Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

High‐resolution cryo‐EM has revolutionized how we look at ABC transporters and membrane proteins in general. An ever‐increasing number of software tools and faster processing now allow dissecting the molecular details of nanomachines at atomic precision. Considering the further benefits of significantly reduced sample demands and increased speed, cryo‐EM will dominate the structure determination of membrane proteins in the near future without compromising on data quality or detail. Moreover, improved and new algorithms make it now possible to resolve the conformational spectrum of macromolecular machines under turnover conditions and to analyze heterogeneous samples at high resolution. The future of cryo‐EM is, therefore, bright, and the growing number of imaging facilities and groups active in this field will amplify this trend even further. Nevertheless, expectations have to be managed, as cryo‐EM alone cannot provide an ultimate answer to all scientific questions. In this review, we discuss the capabilities and limitations of cryo‐EM together with possible solutions for studies of ABC transporters.

[1]  Yong Zi Tan,et al.  Through-grid wicking enables high-speed cryoEM specimen preparation , 2020, bioRxiv.

[2]  J. Frank,et al.  Three‐dimensional reconstruction from a single‐exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli , 1987, Journal of microscopy.

[4]  Martin Grininger,et al.  Protein denaturation at the air-water interface and how to prevent it , 2019, eLife.

[5]  Microscale Fluid Behavior during Cryo-EM Sample Blotting. , 2019, Biophysical journal.

[7]  Jue Chen,et al.  Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1 , 2017, Cell.

[8]  Alex J. Noble,et al.  Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO , 2019, Journal of structural biology: X.

[9]  S. Sligar,et al.  Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins. , 2007, Biochemistry.

[10]  Jue Chen,et al.  ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1 , 2018, Cell.

[11]  Lei Chen,et al.  Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels , 2018, Protein & Cell.

[12]  S H W Scheres,et al.  Processing of Structurally Heterogeneous Cryo-EM Data in RELION. , 2016, Methods in enzymology.

[13]  X. Gong,et al.  Structure of the Human Lipid Exporter ABCA1 , 2017, Cell.

[14]  R. Tampé,et al.  Structure of the human MHC-I peptide-loading complex , 2017, Nature.

[15]  Xing Zhang,et al.  Bioactive Functionalized Monolayer Graphene for High-Resolution Cryo-Electron Microscopy. , 2019, Journal of the American Chemical Society.

[16]  Jose-Maria Carazo,et al.  Faculty Opinions recommendation of 3D Variability Analysis: Directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images. , 2020 .

[17]  Nikolaus Grigorieff,et al.  Structure of the transporter associated with antigen processing trapped by herpes simplex virus , 2016, eLife.

[18]  James M. Bell,et al.  In situ structure and assembly of the multidrug efflux pump AcrAB-TolC , 2019, Nature Communications.

[19]  J. Kowal,et al.  Structure of the human multidrug transporter ABCG2 , 2017, Nature.

[20]  J. Riordan,et al.  Cryo-EM visualization of an active high open probability CFTR anion channel , 2018 .

[21]  B. Shoichet,et al.  Structural identification of a hotspot on CFTR for potentiation , 2019, Science.

[22]  Hongjin Zheng,et al.  Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter , 2020, Science Advances.

[23]  Ashwin Chari,et al.  Atomic-resolution protein structure determination by cryo-EM , 2020, Nature.

[24]  Joseph H. Davis,et al.  Addressing preferred specimen orientation in single-particle cryo-EM through tilting , 2017, Nature Methods.

[25]  Jue Chen,et al.  Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation , 2018, Science.

[26]  T. Walz,et al.  Structural basis of MsbA-mediated lipopolysaccharide transport , 2017, Nature.

[27]  Terrence Frey,et al.  Faculty Opinions recommendation of TRPV1 structures in distinct conformations reveal activation mechanisms. , 2014 .

[28]  N. Gao,et al.  Structure of a Pancreatic ATP-Sensitive Potassium Channel , 2017, Cell.

[29]  W. Kühlbrandt The Resolution Revolution , 2014, Science.

[30]  M. Liao,et al.  ABCG2 transports anticancer drugs via a closed-to-open switch , 2020, Nature Communications.

[31]  Microfluidic protein isolation and sample preparation for high-resolution cryo-EM , 2019, bioRxiv.

[32]  John P. Moore,et al.  Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer , 2013, Science.

[33]  R. Stroud,et al.  Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter , 2014, Nature.

[34]  Sjors H.W. Scheres,et al.  Faculty Opinions recommendation of Real-time cryo-electron microscopy data preprocessing with Warp. , 2019, Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature.

[35]  R. Ravelli,et al.  Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification , 2020, Nature Communications.

[36]  Daniel Picot,et al.  Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins , 2010, Nature Methods.

[37]  A. Cheng,et al.  Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. , 2012, Structure.

[38]  C. Gati,et al.  A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds , 2020, Nature.

[39]  A. Rothnie,et al.  Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer. , 2017, Biochimica et biophysica acta. Biomembranes.

[40]  E. Pardon,et al.  Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM , 2019, Nature Methods.

[41]  P. Penczek,et al.  A Primer to Single-Particle Cryo-Electron Microscopy , 2015, Cell.

[42]  C. Tribet,et al.  Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Glaeser,et al.  Opinion: hazards faced by macromolecules when confined to thin aqueous films , 2016, Biophysics reports.

[44]  Henning Urlaub,et al.  GraFix: sample preparation for single-particle electron cryomicroscopy , 2008, Nature Methods.

[45]  D. Tegunov,et al.  Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells , 2020, Nature Methods.

[46]  Joachim Frank,et al.  Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip. , 2018, Methods in molecular biology.

[47]  David J. Fleet,et al.  Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction , 2019, Nature Methods.

[48]  J. Kowal,et al.  Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1 , 2018, Proceedings of the National Academy of Sciences.

[49]  Jitendra Malik,et al.  Automated multi-model reconstruction from single-particle electron microscopy data. , 2010, Journal of structural biology.

[50]  Q. Luo,et al.  Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism , 2019, Nature Communications.

[51]  Claudio Ciferri,et al.  Cryo-EM in drug discovery: achievements, limitations and prospects , 2018, Nature Reviews Drug Discovery.

[52]  Jun Yu Li,et al.  The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution , 2018, eLife.

[53]  D. Elmlund,et al.  AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar. , 2019, Journal of structural biology.

[54]  B. Carragher,et al.  Distinct conformational spectrum of homologous multidrug ABC transporters. , 2015, Structure.

[55]  C. Russo,et al.  Multifunctional graphene supports for electron cryomicroscopy , 2019, Proceedings of the National Academy of Sciences.

[56]  D. Julius,et al.  Structure of the TRPV1 ion channel determined by electron cryo-microscopy , 2013, Nature.

[57]  J. Kowal,et al.  Structural basis of small-molecule inhibition of human multidrug transporter ABCG2 , 2018, Nature Structural & Molecular Biology.

[58]  Jue Chen,et al.  Conformational Changes of CFTR upon Phosphorylation and ATP Binding , 2017, Cell.

[59]  Michael R. Wasserman,et al.  Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses , 2020, eLife.

[60]  William J. Rice,et al.  A new method for vitrifying samples for cryo-EM , 2017 .

[61]  B. Chait,et al.  Structural basis of substrate recognition by a polypeptide processing and secretion transporter , 2020, eLife.

[62]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[63]  Jue Chen,et al.  Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator , 2016, Cell.

[64]  E. Lindahl,et al.  Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION , 2018, bioRxiv.

[65]  M. Gottesman,et al.  Overview: ABC Transporters and Human Disease , 2001, Journal of bioenergetics and biomembranes.

[66]  Bonnie Berger,et al.  Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs , 2018, Nature Methods.

[67]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[68]  Cong-Zhao Zhou,et al.  Cryo-EM structure of human lysosomal cobalamin exporter ABCD4 , 2019, Cell Research.

[69]  R. Stevens,et al.  Engineered Nanostructured β-Sheet Peptides Protect Membrane Proteins , 2013, Nature Methods.

[70]  J. Briggs,et al.  An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation , 2016, Science.

[71]  M. Sung,et al.  Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM , 2019, eLife.

[72]  D. Gadsby,et al.  Molecular Structure of the Human CFTR Ion Channel , 2017, Cell.

[73]  M. Liao,et al.  Structural basis of lipopolysaccharide extraction by the LptB2FGC complex , 2019, Nature.

[74]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[75]  A. Goldman,et al.  A method for detergent-free isolation of membrane proteins in their local lipid environment , 2016, Nature Protocols.

[76]  Yong Zi Tan,et al.  Structure and Drug Resistance of the Plasmodium falciparum Transporter PfCRT , 2019, Nature.

[77]  N. Unwin,et al.  Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. , 1994, Ultramicroscopy.

[78]  Xueming Li,et al.  Fabs enable single particle cryoEM studies of small proteins. , 2012, Structure.

[79]  Rebecca F Thompson,et al.  Approaches to altering particle distributions in cryo-electron microscopy sample preparation , 2018, Acta crystallographica. Section D, Structural biology.

[80]  Jing Kong,et al.  High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy , 2019, Proceedings of the National Academy of Sciences.

[81]  Jue Chen,et al.  Molecular structure of the ATP-bound, phosphorylated human CFTR , 2018, Proceedings of the National Academy of Sciences.

[82]  R. Henderson,et al.  Comparison of optimal performance at 300 keV of three direct electron detectors for use in low dose electron microscopy , 2014, Ultramicroscopy.

[83]  J. Kowal,et al.  Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states , 2018, Nature.

[84]  William J. Rice,et al.  High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift , 2018, bioRxiv.

[85]  Cong-Zhao Zhou,et al.  Cryo-electron Microscopy Structure and Transport Mechanism of a Wall Teichoic Acid ABC Transporter , 2020, mBio.

[86]  Takanori Nakane,et al.  Single-particle cryo-EM at atomic resolution , 2020, Nature.

[87]  C. Yoshioka,et al.  Anti-diabetic drug binding site in a mammalian KATP channel revealed by Cryo-EM , 2017, eLife.

[88]  A. Meister,et al.  Solubilization of Membrane Proteins into Functional Lipid‐Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer , 2017, Angewandte Chemie.

[89]  Thorsten Wagner,et al.  SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM , 2019, Communications Biology.

[90]  J. Kowal,et al.  Structural insight into substrate and inhibitor discrimination by human P-glycoprotein , 2019, Science.

[91]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[92]  Yong Zi Tan,et al.  Reducing effects of particle adsorption to the air-water interface in cryoEM , 2018, Nature Methods.

[93]  Alexis Rohou,et al.  cisTEM: User-friendly software for single-particle image processing , 2017, bioRxiv.

[94]  Andreas Plückthun,et al.  Covalently circularized nanodiscs for studying membrane proteins and viral entry , 2016, Nature Methods.

[95]  Cong-Zhao Zhou,et al.  Cryo-EM structure of human bile salts exporter ABCB11 , 2020, Cell Research.

[96]  A. Ramamoorthy,et al.  Styrene maleic acid derivates to enhance the applications of bio-inspired polymer based lipid-nanodiscs. , 2018, European polymer journal.

[97]  J. Lyons,et al.  Expression strategies for structural studies of eukaryotic membrane proteins. , 2016, Current opinion in structural biology.

[98]  G. Hummer,et al.  Conformation space of a heterodimeric ABC exporter under turnover conditions , 2019, Nature.

[99]  Tristan Bepler,et al.  Topaz-Denoise: general deep denoising models for cryoEM and cryoET , 2019, Nature Communications.

[100]  D. Vasishtan,et al.  Extracellular Vesicles: A Platform for the Structure Determination of Membrane Proteins by Cryo-EM , 2014, Structure.

[101]  R. MacKinnon,et al.  Molecular structure of human KATP in complex with ATP and ADP , 2017, bioRxiv.

[102]  Lei Chen,et al.  The Structural Basis for the Binding of Repaglinide to the Pancreatic KATP Channel. , 2019, Cell reports.

[103]  J. Kowal,et al.  Structure of the human lipid exporter ABCB4 in a lipid environment , 2019, Nature Structural & Molecular Biology.

[104]  J. Riordan,et al.  Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel. , 2018, Biochemistry.

[105]  Robert M Glaeser,et al.  Laser phase plate for transmission electron microscopy , 2019, Nature Methods.

[106]  Andrej Bieri,et al.  Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts. , 2017, Journal of structural biology.

[107]  J. Rubinstein,et al.  Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device , 2019, bioRxiv.

[108]  Y. Lacasse,et al.  From the authors , 2005, European Respiratory Journal.

[109]  Stephen G. Sligar,et al.  Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins , 2002 .