For Delta-RBD/BA.2–23/BA.2–36/EY6A/mAb-4, delta RBD was incubated with a 1.1 M excess of each Fab on ice for ca. 10 min before application of a 3 μL aliquot of this complex mixture to a freshly glow-discharged (35 s, high with a Plasma Cleaner PDC-002-CE, Harrick Plasma) Quantifoil 2/1 300 mesh grids. Excess liquid was removed by blotting for 6 s with a force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% reported humidity before plunge freezing into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Movies, 20,535 in total, were collected on a Titan Krios operating at 300 kV equipped with a Falcon-IV Selectris at 130 kX magnification, corresponding to a calibrated pixel size of 0.7303 Å2 with a total dose of 50 e−/Å2 using EPU software (ThermoFisher scientific) and defocus range of 0.8–2.6 μm in EER format. Data were pre-processed on-the-fly in the cryoSPARC live interface, using initial 2D classes from blob-picked particles a template for template picking.56 (link) 3,529,798, picked particles, were then 2D classified into 250 classes in cryoSPARC v3.3.2 ‘static’ version, and 335,851 particles that were not obviously ‘junk’ were further classified, resulting in 29,589 particles in classes representing a variety of views. These were then used as input for ab-initio 3D reconstruction to three classes before heterogeneous refinement using the resulting volume set. One class, containing 167,492 particles, clearly commensurate with an RBD decorated with four Fabs, was then refined using non-uniform refinement before unbinning and further refinement to a final reported resolution of 2.9 Å resolution (−93 reported global b-factor).
Dijokaite-Guraliuc A., Das R., Zhou D., Ginn H.M., Liu C., Duyvesteyn H.M., Huo J., Nutalai R., Supasa P., Selvaraj M., de Silva T.I., Plowright M., Newman T.A., Hornsby H., Mentzer A.J., Skelly D., Ritter T.G., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Roemer C., Peacock T.P., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2023). Rapid escape of new SARS-CoV-2 Omicron variants from BA.2-directed antibody responses. Cell Reports, 42(4), 112271.
Publication 2023
B factor Complex mixtureEthane Excess liquid HeterogeneousHumidity Junk PaperPlasma Reconstruction
Corresponding Organization :
Other organizations :
Wellcome Centre for Human Genetics, University of Oxford, Diamond Light Source, Sheffield Teaching Hospitals NHS Foundation Trust, University of Sheffield, Medawar Building for Pathogen Research, Oxford University Hospitals NHS Trust, Medway School of Pharmacy, University of Kent, Mahidol Oxford Tropical Medicine Research Unit, SIB Swiss Institute of Bioinformatics, University of Basel, Imperial College London
Incubation of delta RBD with a 1.1 M excess of each Fab on ice for ca. 10 min
Glow-discharging of Quantifoil 2/1 300 mesh grids (35 s, high with a Plasma Cleaner PDC-002-CE, Harrick Plasma)
Blotting for 6 s with a force of -1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% reported humidity before plunge freezing into liquid ethane using a Vitrobot Mark IV (Thermo Fisher)
dependent variables
Movies, 20,535 in total, collected on a Titan Krios operating at 300 kV equipped with a Falcon-IV Selectris at 130 kX magnification, corresponding to a calibrated pixel size of 0.7303 Å^2 with a total dose of 50 e−/Å^2 using EPU software (ThermoFisher scientific) and defocus range of 0.8–2.6 μm in EER format
3,529,798 picked particles, 2D classified into 250 classes in cryoSPARC v3.3.2 'static' version, and 335,851 particles that were not obviously 'junk' were further classified, resulting in 29,589 particles in classes representing a variety of views
Ab-initio 3D reconstruction to three classes before heterogeneous refinement using the resulting volume set
One class, containing 167,492 particles, clearly commensurate with an RBD decorated with four Fabs, was then refined using non-uniform refinement before unbinning and further refinement to a final reported resolution of 2.9 Å resolution (−93 reported global b-factor)
control variables
Temperature (4.5°C)
Humidity (100% reported)
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to
get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required