Abstract
Helical assemblies of proteins, which consist of a two-dimensional lattice of identical subunits arranged with helical symmetry, are a common structural motif in nature. For membrane proteins, crystallization protocols can induce helical arrangements and take advantage of the symmetry found in these assemblies for the structural determination of target proteins. Modern advances in the field of electron cryo-microscopy (cryo-EM), in particular the advent of direct electron detectors, have opened the potential for structure determination of membrane proteins in such assemblies at high resolution. The nature of the symmetry in helical crystals of membrane proteins means that a single image potentially contains enough information for three-dimensional structural determination. With the current direct electron detectors, we have never been closer to making this a reality. Here, we present a protocol detailing the preparation of helical crystals, with an emphasis on further cryo-EM analysis and structural determination of the sarco(endo)plasmic reticulum Ca2+-ATPase in the presence of regulatory subunits such as phospholamban.
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References
Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257(5521):28–32. https://doi.org/10.1038/257028a0
Henderson R, Unwin PN (1977) Structure of the purple membrane from Halobacterium halobium. Biophys Struct Mech 3(2):121. https://doi.org/10.1007/bf00535804
Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228
Lepault J, Booy FP, Dubochet J (1983) Electron microscopy of frozen biological specimens. J Microsc 129:89–102
Tani K, Fujiyoshi Y (2014) Water channel structures analysed by electron crystallography. Biochim Biophys Acta 1840(5):1605–1613. https://doi.org/10.1016/j.bbagen.2013.10.007
Gonen T, Cheng Y, Sliz P, Hiroaki Y, Fujiyoshi Y, Harrison SC, Walz T (2005) Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438(7068):633–638. https://doi.org/10.1038/nature04321
Abe K, Shimokawa J, Naito M, Munson K, Vagin O, Sachs G et al (2017) The cryo-EM structure of gastric H(+),K(+)-ATPase with bound BYK99, a high-affinity member of K(+)-competitive, imidazo[1,2-a]pyridine inhibitors. Sci Rep 7(1):6632. https://doi.org/10.1038/s41598-017-06698-8
Righetto RD, Biyani N, Kowal J, Chami M, Stahlberg H (2019) Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM. Nat Commun 10(1):1722. https://doi.org/10.1038/s41467-019-09661-5
Brisson A, Unwin PNT (1984) Tubular crystals of acetylcholine receptor. J Cell Biol 99:1202–1211
Toyoshima C, Unwin N (1990) Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction. J Cell Biol 111:2623–2635
Toyoshima C, Sasabe H, Stokes DL (1993) Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane. Nature 362:469–471
Zhang P, Toyoshima C, Yonekura K, Green N, Stokes D (1998) Structure of the calcium pump from sarcoplasmic reticulum at 8 Angstroms resolution. Nature 392:835–839
Galkin VE, Orlova A, Vos MR, Schroder GF, Egelman EH (2015) Near-atomic resolution for one state of F-actin. Structure 23(1):173–182. https://doi.org/10.1016/j.str.2014.11.006
Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ et al (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547(7662):185–190. https://doi.org/10.1038/nature23002
Wang F, Burrage AM, Postel S, Clark RE, Orlova A, Sundberg EJ et al (2017) A structural model of flagellar filament switching across multiple bacterial species. Nat Commun 8(1):960. https://doi.org/10.1038/s41467-017-01075-5
Kollmer M, Close W, Funk L, Rasmussen J, Bsoul A, Schierhorn A et al (2019) Cryo-EM structure and polymorphism of Abeta amyloid fibrils purified from Alzheimer's brain tissue. Nat Commun 10(1):4760. https://doi.org/10.1038/s41467-019-12683-8
Fromm SA, Bharat TA, Jakobi AJ, Hagen WJ, Sachse C (2015) Seeing tobacco mosaic virus through direct electron detectors. J Struct Biol 189(2):87–97. https://doi.org/10.1016/j.jsb.2014.12.002
He S, Scheres SHW (2017) Helical reconstruction in RELION. J Struct Biol 198(3):163–176. https://doi.org/10.1016/j.jsb.2017.02.003
Egelman E (2000) A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85:225–234
Young HS, Rigaud JL, Lacapere JJ, Reddy LG, Stokes DL (1997) How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase. Biophys J 72(6):2545–2558. https://doi.org/10.1016/S0006-3495(97)78898-2
Stokes DL, Pomfret AJ, Rice WJ, Glaves JP, Young HS (2006) Interactions between Ca2+-ATPase and the pentameric form of phospholamban in two-dimensional co-crystals. Biophys J 90(11):4213–4223. https://doi.org/10.1529/biophysj.105.079640
Coudray N, Lasala R, Zhang Z, Clark KM, Dumont ME, Stokes DL (2016) Deducing the symmetry of helical assemblies: applications to membrane proteins. J Struct Biol 195(2):167–178. https://doi.org/10.1016/j.jsb.2016.05.011
Coudray N, Lasala R, Zhang Z, Clark KM, Dumont ME et al (2017) Structure of the SLC4 transporter Bor1p in an inward-facing conformation. Protein Sci 26(1):130–145. https://doi.org/10.1002/pro.3061
Beroukhim R, Unwin N (1997) Distortion correction of tubular crystals: Improvements in the acetylcholine receptor structure. Ultramicroscopy 70:57–81
Avery AW, Fealey ME, Wang F, Orlova A, Thompson AR, Thomas DD et al (2017) Structural basis for high-affinity actin binding revealed by a beta-III-spectrin SCA5 missense mutation. Nat Commun 8(1):1350. https://doi.org/10.1038/s41467-017-01367-w
Stokes DL, Green NM (1990) Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys J 57:1–14.
Douglas JL, Trieber CA, Afara M, Young HS (2005) Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies. Protein Expr Purif 40(1):118–125. https://doi.org/10.1016/j.pep.2004.11.015
Booth, DS, Avila-Sakar, A, & Cheng, Y (2011) Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. JoVE (58):3227
Grassucci, RA, Taylor, DJ, & Frank, J (2007) Preparation of macromolecular complexes for cryo-electron microscopy. Nature protocols 2(12): 3239–3246
Nicholson WV, White H, Trinick J (2010) An approach to automated acquisition of cryoEM images from lacey carbon grids. J Struct Biol 172(3):395–399. https://doi.org/10.1016/j.jsb.2010.08.014
Shi J, Williams DR, Stewart PL (2008) A Script-Assisted Microscopy (SAM) package to improve data acquisition rates on FEI Tecnai electron microscopes equipped with Gatan CCD cameras. J Struct Biol 164(1):166–169. https://doi.org/10.1016/j.jsb.2008.05.011
Yonekura K, Toyoshima C (2000) Structure determination of tubular crystals of membrane proteins. II. Averaging of tubular crystals of different helical classes. Ultramicroscopy 84:15–28
Fernandez-Leiro, R, & Scheres, SHW (2017) A pipeline approach to single-particle processing in RELION. Acta Crystallogr D Struct Biol 73(Pt 6):496–502. https://doi.org/10.1107/S2059798316019276
Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14(3):290–296. https://doi.org/10.1038/nmeth.4169
Fromm SA, Sachse C (2016) Cryo-EM structure determination using segmented helical image reconstruction. Methods Enzymol 579:307–328. https://doi.org/10.1016/bs.mie.2016.05.034
Huber ST, Kuhm T, Sachse C (2018) Automated tracing of helical assemblies from electron cryo-micrographs. J Struct Biol 202(1):1–12. https://doi.org/10.1016/j.jsb.2017.11.013
Pothula KR, Smyrnova D, Schroder GF (2019) Clustering cryo-EM images of helical protein polymers for helical reconstructions. Ultramicroscopy 203:132–138. https://doi.org/10.1016/j.ultramic.2018.12.009
Glaves JP, Primeau JO, Espinoza-Fonseca LM, Lemieux MJ, Young HS (2019) The phospholamban pentamer alters function of the sarcoplasmic reticulum calcium pump SERCA. Biophys J 116(4):633–647. https://doi.org/10.1016/j.bpj.2019.01.013
Young HS, Jones LR, Stokes DL (2001) Locating phospholamban in co-crystals with Ca2+-ATPase by cryoelectron microscopy. Biophys J 81(2):884–894. https://doi.org/10.1016/S0006-3495(01)75748-7
Anderson DM, Makarewich CA, Anderson KM, Shelton JM, Bezprozvannaya S, Bassel-Duby R, Olson EN (2016) Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides. Sci Signal 9(457):ra119. https://doi.org/10.1126/scisignal.aaj1460
Primeau JO, Armanious GP, Fisher ME, Young HS (2017) The sarcoendoplasmic reticulum calcium ATPase. Subcell Biochem. https://doi.org/10.3389/fmicb.2019.00209
Dyla M, Kjaergaard M, Poulsen H, Nissen P (2019) Structure and mechanism of P-type ATPase ion pumps. Annu Rev Biochem. https://doi.org/10.1146/annurev-biochem-010611-112801
Glaves JP, Fisher L, Ward A, Young HS (2010) Helical crystallization of two example membrane proteins MsbA and the Ca(2+)-ATPase. Methods Enzymol 483:143–159. https://doi.org/10.1016/S0076-6879(10)83007-1
Russo CJ, Henderson R (2018) Charge accumulation in electron cryomicroscopy. Ultramicroscopy 187:43–49. https://doi.org/10.1016/j.ultramic.2018.01.009
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Mary D. Hernando and Joseph O. Primeau contributed equally to this work.
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Hernando, M.D., Primeau, J.O., Young, H.S. (2021). Helical Membrane Protein Crystallization in the New Era of Electron Cryo-Microscopy. In: Schmidt-Krey, I., Gumbart, J.C. (eds) Structure and Function of Membrane Proteins. Methods in Molecular Biology, vol 2302. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1394-8_10
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DOI: https://doi.org/10.1007/978-1-0716-1394-8_10
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