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Rubinstein Laboratory
Добавлен 25 дек 2015
This channel is a repository for videos from John Rubinstein's research group at the Hospital for Sick Children and University of Toronto (Departments of Biochemistry and Medical Biophysics).
Mechanism of ATP hydrolysis inhibition in mycobacterial ATP synthase
Extensions from the alpha-subunits of ATP synthase in mycobacteria prevent the enzyme from running in reverse to hydrolyze ATP.
From:
Guo*, Courbon*,Bueler, Mai, Liu, and Rubinstein (2021). Nature 589, 143-7.
www.nature.com/articles/s41586-020-3004-3
From:
Guo*, Courbon*,Bueler, Mai, Liu, and Rubinstein (2021). Nature 589, 143-7.
www.nature.com/articles/s41586-020-3004-3
Просмотров: 427
Видео
Mycobacterial ATP synthase with the TB drug bedaquiline
Просмотров 7313 года назад
Structure and conformational changes induced by binding of bedaquiline to mycobacterial ATP synthase. From: Guo*, Courbon*,Bueler, Mai, Liu, and Rubinstein (2021). Nature 589, 143-7. www.nature.com/articles/s41586-020-3004-3
Through-grid wicking
Просмотров 2,2 тыс.3 года назад
Yong Zi Tan explains the through-grid wicking approach for cryoEM specimen grid preparation. See scripts.iucr.org/cgi-bin/paper?S2059798320012474 for more information.
Rotary catalytic cycle of ClpXP
Просмотров 1244 года назад
Structure and rotational states of the ClpXP complex from Neisseria meningitidis. From Ripstein, Vahidi, Houry, Rubinstein, and Kay (2020). eLife 9, e52158 elifesciences.org/articles/52158
Proposed mechanism for substrate unfolding by the AAA+ unfoldase VAT
Просмотров 3277 лет назад
The structure of the substrate-bound form of ΔN-VAT by cryo-EM suggests how AAA unfoldases processively unfold substrate. With ATP binding and hydrolysis each subunit release the substrate from the bottom of the helical structure and rebinds near the top, gripping and pulling apart the target protein. The panel on the right shows the conserved pore loop residues th. From Ripstein, Huang, August...
Conformation changes that occur upon pore loop closing when ΔN-VAT binds substrate
Просмотров 877 лет назад
Comparison of NBD1 and NBD2 structures of the AAA unfoldase ΔN-VAT with and without substrate bound. From Ripstein, Huang, Augustyniak, Kay, and Rubinstein (2017) biorxiv.org/content/early/2017/02/04/105866
ΔN-VAT with and without substrate bound
Просмотров 647 лет назад
Comparison of the 6-fold symmetric structure of the AAA unfoldase ΔN-VAT, and ΔN-VAT with substrate bound. The panel on the right shows the pore loop residues. From Ripstein, Huang, Augustyniak, Kay, and Rubinstein (2017) biorxiv.org/content/early/2017/02/04/105866
Model for rotation by a Brownian ratche for the mitochondrial ATP synthase
Просмотров 1 тыс.8 лет назад
Model for proton translocation with a Brownian ratchet in the mitochondrial ATP synthase. This model is based on the cryo-EM stucture of the ATP synthase and evolutionary covariance analysis, and includes features from the observed rotational states and substates, as well as side chain orientations from X-ray crystallography. This work is described in detail in Zhou A, Rohou A, Schep DG, Bason ...
The three main rotational states of the mammalian mitochondrial ATP synthase
Просмотров 3418 лет назад
This work is described in detail in Zhou A, Rohou A, Schep DG, Bason JV, Montgomery MG, Walker JE, Grigorieff N, Rubinstein JL (2015). Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. eLife e10180. doi: 10.7554/eLife.10180. [download and view as loop for best results]
Mitochondrial ATP synthase a subunit structure by cryo-EM and evolutionary covariance
Просмотров 3468 лет назад
A model of the proton conducting a subunit was constructed by tracing the protein sequence through a 6-7 Å resolution cryo-EM map while satisfying constraints from evolutionary covariance analysis. The green density is from the a subunit, the red density is from the b subunit and the blue density is from subunit A6L. The ribbon diagram represents the a subunit sequence and the red lines show pa...
SickKids Video about Zhao, Benlekbir, and Rubinstein
Просмотров 4558 лет назад
John Rubinstein describes the work published in Zhao, Benlekbir, and Rubinstein (2015). Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241-5. By control of "internal pH", he really means control of the "pH of internal _compartments_". Cytosolic pH is primarily controlled by the plasma membrane P-type ATPase.
Cryo-EM grid nanofabrication
Просмотров 3,7 тыс.8 лет назад
Rubinstein Laboratory's nanofabrication protocol for making holey-carbon coated grids for cryo-EM. Evaporation of carbon onto the formvar layer can be substituted for evaporation of gold to nanofabricate holey-gold coated grids. Described in Marr, Benlekbir, and Rubinstein (2014). Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device. Journal of Structural Bio...
Rotational states of the S. cerevisiae V-ATPase
Просмотров 2 тыс.8 лет назад
Cryo-EM allowed us to model the rotational states of the Saccharomyces cerevisiae V-ATPase. Described in Zhao, Benlekbir, and Rubinstein (2015). Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241-5.
Our current protocol for stamping is ~4-5 lbs for 2 minutes (with the 1.2 um pattern)
I love how the animation shows the secondary structure and not just blobs. Those are some beautiful alpha helices (the beta sheets in the "top" of the molecule in red are pretty neat too, but something about those magenta spinning alpha helices at the "bottom" just blows my mind)!
What is the height of the features on the silicon master?
We now let the PDMS cure for several days before removing it from the silicon wafer. It should not be at all tacky when removed.
We attach the PDMS stamp to the pressure test stand with double sided tape.
I tend to let PDMS polymerize for a few days before removing stamps from the silicon master pattern. The surface of the PDMS should not be tacky at all. We attach stamps to the pressure test stand with double sided tape.
Most people in the laboratory now use a sputter coater to deposit ~35 nm of gold on grids, rather than evaporating gold (or carbon)
Members of the laboratory now inspect the plastic pattern regularly during baking to ensure desired hole sizes are obtained.
We now use a release agent on the silicon master patter (TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)-1-TRICHLOROSILANE [WARNING: dangerous substance - use appropriate precautions]. This only has to be done once per master pattern. Deposit ~ 30 uL into an open top container inside a dedicated desiccator. Apply vacuum with wafer in chamber for ~ 15 min to apply release agent to wafer. Too much release agent will inhibit PDMS binding to glass during stamp making. (NB: the release agent is not added directly on top of the master pattern, it transfers through the atmosphere in the dessicator)