Eva Nogales, Professor

Closed (1) Structural analyses of novel ribonucleoproteins that function in antiviral immunity and genome transposition and their development into genome engineering tools

Applications for fall 2021 are now closed for this project.

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. Recent bioinformatics work has discovered an immense array of novel antiviral defense genes in prokaryotes (Linyi Gao … Feng Zhang et al, Science 369, 1077–1084 (2020)). One of these newly discovered systems encodes for a set of novel ribonucleoproteins containing reverse transcriptase domains in an unprecedented pairing with other important domains. While the molecular and structural mechanisms of these enzymes remain unclear, their domain organization suggests targeted genetic transposition as a possible mechanism for antiviral defense. Towards understanding the molecular and structural basis of antiviral immunity by these novel enzymes, we will reconstitute these ribonucleoproteins complexes in vitro, perform biochemical analyses for protein interaction and enzymatic activity, and determine molecular structures using cryo-EM. The hypothetical function of these enzymes suggest that they could be harnessed into a powerful biotechnology for targeted insertion of transgenes into the host genome.

The candidate for this position will have an opportunity to work on a new and exciting independent project under the mentorship of the supervisor.

The candidate would: 1) express and purify novel transposition proteins recombinantly from bacteria and insect cells, 2) perform biochemical analyses to reveal how these proteins impart antiviral immunity and recognize their specific nucleic acid targets, 3) prepare cryo-EM samples and use single-particle reconstruction methods to determine the molecular structures at high-resolution, and 4) utilize engineering these proteins into new biotechnology tools with applications in targeted gene insertion and antiviral immunity.

Day-to-day supervisor for this project: Akanksha Thawani, Post-Doc

Qualifications: Students with some familiarity of experimental molecular biology, protein expression and purification, biochemical assays, structural biology and/or genome engineering. Good analytical and organizational skills, critical thinking, and a desire to participate in cutting-edge research are totally required!!

Weekly Hours: 12 or more hours

Related website: http://cryoem.berkeley.edu

Closed (2) Structural analysis of the Ska complex and its role in mediating end-on chromosome attachment with Ndc80 at microtubules

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

Proper segregation of chromosomes during cell division is fundamental for all living organisms to maintain genome integrity. Kinetochores are large protein assemblies that connect chromosomes to microtubule (MT) structures that drive chromosome separation across the mitotic spindle. Understanding how kinetochores maintain stable attachment at MT ends, and how these interactions are modified to support the addition and removal of tubulin molecules has been a long-standing question in the field. While the molecular and structural mechanisms of these attachments remain unclear, it is clear that two important protein complexes, Ska and Ndc80, play a critical role. Towards understanding the molecular and structural basis of chromosome end-on attachment at the MT, we will reconstitute Ska-Ndc80-MT complexes in vitro and determine molecular structures using cryo-EM.

The candidate for this position would: 1) Participate in the cloning, expression, and purification of recombinant protein Ska/Ndc80 complexes from bacteria and insect cells, 2) Learn to prepare and freeze samples for cryo-EM, and 3) use single-particle reconstruction methods or sub-tomogram averaging toward determining molecular structures at high-resolution.

Day-to-day supervisor for this project: Anthony Schuller, Post-Doc

Qualifications: Qualifications: Students with some familiarity of experimental molecular biology, protein expression and purification, and/or structural biology. Good analytical and organizational skills, critical thinking, and a desire to participate in cutting-edge research are totally required!

Weekly Hours: 12 or more hours

Related website: http://cryoem.berkeley.edu

Closed (3) Cryo-electron microscopy studies of DNA methylation complexes

Applications for fall 2021 are now closed for this project.

DNA methylation at CpG sites plays an essential role in maintaining genome stability and regulating gene expression and it is strictly monitored and controlled by a series of molecular machines. Defects in DNA methylation are frequently found in severe diseases, such as cancer and Alzheimer's. DNMT1(DNA methyltransferase 1) with the help of UHRF1(Ubiquitin-like, containing PHD and RING finger domains 1) can be recruited to the DNA replication site and methylate the target loci. Despite intense study, the structure of the complex of DNMT1-UHRF1, and their interaction with nucleosomes are still unknown. Thus, it is still unclear how UHRF1 interacts with a nucleosome through its multi histone reader domains, how it recruits DNMT1, or how DNMT1 methylate DNA in the context of a nucleosome. Our goal is to answer these questions using cryo-EM.

The DNMT1-UHRF1 project will involve molecular cloning, protein purification, nucleosome reconstitution, and high-resolution cryo-EM structure analysis. Advanced technology will be used for cryo-EM sample preparation, data collection, and high-resolution structure determination. Biochemistry assays will be performed to validate the cryo-EM findings.

Day-to-day supervisor for this project: Zhenlin Yang, Post-Doc

Qualifications: Qualifications: Students with passion and enthusiasm for cutting edge research. The candidate is expected to have basic knowledge of biochemistry, such as molecular cloning and protein purification. Benchwork experience in biochemistry is preferred but not required. Ideally, the student commits to at least six months of work with the possibility of extension.

Weekly Hours: 12 or more hours

Related website: http://cryoem.berkeley.edu/cryoem

Closed (4) Structural analyses of microtubule plus end binding proteins and their role in regulating microtubule dynamics

Applications for fall 2021 are now closed for this project.

Accurate segregation of chromosomes during cell division is essential. Microtubules are tube-shaped, dynamic polymers that are a fundamental part of this process. A hallmark of microtubules is their dynamic polymerization and depolymerization behavior at the microtubule plus end known as dynamic instability. For microtubules to function correctly inside the cell, those dynamics must be properly regulated. The regulatory mechanisms are complex and far from being completely understood. It is however known that a range of proteins interacting specifically with the dynamic microtubule plus end play an essential role but the molecular mechanisms behind their regulatory mechanisms remain unclear. Here we use cryo-electron microscopy and cryo-electron tomography to study the structure of the microtubule plus end bound by multiple proteins to understand the molecular mechanisms of how they affect microtubule dynamics.

The candidate would 1) Participate in in vitro microtubule assays and purification of recombinant proteins 2) Learn and participate in sample preparation for cryo-electron microscopy including plunge freezing and 3) learn about cryo-electron microscopy/tomography and data processing of cryo-electon microscopy/tomography data for structure determination including subtomogram averaging.

Day-to-day supervisor for this project: Julia Peukes, Post-Doc

Qualifications: The applicant ideally would have some basic familiarity of experimental molecular biology, biochemistry, protein purification or structural biology. Strong motivation to learn about cryo-electron microscopy and cryo-electron tomography, good analytical and organizational skills, critical thinking, and a desire to participate in cutting-edge research as a team are required!

Weekly Hours: 12 or more hours

Related website: http://cryoem.berkeley.edu