Success Story, May 7, 2019

May 7, 2019  |  Jola Glotzer

Solving a part of CRISPR structure

Part of the CRISPR complex structure resolved with help of Gatan 2, a direct electron detector purchased by NU with CBC funds

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Alfonso Mondragón, Northwestern

Congratulations to Alfonso Mondragón, NU, for his recent publication in Nucleic Acids Research, titled “Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.” The authors acknowledge CBC support through a CBC Infrastructure Award (2014), which allowed NU to acquire an expensive next-generation electron detector – Gatan 2 – for its $5 million cryo-electron microscope (cryoEM) core facility. Alfonso Mondragón and Jay Welsh, NU, were co-PIs on the CBC grant. Mondragón has several additional ties to the CBC. He received two CBC Catalyst Awards (2006 and 2016), CBC Postdoctoral Research Award (2015) and a CBC Lever Award Pilot Project (2014, renewed for 2015). He also served as a chairman on the 16^th Annual CBC Symposium (2018) and was a member of the CBC Catalyst Board (2007-2011). CBC is proud to have provided support to Mondragón’s research and is grateful for his time and dedication while working with the CBC on its programs.

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Publication attributed to CBC funding*:

Dorsey BW, Huang L, Mondragón A. Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM. Nucleic Acids Res. 2019 Apr 23;47(7):3765-3783. (PubMed)

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ABSTRACT

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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR–Cas systems are classified into different classes and types. Class 1 CRISPR–Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

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ACKNOWLEDGMENTS

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National Institutes of Health (NIH) [R35 GM118108 to A.M.]; LS-CAT/Sector 21 at the Advanced Photon Source, Argonne National Laboratory was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor. Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Structural Biology, Keck Biophysics, and High Throughput Analysis Facilities is acknowledged. The Gatan K2 DDE at Northwestern University was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. The GE Healthcare Typhoon 9400 Fluorimager at Northwestern University was purchased with funds provided by award [S10RR027842] from the National Center for Research Resources. Funding for open access charge: National Institutes of Health [R35 GM118108].

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