Category Archives: DEER

Research Fellow in Membrane Protein Structural Biology #EPR

Additional Information at: https://jobs.leeds.ac.uk/vacancy.aspx?ref=FBSBM1119

Are you an ambitious researcher looking for your next challenge? Do you have an established background in structural biology of membrane proteins? Are you interested in ion channels and mechanical sensing? Do you want to further your career in one of the UKs leading research intensive Universities?

Applications are invited to conduct research investigating the structure and function of mechanosensitive ion channels. These systems form pores in the cell membrane and allow the passage of molecules in response to membrane tension. 

A multi-disciplinary approach combining molecular/chemical (cloning, recombinant expression and purification), structural (CryoEM and PELDOR/DEER spectroscopy), functional (Electrophysiology) and computational (MD simulations) methods will be employed to investigate gating of these systems and characterise their unique states. CryoEM facilities in the Astbury Centre at University of Leeds are state-of-the-art, including 2 x Titan Krios 300keV electron microscopes. The project will also involve trips to the St Andrews and Manchester EPR facilities for PELDOR/DEER experiments.

We are seeking a highly motivated and talented individual interested in undertaking a challenging and exciting 3 year BBSRC-funded post in the laboratory of Dr Christos Pliotas. The project aims at obtaining and solving novel CryoEM structures of mechanosensitive ion channels in distinct conformational states. Dynamics will be interrogated by PELDOR/DEER and MD within lipid/native environment and function will be assessed by single molecule electrophysiology.

You should have a PhD (or close to completion) in Molecular/Structural Biology, Biochemistry, Biophysics or a closely allied discipline; with significant experience in expression, purification and structural biology of membrane proteins and ion channels.

To explore the post further or for any queries you may have, please contact: 

Dr Christos Pliotas, Lecturer in Integrative Membrane Biology

Tel: +44 (0)113 343 1229, email: C.Pliotas@leeds.ac.uk, University Profile, twitter: https://twitter.com/PliotasGroup

Location:Leeds – Main Campus

Faculty/Service: Faculty of Biological Sciences

School/Institute: School of Biomedical Sciences

Category: Research

Grade: Grade 7

Salary: £33,199 to £39,609 p.a.

Due to funding limitations it is unlikely an appointment will be made above £35,211 p.a.

Working Time: 100%

Post Type: Full Time

Contract Type: Fixed Term (3 years – external funding)

Release Date: Monday 03 June 2019

Closing Date: Sunday 14 July 2019

Reference: FBSBM1119

[NMR] postdoctoral position at UC Santa Cruz

Dear Colleagues,

I am please to announce the opening of a postdoctoral researcher position in the Department of Chemistry and Biochemistry at UC Santa Cruz. The selected individual should have demonstrated expertise in Double Electron-Electron Resonance (DEER) EPR. Research will focus on structural biology issues related to neurodegenerative diseases, as well as the facilitation of diverse collaborative studies among California Bay Area labs including UCSF, UC Merced, UC Davis and Stanford. Please see our website: 

https://millhauser.chemistry.ucsc.edu

and the job posting: 

https://millhauser.chemistry.ucsc.edu/Millhauser_pages/EPR_postdoc_ad_2018.pdf.

With best regards,

glenn

Glenn L. Millhauser

Department of Chemistry & Biochemistry

UC Santa Cruz

Santa Cruz, CA 95064

831 459 2176 voice

831 566 3337 cell

831 459 2935 fax

glennm@ucsc.edu

http://millhauser.chemistry.ucsc.edu

https://www.chemistry.ucsc.edu/about/directory-page.php?uid=glennm

====================================

This is the AMPERE MAGNETIC RESONANCE mailing list:

http://www.drorlist.com/nmrlist.html

NMR web database:

http://www.drorlist.com/nmr.html

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

Another article that does not directly cover DNP spectroscopy. However, sample preparation, especially the right choice of a solvent that makes a good glass at cryogenic temperatures is critical for optimum performance in DNP experiments.

Georgieva, E.R., et al., Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme. J. Magn. Reson., 2012. 216(0): p. 69-77.

http://dx.doi.org/10.1016/j.jmr.2012.01.004

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (⩽100μs) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

Another article that does not directly cover DNP spectroscopy. However, sample preparation, especially the right choice of a solvent that makes a good glass at cryogenic temperatures is critical for optimum performance in DNP experiments.

Georgieva, E.R., et al., Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme. J. Magn. Reson., 2012. 216(0): p. 69-77.

http://dx.doi.org/10.1016/j.jmr.2012.01.004

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (⩽100μs) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

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