Journal: Acta Crystallogr D Struct Biol / Year: 2024 Title: VitroJet: new features and case studies. Authors: Rene J M Henderikx / Daniel Mann / Aušra Domanska / Jing Dong / Saba Shahzad / Behnam Lak / Aikaterini Filopoulou / Damian Ludig / Martin Grininger / Jeffrey Momoh / Elina Laanto / Hanna M ...Authors: Rene J M Henderikx / Daniel Mann / Aušra Domanska / Jing Dong / Saba Shahzad / Behnam Lak / Aikaterini Filopoulou / Damian Ludig / Martin Grininger / Jeffrey Momoh / Elina Laanto / Hanna M Oksanen / Kyrylo Bisikalo / Pamela A Williams / Sarah J Butcher / Peter J Peters / Bart W A M M Beulen / Abstract: Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before ...Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: OTHER Details: External glow discharge with Pelco EasiGlow plus internal glow discharging in VitroJet. VitroJet pins were cleaned with detergent and 70% EtOH in an ultrasonic bath (5 minutes each), ...Details: External glow discharge with Pelco EasiGlow plus internal glow discharging in VitroJet. VitroJet pins were cleaned with detergent and 70% EtOH in an ultrasonic bath (5 minutes each), followed by 1 min drying under nitrogen stream. Freshly purified FAS was pin printed at 4 degC climate chamber temperature with a 70 micrometer spiral, 15 micrometer standoff with a velocity of 5 mm per second.. The value given for _em_vitrification.instrument is CRYOSOL VITROJET. This is not in a list of allowed values {'HOMEMADE PLUNGER', 'FEI VITROBOT MARK III', 'FEI VITROBOT MARK II', 'FEI VITROBOT MARK I', 'GATAN CRYOPLUNGE 3', 'LEICA PLUNGER', 'LEICA EM GP', 'REICHERT-JUNG PLUNGER', 'LEICA EM CPC', 'OTHER', 'FEI VITROBOT MARK IV', 'SPOTITON', 'EMS-002 RAPID IMMERSION FREEZER', 'LEICA KF80'} so OTHER is written into the XML file.
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Electron microscopy
Microscope
TFS TALOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 3696 / Average electron dose: 48.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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