I would truncate the terminus and crystallize the globular 
domain in parallel.


Good luck

Klemens




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[ccp4bb] Postdoctoral fellowships in structural biology at Heidelberg University Biochemistry Center

[Klemens Wild]

*Postdoctoral Fellowships: Structural Biology / Molecular machines in 
protein targeting and ribosome biogenesis*


The lab of Prof. Irmi Sinning at the Biochemistry Center (BZH) of 
Heidelberg University seeks to recruit 4 outstanding postdoctoral 
researchers for a period of 2--5 years.


BZH is a leading research center with a collaborative and 
interdisciplinary atmosphere. Prof. Sinning is an investigator of the 
Cluster of Excellence:Cellnetworks (http://www.cellnetworks.uni-hd.de/) 
and studies molecular machines involved in cellular key processes using 
structural biology (http://www.uni-heidelberg.de/zentral/bzh/).


We are interested in co- or post-translational protein targeting 
pathways, membrane protein biogenesis and ribosome associated enzymes 
and chaperones. Together with the team of Prof. Ed Hurt 
(http://www.uni-heidelberg.de/zentral/bzh/) we currently develop a 
strong interest in the structural framework of ribosome biogenesis. The 
successful candidates will be encouraged to pursue these projects well 
integrated into a highly collaborative team of researchers.


Depending on previous experience and scientific interest, the projects 
will involve a combination of advanced techniques in structural biology 
(mainly X-ray crystallography, SAXS), biochemistry, biophysics (ITC, 
SLS, fluorescence spectroscopy, HX-MS), and/or molecular cell biology. 
We routinely exploit the genome of a thermophilic fungus for structural 
studies; for protein crystallization we are equipped with a 
crystallization platform (http://xtals.bzh.uni-heidelberg.de/) and we 
have steady access to major European synchrotron high brilliance X-ray 
sources.


Qualifications and experience:

We are seeking skilled and passionate biochemists/structural biologists 
who are enthusiastic to venture into this area of research. You will 
join an energetic team in the resourceful environment of BZH, surrounded 
by world-class scientists from a wide variety of fields. Applicants 
should hold a Ph.D. in structural biology, biochemistry, biophysics, or 
in a closely related area. Research experience in structural biology, 
biochemistry, biophysics or molecular cell biology is desired. Good 
knowledge of standard cloning techniques, protein expression and 
purification is required, and experience in structural biology on 
macromolecular complexes is a plus. The ability to work independently as 
well as in a team, good communication skills, and excellent knowledge of 
English are also essential.


Documents:

Please submit a pdf file with the following information: a cover letter 
describing your motivation and experience, a scientific CV including a 
list of your publications, and the names and contact information of 3 
referees.


Address for application: irmi.sinn...@bzh.uni-heidelberg.de

Contract: 2 -- 5 years (extension possible); starting date: as soon as 
possible.


Applications will be considered until the positions are filled.

Best regards,

Irmi Sinning

Prof. Dr. Irmi Sinning

Dept. of Structural Biology

Heidelberg University Biochemistry Center

INF 328

69120 Heidelberg, Germany

Re: [ccp4bb] Synthetic RNA for Crystallization

[Klemens Wild]

Hi folks,

crystallized a lot of SRP RNAs - never had success with synthetic RNA, 
even with small 30mers T7 RNA pol in vitro transcribed was always 
extremely better - moreover: I always use a 3' hammerhead which yields 
in the end 100% pure material in mg amounts - Kiyoshi Nagais protocol is 
a perfect basis.


Greetings

Klemens

Martin Hällberg wrote:

Hi,

I'll second Israels's comment. Since the yield per coupling in synthesis is 
lower for RNA than for DNA it gets really expensive over 30-35 nucleotides.
However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). 
Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. 

For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. 


See:
Batey, R.T.  Kieft, J.S. Improved native affinity purification of RNA (2007) 
RNA, 13, 1384-1389.

You are going to need milligrams of T7 RNA polymerase in the end so forget 
about transcription kits, make your own.

Cheers,

Martin


On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote:

  

Hi Michael,



we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. 
Hope it helps






J Mol Biol. 1995 Jun 2;249(2):398-408.

Crystallization of RNA-protein complexes. I. Methods for the large-scale 
preparation of RNA suitable for crystallographic studies.

Price SR, Ito N, Oubridge C, Avis JM, Nagai K.

MRC Laboratory of Molecular Biology, Cambridge, UK.

Abstract

In vitro transcription using bacteriophage RNA polymerases and linearised 
plasmid or oligodeoxynucleotide templates has been used extensively to produce 
RNA for biochemical studies. This method is, however, not ideal for generating 
RNA for crystallisation because efficient synthesis requires the RNA to have a 
purine rich sequence at the 5' terminus, also the subsequent RNA is 
heterogenous in length. We have developed two methods for the large scale 
production of homogeneous RNA of virtually any sequence for crystallization. In 
the first method RNA is transcribed together with two flanking 
intramolecularly-, (cis-), acting ribozymes which excise the desired RNA 
sequence from the primary transcript, eliminating the promoter sequence and 
heterogeneous 3' end generated by run-off transcription. We use a combination 
of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The 
RNA-enzyme activity generates few sequence restrictions at the 3' terminus and 
none at the 5' terminus, a considerable improvement on current methodologies. 
In the second method the BsmAI restriction endonuclease is used to linearize 
plasmid template DNA thereby allowing the generation of RNA with any 3' end. In 
combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be 
generated by in vitro transcription. This has proven to be extremely useful for 
the synthesis of short RNAs.



2011/3/13 Michael Thompson mi...@chem.ucla.edu
Hello All,

I am looking for some advice from some experienced RNA crystallographers. I would 
like to order some relatively short (90 bases) synthetic RNAs for 
crystallization trials. I was wondering if anyone could comment on the use of 
synthetic RNAs for crystallization. Specifically, what is the longest synthetic 
RNA that can be used for crystallization trials? I've seen some structures in the 
PDB that are up to 88 bases and are reported to have been obtained with synthetic 
constructs (3OWI - glycine riboswitch), but I don't really know if that's routine 
or if it's an exceptional case. Also, for those who have experience with the use 
of synthetic RNAs, I was wondering where people generally order their synthetic 
constructs from? Our resident expert in RNA crystallography recommended a company 
called Dharmacon (part of ThermoFisher), but I was hoping that I might get some 
other opinions as to which companies make the best quality oligonucleotides, 
provide samples with the highest purity, and have the most reasonable prices.

Thanks in advance for the help!

Mike



--
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu



--
 Israel Sanchez Fernandez PhD
Ramakrishnan-lab
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 0QH, UK





  

[ccp4bb] Hg rippled summary

[Klemens Wild]

Dear colleagues,

here some comments about our Hg ripple problem - many thanks to all your 
answers:


Indeed we were not able to completely get rid of the ripples (as stated 
by Bart Hazes: not really affecting the biological conclusions - but 
anyway!). Summarizing the suggested problems are: summation truncation, 
anomalous scattering, anisotropy, multiple conformations, radiation 
damage, occupancy.
We already did the occupancy refinement beforehand, which helped as well 
as the anisotropic B refinement. Changing resolution limits did not help 
much - anyhow unfortunately we do not have higher resolution collected 
and data stops at the overall I/sigI at 3  sigma. Changing from Hg to 
Hg+1 (compound originally was ClHgCH3) helped in decreasing R-factors, 
although I always thought there should be covalent binding to Cys. 
Changing f' did not seem to have a big impact, although it is clearly 
different (approx. -11) from the standard CCP4 library for CuKa (-5.0)?! 
BTW: the Hg peaks are only 25 sigmas, and not 80 and dual conformations 
were not obvious.


Thanks to: ClemensV, KayD, GeorgeS,  BartH, DavidS,  KevinJ,  EleanorD, 
TassosP, TommiK, PeterM


Klemens Wild

[ccp4bb] Open position at the crystallography platform, Heidelberg University Biochemistry Center

[Klemens Wild]

Institution: Heidelberg University Biochemistry Center (BZH) and Cluster 
of Excellence (CellNetworks)

Location: Heidelberg, Germany
URL: www.uni-heidelberg.de/zentral/bzh/
Start: As soon as possible
Duration: 4 years
Salary: According to the German public service tariff scale TV-L.

Description: We are establishing a medium-throughput crystallization 
facility for biological macromolecules as a central technology platform. 
We are looking for a scientific leader of the facility, to both run the 
platform and organize the user support. The successful candidate is an 
expert in protein biochemistry with a profound knowledge in recombinant 
protein expression, purification, and crystallization. Experience in 
X-ray crystallography and/or automation would be an advantage.


Prerequisites: PhD or equivalent, basic knowledge in German
Please submit: CV, 2 letters of reference until July 20
Contact: Prof. Dr. Irmgard Sinning
Address: Heidelberg University Biochemistry Center (BZH), INF328, 
D-69120 Heidelberg, Germany.

Email: irmi.sinning(at)bzh.uni-heidelberg.de, Phone: +49 6221 544780