A5026689567- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- RNA Research and Splicing
- Advanced Electron Microscopy Techniques and Applications
- Electron and X-Ray Spectroscopy Techniques
- DNA Repair Mechanisms
- Genomics and Chromatin Dynamics
- Microtubule and mitosis dynamics
- Enzyme Structure and Function
- Ubiquitin and proteasome pathways
- DNA and Nucleic Acid Chemistry
- Molecular Biology Techniques and Applications
- Fungal and yeast genetics research
- Cancer, Hypoxia, and Metabolism
- Advanced biosensing and bioanalysis techniques
- Glycosylation and Glycoproteins Research
- Bacterial Genetics and Biotechnology
- Nanopore and Nanochannel Transport Studies
- Metabolomics and Mass Spectrometry Studies
- Ion-surface interactions and analysis
- melanin and skin pigmentation
- Protein Kinase Regulation and GTPase Signaling
- Advanced X-ray Imaging Techniques
- Biochemical Analysis and Sensing Techniques
- Genomics and Phylogenetic Studies
MRC Laboratory of Molecular Biology
2016-2025
Medical Research Council
2014-2023
University of Cambridge
2023
Wake Forest University
2008
Pennsylvania State University
2007
Institute of Cancer Research
2004-2005
Institute of Structural and Molecular Biology
2003
University of British Columbia
1999-2000
Université Laval
2000
BC Cancer Agency
1998
Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because individual move during irradiation. This blurs images so that they aligned with each other to calculate a three-dimensional density. Much this movement stems from instabilities in carbon substrates used support frozen samples microscope. Here we demonstrate gold specimen nearly eliminates substrate motion increases subnanometer image contrast such α helices are resolved. With...
Recent innovations in specimen preparation, data collection, and image processing have led to improved structure determination using single-particle electron cryomicroscopy (cryo-EM). Here we explore some of these advances improve structures determined cryotomography (cryo-ET) sub-tomogram averaging. We implement a new three-dimensional model for the contrast transfer function, use this regularized likelihood optimization algorithm as implemented RELION program. Using direct detector data,...
As part of a cDNA library screen for clones that induce transformation NIH 3T3 fibroblasts, we have isolated encoding the murine homolog guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished ability RasGRP (mRasGRP) transform fibroblasts and activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression H-, K-, N-Ras partially suppressed dominant negative forms H- K-Ras. The C...
The comparison of a pair electron microscope images recorded at different specimen tilt angles provides powerful approach for evaluating the quality images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair from range specimens with symmetries and molecular masses show how analysis can produce valuable information not easily obtained otherwise. We that accuracy orientation determination individual single particles depends on mass, as expected...
Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, triggers transcription termination, but it is unclear how its various enzymes coordinated assembled. Here, we show that nuclease, polymerase, phosphatase activities of yeast organized into three modules. Using electron cryomicroscopy, determined 3.5-angstrom-resolution...
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA an impaired repair pathway. critical event activating this pathway monoubiquitination of FANCD2. In vivo, multisubunit core complex catalyzes step, but its mechanism unclear. Here, we report purification native avian and biochemical reconstitution FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must...
We show that central components of the Fanconi anemia (FA) DNA repair pathway, tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The complex is a manner depends on ATR kinase, FA ubiquitin ligase core complex, SUMO E3 ligases PIAS1/PIAS4 antagonized by protease SENP6. SUMOylation drives substrate selectivity triggering its polyubiquitylation SUMO-targeted RNF4 promote removal from sites damage via DVC1-p97 segregase complex....
Electron cryomicroscopy (cryo-EM) allows structure determination of a wide range biological molecules and specimens. All-gold supports improve cryo-EM images by reducing radiation-induced motion image blurring. Here we compare the mechanical electrical properties all-gold to amorphous carbon foils. Gold are more conductive, have suspended foils that not compressed differential contraction when cooled liquid nitrogen temperatures. These measurements show how choice support material geometry...
Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at specific site adds poly(A) tail. The cleavage reaction defines the end mature mRNA, thus activity endonuclease highly regulated. Here, we show that reconstitution pre-mRNA with recombinant yeast proteins requires incorporation Ysh1 into an eight-subunit "CPFcore" complex. also accessory factors IA IB, which bind substrate CPF, likely...
The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic stability and translation. RNA-binding proteins contribute specificity by interacting with both target mRNAs, but this is not fully understood. Here, we reconstitute accelerated selective deadenylation of RNAs containing AU-rich elements (AREs) Pumilio-response (PREs). We find that the fission yeast homologues Tristetraprolin/TTP Pumilio/Puf (Zfs1 Puf3) interact via multiple regions within low-complexity sequences,...
3' end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic nascent pre-mRNA defines mature transcript, which important for mRNA localization, translation, stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific efficient endonuclease activity CPSF with purified proteins. This required seven-subunit as well three additional protein factors: stimulatory (CStF), IIm (CFIIm),...