STAR Protocols (Mar 2022)
Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography
- Fatma Betul Ertem,
- Omur Guven,
- Cengizhan Buyukdag,
- Oktay Gocenler,
- Esra Ayan,
- Busra Yuksel,
- Mehmet Gul,
- Gozde Usta,
- Barıs Cakılkaya,
- J. Austin Johnson,
- E. Han Dao,
- Zhen Su,
- Frederic Poitevin,
- Chun Hong Yoon,
- Christopher Kupitz,
- Brandon Hayes,
- Mengning Liang,
- Mark S. Hunter,
- Alexander Batyuk,
- Raymond G. Sierra,
- Gihan Ketawala,
- Sabine Botha,
- Çağdaş Dağ,
- Hasan DeMirci
Affiliations
- Fatma Betul Ertem
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Omur Guven
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Cengizhan Buyukdag
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Oktay Gocenler
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Esra Ayan
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Busra Yuksel
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Mehmet Gul
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Gozde Usta
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- Barıs Cakılkaya
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- J. Austin Johnson
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey
- E. Han Dao
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Zhen Su
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA; Department of Applied Physics, Stanford University, Stanford, CA, USA
- Frederic Poitevin
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Chun Hong Yoon
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Christopher Kupitz
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Brandon Hayes
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Mengning Liang
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Mark S. Hunter
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Alexander Batyuk
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Raymond G. Sierra
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, 94025 CA, USA
- Gihan Ketawala
- Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA; Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA
- Sabine Botha
- Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA; Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA
- Çağdaş Dağ
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey; Koc University, Nanofabrication and Nanocharacterization Center for Scientific and Technological Advanced Research (n2STAR), Istanbul, Turkey; Koc University Isbank Center for Infectious Diseases (KUISCID), Istanbul, Turkey
- Hasan DeMirci
- Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey; Koc University Isbank Center for Infectious Diseases (KUISCID), Istanbul, Turkey; Stanford PULSE Institute, SLAC National Laboratory, Menlo Park, CA, USA; Corresponding author
- Journal volume & issue
-
Vol. 3,
no. 1
p. 101158
Abstract
Summary: The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography.For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).
