英国弗朗西斯·克里克研究所博士后职位—生化重建/光和冷冻电镜

2022年09月28日

来源：知识人网整理

摘要：

英国弗朗西斯·克里克研究所博士后职位—生化重建/光和冷冻电镜

Postdoctoral Training Fellow

Francis Crick Institute

Postdoctoral Training Fellow

This is a full time, fixed term (4 years) position, with possibility of extension, on Crick terms and conditions of employment.

The research group

The laboratory focuses on the mechanism of chromosome replication using biochemical reconstitution, light and cryo-electron microscopy (cryo-EM).

Using yeast and human proteins, we study the initiation step of DNA replication, which is highly regulated to ensure that every chromosome is copied only once per cell cycle1. It involves a cascade of molecular events reflected in discrete structural intermediates, such as helicase loading2, phosphorylation3, DNA melting4 and replication fork establishment5.

We currently focus on how DNA unwinding and synthesis are coordinated, and how the replication machinery efficiently replicates DNA molecules that are packaged in nucleosome arrays6.

To perform our research, we develop new tools in three areas.

Kinetics. We pioneered time resolved electron microscopy approaches, enabling us to describe the sequential, concerted mechanism for loading pairs of helicase molecules onto origin DNA2. Image analysis. We develop RECONSIL, a pipeline to extract information on physiological context from cryo-electron micrographs, which is otherwise lost in single-particle image processing7. Correlative microscopy. We develop methods to follow DNA replication with single-molecule fluorescence and electron microscopy using one cryo-EM grid support.

More information can be found on the Francis Crick Institute web page:

https://www.crick.ac.uk/research/labs/alessandro-costa

or our lab website

https://www.alecostalab.net/

Please direct any questions to Alessandro Costa (alessandro.costa@crick.ac.uk).

References

1 Costa, A. & Diffley, J. F. X. The Initiation of Eukaryotic DNA Replication. Annual Review of Biochemistry, (2022).

2 Miller, T. C. R., Locke, J., Greiwe, J. F., Diffley, J. F. X. & Costa, A. Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Nature 575, 704-710, (2019).

3 Greiwe, J. F., … Diffley JF.X. & Costa, A. Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Nature Structural & Molecular Biology 29, 10-20, (2022).

4 Lewis, J. S., … Diffley JF.X. & Costa, A. Mechanism of replication origin melting nucleated by CMG helicase assembly. Nature 606, 1007-1014, (2022).

5 Douglas, M. E., Ali, F. A., Costa, A. & Diffley, J. F. X. The mechanism of eukaryotic CMG helicase activation. Nature 555, 265-268, (2018).

6 Willhoft, O. & Costa, A. A structural framework for DNA replication and transcription through chromatin. Current Opinion in Structural Biology 71, 51-58, (2021).

7 Puhringer, T., Greiwe, J. F., Miller, T. C. R. & Costa, A. ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication. Methods in Enzymology 672, 203-231, (2022).

Project summary

The replication machinery is powered by a helicase motor that unwinds the double helix and replicative polymerases that duplicate DNA. How are DNA unwinding and synthesis coordinated? How is continuous DNA synthesis on the leading strand coupled with discontinuous Okazaki fragment synthesis on the lagging strand? How are nucleosomes that protect parental DNA uncoiled and how are they re-assembled on duplicated DNA?

To address these questions the postdoctoral training fellow will perform in vitro reconstitution of chromatin replication reactions using established protocols. Reactions will be imaged by cryo-EM for single particle analysis using state of the art instruments available at the Crick Institute. These include a Thermofisher Talos Arctica and a Glacios microscopes equipped with Falcon 3 detectors and two Titan Krios equipped with Falcon 4 detectors and Selectris Imaging filter. Computing facilities include the Crick CPU and GPU clusters, as well as stand alone workstations available in the lab. To monitor the kinetics of chromatin replication, single-molecule fluorescence imaging will be employed using correlative microscopy approaches recently established in our group. Three-dimensional structures will be solved and atomic models built to understand replication mechanisms at the molecular level. Structural mechanisms will be validated using site-directed mutagenesis and biochemical assays.

Postdoctoral Training Fellows are expected to lead their own projects and contribute to other studies by working together with other members of the lab or external collaborators.

Key experience and competencies

This position is ideal for a biochemist or single-molecule fluorescence microscopist who wants to learn cryo-EM. Alternatively, this position would suit cryo-EM scientists interested in developing their biochemistry and working on correlative light and electron microscopy, at the single molecule level.

Essential

PhD in Biochemistry (and related fields) or in the final stages of PhD submission

Expertise at least one of three areas: protein biochemistry, structural biology or single-molecule fluorescence microscopy.

Research articles published or submitted for publication in peer-reviewed journals.

Experience of experimental design.

Ability to work independently and as part of a team.

Desirable

Experience in the biochemistry of nucleic acid interacting proteins.

Experience in single-particle cryo-EM and atomic-model building.

Expertise in single-molecule fluorescence microscopy.

Find out what benefits the Crick has to offer:

For more information on our great pay and benefits package please click here: https://www.crick.ac.uk/careers-and-study/life-at-the-crick/pay-and-benefits

Equality, Diversity & Inclusion:

We welcome applications from all backgrounds. We are committed to providing equal employment opportunities, regardless of ethnicity, nationality, gender, sexual orientation, gender identity, religion, pregnancy, age, disability, or civil partnership, marital or family status. We particularly welcome applications from people who are Minority Ethnic as they are currently underrepresented in the Crick at this level.

Diversity is essential to excellence in scientific endeavour. It increases breadth and perspective, leading to more innovation and creativity. We want the Crick to be a place where everyone feels valued and where diversity is celebrated and seen as part of the foundation for our Institute’s success.

The Crick is committed to creating equality of opportunity and promoting diversity and inclusivity. We all share in the responsibility to actively promote dignity, respect, inclusivity and equal treatment and it is our aim to ensure that these principles are reflected and implemented in all strategies, policies and practices.