In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a 'digital twin' software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.

Department of Materials University of Oxford Parks Road Oxford OX1 3PH UK

Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0DE UK

Division of Structural Biology University of Oxford Roosevelt Drive Oxford OX3 7BN UK

Electron Physical Science Imaging Centre Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0DE UK

Rosalind Franklin Institute Harwell Science and Innovation Campus Didcot OX11 0FA UK

Thermo Fisher Scientific Vlastimila Pecha Brno Czech Republic

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Bracewell RN. 1956. Strip integration in radio astronomy. Aust. J. Phys. 9, 198-217. (10.1071/PH560198) DOI

Crowther RA, DeRosier DJ, Klug A. 1970. The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. Proc. R. Soc. A 317, 319-340. (10.1098/rspa.1970.0119) DOI

Hagen WJH, Wan W, Briggs JAG. 2017. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191-198. (10.1016/j.jsb.2016.06.007) PubMed DOI PMC

Eisenstein F, Danev R, Pilhofer M. 2019. Improved applicability and robustness of fast cryo-electron tomography data acquisition. J. Struct. Biol. 208, 107-114. (10.1016/j.jsb.2019.08.006) PubMed DOI PMC

Weis F, Hagen WJH. 2020. Combining high throughput and high quality for cryo-electron microscopy data collection. Acta Crystallogr. Section D 76, 724-728. (10.1107/S2059798320008347) PubMed DOI PMC

Bouvette J, et al. 2021. Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography. Nat. Commun. 12, 1957. (10.1038/s41467-021-22251-8) PubMed DOI PMC

Schorb M, Haberbosch I, Hagen WJ, Schwab Y, Mastronarde DN. 2020. Europe PMC funders group software tools for automated transmission electron microscopy. Nat. Methods 16, 471-477. (10.1038/s41592-019-0396-9.Software) PubMed DOI PMC

Zivanov J, Nakane T, Forsberg B, Kimanius D, Hagen WJH, Lindahl E, Scheres SHW. 2018. RELION-3: new tools for automated high-resolution cryo-EM structure determination. BioRxiv, 1–22. (10.1101/421123) PubMed DOI PMC

Dubochet J, Adrian M, Chang J-J, Homo J-C, Lepault J, McDowall AW, Schultz P. 1988. Cryo-electron microscopy of vitrified specimens. Quarterly Reviews of Biophysics, 21. PubMed

Cowley JM, Moodie AF. 1957. The scattering of electrons by atoms and crystals. I. A new theoretical approach. Acta Crystallogr. 10, 609-619. (10.1107/s0365110X57002194) DOI

Goodman P, Moodie AF. 1974. Numerical evaluations of N-beam wave functions in electron scattering by the multi-slice method. Acta Crystallogr. Section A 30, 280-290. (10.1107/S056773947400057X) DOI

Zhang Y, Tammaro R, Peters PJ, Ravelli RBG. 2020. Could egg white lysozyme be solved by single particle cryo-EM? J. Chem. Information Model. 60, 2605-2613. (10.1021/acs.jcim.9b01176) PubMed DOI PMC

Palmer CM, Löwe J. 2014. A cylindrical specimen holder for electron cryo-tomography. Ultramicroscopy 137, 20-29. (10.1016/j.ultramic.2013.10.016) PubMed DOI PMC

Harauz G, van Heel M. 1985. Exact filters for general geometry three dimensional reconstruction. Optik 78, 1986.

Midgley PA, Weyland M. 2003. 3D electron microscopy in the physical sciences: the development of Z-contrast and EFTEM tomography. Ultramicroscopy 96, 413-431. (10.1016/S0304-3991(03)00105-0) PubMed DOI

Penczek P, Marko M, Buttle K, Frank J. 1995. Double-tilt electron tomography. Ultramicroscopy 60, 393-410. (10.1016/0304-3991(95)00078-X) PubMed DOI

Tong JR, Weyland M, Midgley PA. 2003. Benefits and drawbacks of dual-axis STEM tomography. See www.rafaldb.com/papers/C-2004-08-Antwerp-EMC-dual-axis-STEM-tomography.pdf.

Tong J, Arslan I, Midgley P. 2006. A novel dual-axis iterative algorithm for electron tomography. J. Struct. Biol. 153, 55-63. (10.1016/j.jsb.2005.10.005) PubMed DOI

Turoňová B, et al. 2020. Benchmarking tomographic acquisition schemes for high-resolution structural biology. Nat. Commun. 11, 1-9. (10.1038/s41467-020-14535-2) PubMed DOI PMC

Iancu CV, et al. 2005. A ‘flip-flop’ rotation stage for routine dual-axis electron cryotomography. J. Struct. Biol. 151, 288-297. (10.1016/j.jsb.2005.07.004) PubMed DOI

Arslan I, Tong JR, Midgley PA. 2006. Reducing the missing wedge: high-resolution dual axis tomography of inorganic materials. Ultramicroscopy 106(11–12 SPEC. ISS.), 994-1000. (10.1016/j.ultramic.2006.05.010) PubMed DOI

Cantele F, Paccagnini E, Pigino G, Lupetti P, Lanzavecchia S. 2010. Simultaneous alignment of dual-axis tilt series. J. Struct. Biol. 169, 192-199. (10.1016/j.jsb.2009.10.003) PubMed DOI

Bals S, Goris B, Altantzis T, Heidari H, Van Aert S, Van Tendeloo G. 2014. Seeing and measuring in 3D with electrons. Comptes Rendus Physique 15, 140-150. (10.1016/j.crhy.2013.09.015) DOI

Batenburg KJ, et al. 2009. 3D imaging of nanomaterials by discrete tomography. Ultramicroscopy 109, 730-740. (10.1016/j.ultramic.2009.01.009) PubMed DOI

Goris B, Van den Broek W, Batenburg KJ, Heidari Mezerji H, & Bals S. 2012. Electron tomography based on a total variation minimization reconstruction technique. Ultramicroscopy 113, 120-130. (10.1016/j.ultramic.2011.11.004) DOI

Bals S, Van Aert S, Van Tendeloo G. 2013. High resolution electron tomography. Curr. Opin. Solid State Materials Sci. 17, 107-114. (10.1016/j.cossms.2013.03.001) DOI

Saghi Z, Benning M, Leary R, Macias-Montero M, Borras A, Midgley PA. 2015. Reduced-dose and high-speed acquisition strategies for multi-dimensional electron microscopy. Adv. Struct. Chem. Imag. 1, 1-10. (10.1186/s40679-015-0007-5) DOI

Liu J, et al. 2018. A modified discrete tomography for improving the reconstruction of unknown multi-gray-level material in the ‘missing wedge’ situation. J. Synchrotron Radiation 25, 1847-1859. (10.1107/S1600577518013681) PubMed DOI

Cheng A, et al. 2015. MRC2014: extensions to the MRC format header for electron cryo-microscopy and tomography. J. Struct. Biol. 192, 146-150. (10.1016/j.jsb.2015.04.002) PubMed DOI PMC

Burnley T, Palmer CM, Winn M. 2017. Recent developments in the CCP-EM software suite. Acta Crystallogr. Section D 73, 469-477. (10.1107/S2059798317007859) PubMed DOI PMC

Parkhurst J. 2021. Software for parakeet (Program for Analysis and Reconstruction of Artificial data for Kryo ElEctron Tomography): a digital twin for cryo electron tomography. (10.5281/zenodo.5185910Peet) DOI

Berman HM, et al. 2000. The Protein Data Bank. Nucleic Acids Res. 28, 235-242. (10.1093/nar/28.1.235) PubMed DOI PMC

Lobato I, Van Dyck D. 2015. MULTEM: a new multislice program to perform accurate and fast electron diffraction and imaging simulations using Graphics Processing Units with CUDA. Ultramicroscopy 156, 9-17. (10.1016/j.ultramic.2015.04.016) PubMed DOI

Jakob W, Rhinelander J, Moldovan D. 2017. pybind11—Seamless operability between C++11 and Python. Retrieved from https://github.com/pybind/pybind11.

Carugo O. 2019. Maximal B-factors in protein crystal structures. Zeitschrift Fur Kristallographie—Crystalline Materials 234, 73-77. (10.1515/zkri-2018-2057) DOI

Owen RL, Rudiño-Piñera E, Garman EF. 2006. Experimental determination of the radiation dose limit for cryocooled protein crystals. Proc. Natl Acad. Sci. USA 103, 4912-4917. (10.1073/pnas.0600973103) PubMed DOI PMC

Garman EF. 2010. Radiation damage in macromolecular crystallography: what is it and why should we care? Acta Crystallogr. Section D 66, 339-351. (10.1107/S0907444910008656) PubMed DOI PMC

Shelley KL, Dixon TPE, Brooks-Bartlett JC, Garman EF. 2018. RABDAM: quantifying specific radiation damage in individual protein crystal structures. J. Appl. Crystallogr. 51, 552-559. (10.1107/S1600576718002509) PubMed DOI PMC

Peet MJ, Henderson R, Russo CJ. 2019. The energy dependence of contrast and damage in electron cryomicroscopy of biological molecules. Ultramicroscopy 203, 125-131. (10.1016/j.ultramic.2019.02.007) PubMed DOI PMC

Zivanov J, Nakane T, Scheres SHW. 2019. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ, 6, 5-17. (10.1107/S205225251801463X) PubMed DOI PMC

Thermo Fisher Scientific. 2020. Krios G4 pre-installation manual. Waltham, MA: Thermo Fisher Scientific.

Lobato I, Van Dyck D. 2014. An accurate parameterization for scattering factors, electron densities and electrostatic potentials for neutral atoms that obey all physical constraints. Acta Crystallogr. Section A 70, 636-649. (10.1107/S205327331401643X) DOI

Downing KH, Glaeser RM. 2008. Restoration of weak phase-contrast images recorded with a high degree of defocus: the ‘twin image’ problem associated with CTF correction. Ultramicroscopy 108, 921-928. (10.1016/j.ultramic.2008.03.004) PubMed DOI PMC

Haigh SJ, Kirkland AI. 2012. High resolution exit wave restoration. In Nanostructure science and technology (ed. Lockwood DJ), pp. 1-362. Berlin, Germany: Springer.

Zanetti G, Riches JD, Fuller SD, Briggs JAG. 2009. Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging. J. Struct. Biol. 168, 305-312. (10.1016/j.jsb.2009.08.002) PubMed DOI PMC

Turoňová B, Schur FKM, Wan W, Briggs JAG. 2017. Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4 Å. J. Struct. Biol. 199, 187-195. (10.1016/j.jsb.2017.07.007) PubMed DOI PMC

Wan W, Briggs JAGG. 2016. Cryo-electron tomography and subtomogram averaging. Amsterdam, Netherlands: Elsevier. PubMed

van Aarle W, Palenstijn WJ, De Beenhouwer J, Altantzis T, Bals S, Batenburg KJ, Sijbers J. 2015. The ASTRA Toolbox: a platform for advanced algorithm development in electron tomography. Ultramicroscopy 157, 35-47. (10.1016/j.ultramic.2015.05.002) PubMed DOI

Gürsoy D, De Carlo F, Xiao X, Jacobsen C. 2014. TomoPy: a framework for the analysis of synchrotron tomographic data. J. Synchrotron Radiation 21, 1188-1193. (10.1107/S1600577514013939) PubMed DOI PMC

Pelt DM, Gürsoy D, Palenstijn WJ, Sijbers J, De Carlo F, Batenburg KJ. 2016. Integration of TomoPy and the ASTRA toolbox for advanced processing and reconstruction of tomographic synchrotron data. J. Synchrotron Radiation 23, 842-849. (10.1107/S1600577516005658) PubMed DOI PMC

Radermacher M. 2005. Weighted back projection methods. In Electron tomography: methods for three-dimensional visualization of structures in the cell (ed. Frank J), pp. 245-274. Berlin, Germany: Springer.

Penczek PA. 2010. Fundamentals of three-dimensional reconstruction from projections. Methods Enzymol. 482, 1-33. (10.1016/S0076-6879(10)82001-4) PubMed DOI PMC

Heidari Mezerji H, Van den Broek W, Bals S. 2011. A practical method to determine the effective resolution in incoherent experimental electron tomography. Ultramicroscopy 111, 330-336. (10.1016/j.ultramic.2011.01.021) PubMed DOI

Chen D, et al. 2014. The properties of SIRT, TVM, and DART for 3D imaging of tubular domains in nanocomposite thin-films and sections. Ultramicroscopy 147, 137-148. (10.1016/j.ultramic.2014.08.005) PubMed DOI

Paavolainen L, et al. 2014. Compensation of missing wedge effects with sequential statistical reconstruction in electron tomography. PLoS ONE 9, 10. (10.1371/journal.pone.0108978) PubMed DOI PMC

Kupsch A, et al. 2016. Missing wedge computed tomography by iterative algorithm DIRECTT. J. Microsc. 261, 36-45. (10.1111/jmi.12313) PubMed DOI

Wadeson N, Basham M. 2016. Savu: a Python-based, MPI framework for simultaneous processing of multiple, N-dimensional, large tomography datasets. ArXiv:1610.08015 [Cs]. Retrieved from http://arxiv.org/abs/1610.08015%0Ainternal-pdf://0.0.0.96/1610.html.

Murshudov GN, et al. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Section D 67, 355-367. (10.1107/S0907444911001314) PubMed DOI PMC

Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G. 2015. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. Section D 71, 136-153. (10.1107/S1399004714021683) PubMed DOI PMC

Midgley PA, Ward EPW, Hungría AB, Thomas JM. 2007. Nanotomography in the chemical, biological and materials sciences. Chem. Soc. Rev. 36, 1477-1494. (10.1039/b701569k) PubMed DOI

Kawase N, Kato M, Nishioka H, Jinnai H. 2007. Transmission electron microtomography without the ‘missing wedge’ for quantitative structural analysis. Ultramicroscopy 107, 8-15. (10.1016/j.ultramic.2006.04.007) PubMed DOI

Le Gros MA, et al. 2014. Biological soft X-ray tomography on beamline 2.1 at the advanced light source. J. Synchrotron Radiation 21, 1370-1377. (10.1107/S1600577514015033) PubMed DOI PMC

Baumeister W, Grimm R, Walz J. 1999. Electron tomography of molecules and cells. Trends Cell Biol. 9, 81-85. (10.1016/S0962-8924(98)01423-8) PubMed DOI

Moor H. 1987. Theory and practice of high pressure freezing. In Cryotechniques in biological electron microscopy (eds Steinbrecht RA, Zierold K), pp. 175-191. Berlin, Germany: Springer.

Studer D, Humbel BM, Chiquet M. 2008. Electron microscopy of high-pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem. Cell Biol. 130, 877-889. (10.1007/s00418-008-0500-1) PubMed DOI

Thompson RF, Walker M, Siebert CA, Muench SP, Ranson NA. 2016. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology. Methods 100, 3-15. (10.1016/j.ymeth.2016.02.017) PubMed DOI PMC

Van Doren EAF, De Temmerman PJRH, Francisco MAD, Mast J. 2011. Determination of the volume-specific surface area by using transmission electron tomography for characterization and definition of nanomaterials. J. Nanobiotechnol. 9, 1-8. (10.1186/1477-3155-9-17) PubMed DOI PMC

Huang J, Wang X, Grandfield K. 2014. FIB preparation of bone-implant interfaces for correlative on-axis rotation electron tomography and atom probe tomography. Microsc. Microanal. 20, 352-353. (10.1017/S1431927614003481) DOI

Yip KM, Fischer N, Paknia E, Chari A, Stark H. 2020. A structure of human apoferritin obtained from Titan Mono-BCOR microscope. See www.wwpdb.org/pdb?id=pdb_00006z6u. (10.2210/pdb6z6u/pdb) DOI

Voorhees RM, Weixlbaumer A, Loakes D, Kelley AC, Ramakrishnan V. 2009. Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat. Struct. Mol. Biol. 16, 528-533. (10.1038/nsmb.1577) PubMed DOI PMC

Nakane T, et al. 2020. Single-particle cryo-EM at atomic resolution. bioRxiv. (10.1101/2020.05.22.110189) DOI

Kmetko J, Husseini NS, Naides M, Kalinin Y, Thorne RE. 2006. Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures. Acta Crystallogr. Section D 62, 1030-1038. (10.1107/S0907444906023869) PubMed DOI

Egerton RF. 2015. Outrun radiation damage with electrons? Adv. Struct. Chem. Imaging 1, 1-11. (10.1186/s40679-014-0001-3) DOI

Langmore JP, Smith MF. 1992. Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultramicroscopy 46, 349-373. (10.1016/0304-3991(92)90024-E) PubMed DOI

Pillar data-acquisition strategies for cryo-electron tomography of beam-sensitive biological samples