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Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

Mukherjee, Somnath and Erramilli, Satchal K. and Ammirati, Mark and Alvarez, Frances J. D. and Fennell, Kimberly F. and Purdy, Michael D. and Skrobek, Blazej M. and Radziwon, Katarzyna and Coukos, John and Kang, Yanyong and Dutka, Przemysław and Gao, Xiang and Qiu, Xiayang and Yeager, Mark and Xu, H. Eric and Han, Seungil and Kossiakoff, Anthony A. (2020) Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins. Nature Communications, 11 . Art. No. 1598. ISSN 2041-1723. PMCID PMC7101349.

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We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

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URLURL TypeDescription CentralArticle
Skrobek, Blazej M.0000-0001-8487-6293
Coukos, John0000-0001-7860-7847
Dutka, Przemysław0000-0003-3819-1618
Han, Seungil0000-0002-1070-3880
Additional Information:© 2020 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Received 01 November 2019; Accepted 03 March 2020; Published 27 March 2020. We thank Shohei Koide for providing the DNA of phage Library E. We acknowledge the Advanced Electron Microscopy Facility, University of Chicago, Molecular Electron Microscopy Core (MEMC), University of Virginia, David Van Andel Advanced Cryo-Electron Microscopy Suite, VARI for data collection. Assistance of staff from 24-ID beamline, Advanced Photon Source, in diffraction data collection is duly acknowledged. We thank Ravi Kurumbail, Jayasankar Jasti, Jotham Coe from Pfizer for α4β2 construct design, early reagent generation and supply of varenicline, respectively. We thank Minglei Zhao, University of Chicago for helpful discussions. This work was supported by National Institutes of Health grants GM117372 (to A.A. Kossiakoff), CHEETAH Collaborative Development Program P50 GM082545 (to W.I. Sundquist) and Pfizer Inc. Data availability: The plasmids of the synthetic antibodies will be available from the corresponding author upon reasonable request. The crystal structure of the BRIL bound to the synthetic antibody BAG2 has the accession code 6CBV in Protein Data Bank. EM maps and atomic models of the cryoEM structure of the nicotinic acid receptor with the bound antibody BAK5 have been deposited at the Electron Microscopy data bank (Accession code: EMD-20863, EMD-20857) and the Protein Data Bank (Accession code: 6USF, 6UR8). Author Contributions: A.A.K. designed research and supervised the project. S.M. initiated the project, performed cloning, expression, purification, characterization of the targets, phage display selection, affinity maturation, and crystal structure determination. S.K.E., Y.K., and X.G. prepared samples for NS data collection, collected NS data, and analysis. S.M. prepared samples from cryoEM and M.D.P. collected, analyzed cryoEM data of KcsA-BAG2 complex. B.S. purified and crystallized BRIL–BAG2 complex. K.R. performed cloning, expression, purification, and characterization of alanine mutants. J.C, K.R., and P.D. were involved in purification and characterization of targets and sABs. K.F.F. did the cloning and expression of α4β2 constructs. M.A. was involved in α4β2 purification, characterization, cryoEM screening/data collection. F.J.D.A. determined the cryo-EM structures of α4(BRIL)β2 nicotinic acetylcholine receptor. M.Y., H.E.X., S.H., X.Q., and A.A.K. were involved in supervision and provided resource. S.M. and A.A.K. wrote the manuscript with contributions from S.K.E., K.F.F., M.A., F.J.D.A., and S.H. The authors declare no competing interests.
Funding AgencyGrant Number
NIHP50 GM082545
PubMed Central ID:PMC7101349
Record Number:CaltechAUTHORS:20200331-125405003
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Official Citation:Mukherjee, S., Erramilli, S.K., Ammirati, M. et al. Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins. Nat Commun 11, 1598 (2020).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102203
Deposited By: Tony Diaz
Deposited On:31 Mar 2020 20:07
Last Modified:06 Jul 2020 21:09

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