Computational Methods for Interpretation of EM Maps at Subnanometer Resolution

Abstract

Driven by a remarkable number of technological advances in recent years, electron cryo‐microscopy (cryo‐EM) has played an increasingly important role in deciphering macromolecular structure and function. Today, cryo‐EM routinely achieves subnanometer resolutions, with current state‐of‐the‐art reconstructions approaching atomic resolutions. Though achieving high‐resolution density maps is still a challenging aspect of cryo‐EM, it is possible to isolate individual subunits, identify secondary structures elements and accurately fit atomic models in reconstructions of large macromolecular assemblies. Additionally, computational modelling and feature recognition tools are routinely employed to construct backbone and atomic models of entire assemblies directly from a density map. Together, these tools provide researchers with unprecedented insight into structure and function, revealing everything from subunit arrangements to complex sets of interactions in macromolecular assemblies.

Key Concepts:

  • Cryo‐EM is capable of imaging large macromolecular assemblies in order to understand their structure and function.

  • Analysing macromolecular assemblies requires a variety of complex computational tools.

  • In subnanometer resolution cryo‐EM density maps, it is possible to see individual protein subunits, as well as higher‐resolution structural features.

  • It is now possible to construct backbone and atomic models directly from cryo‐EM density maps at near‐atomic resolutions.

Keywords: cryo‐EM; segmentation; fitting; modelling; macromolecular assemblies

Figure 1.

Resolution in cryo‐EM. Using cryo‐EM, GroEL has been imaged and reconstructed at a variety of resolutions. Shown in the top row are three reconstructions of GroEL at 10.4, 7.8 and 4.2 Å resolution (from left to right EMDB Ids: 1040, 1200 and 5001, respectively). In the bottom row, a single subunit is shown with the X‐ray crystal structure (PDB ID: 1SS8) of fit to the density map. At ∼10 Å resolution, the overall size and shape of the assembly is evident and allows for the segmentation of individual protein subunits. At ∼8 Å resolution, secondary structure elements begin to be resolved. Helices appear as density rods and β‐sheets appear as thin, flat planes. However, connections between the SSEs are not clearly visible. At ∼4 Å, higher resolution features, such as the pitch of the α‐helix and separation of β‐strands is seen. At this resolution, it is possible to observe the connections between SSEs and construct a model directly from the density map with the aid of computational modelling tools.

Figure 2.

Segmentation of a cryo‐EM density map. Four representative cryo‐EM density maps are shown in which individual proteins or domains have been isolated using different techniques. (a) Segger, a semi‐automated utility for segmentation, was used to identify the 16 subunits in the 4.3 Å resolution structure of MM‐CPN (EMDB ID: 5137). (b) Segment3D, a utility in EMAN, was used to automatically identify potential domains in one subunit of the homotetrameric Ca2+ release channel (EMDB ID: 1275, 9.6 Å resolution). (c) VolRover was used to automatically identify the two subunits in the Thermus thermophilus 70S ribosome along with the t‐RNA (EMDB ID: 5030, 6.4 Å resolution). (d) The tail‐spike and its components were isolated from the bacteriophage P‐SSP7 reconstruction manually using Amira (EMDB ID: 1713, 4.6 Å resolution). Individual images were provided by Greg Pintile (Figure a), Irina Serysheva (Figure b) and Xiangan Liu (Figure d).

Figure 3.

Analysis of a cryo‐EM density map. Over the past decade, a number of computational tools have been developed to extract information from a cryo‐EM density map. (a) SSEHunter was used to identify SSEs in the 9.5 Å resolution reconstruction of Rotavirus. Two copies of the inner capsid protein P3 are shown along with their respective SSEs; helices are shown as green cylinders and sheets are represented as cyan planes. (b) CP from Venezuelan Encephalitis Virus (PDB ID: 1EP5) was solved by X‐ray crystallography and fit to the 4.4 Å density map using Foldhunter. (c) An example of flexible fitting is shown in which Flex‐EM was used to flexibly fit the crystal structure of the N‐terminal portion of the human apoptosome (PDB IDs: 1Z6T, 2B4Z) in the 9.5 Å resolution cryo‐EM structure of the apoptosome–procaspase CARD complex (EMDB ID: 5186, PDB ID: 3IYT). (d) Constrained comparative modelling with Modeller was used to generate a model for the 80S ribosome (from Thermomyces lanuginosus) (EMDB IDs: 1067, 1345, PDB IDs: 3JYV, 3JYW, 3JYX), including the rpS19e protein (red) from the 40S subunit. In (e), a model for VP26, a small capsid protein in HSV‐1 (8.9 Å resolution), was constructed using Rosetta with density constraints. Individual images were provided by Corey Hryc (Figure b) and Maya Topf (Figure c, d).

Figure 4.

Modelling in cryo‐EM. As cryo‐EM is now approaching atomic resolutions, it is possible to construct protein structural models directly from a cryo‐EM density map. (a) A basic protocol for de novo model building is shown using the group II chaperonin, MM‐CPN, which was imaged to 4.3 Å resolution (EMDB ID: 5137). (b) A model for the bacteriophage P22 capsid protein constructed from the 3.8 Å resolution density map of the infectious virus is shown (PDB ID: 2XYZ, EMDB ID: 1824). This model was constructed using the de novo modelling approach in (a), in combination with a constrained homology modelling approach (Figure e). In the P22 structure, not all side chains could be resolved; the resulting model is Cα‐only model. (c) A full atomic model for the human adenovirus penton protein was constructed directly from the 3.6 Å resolution density map with a similar protocol (PDB ID: 3IYN, EMDB ID: 5172). Images for panels b and c were provided by Corey Hryc.

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References

Abeysinghe S, Baker ML, Chiu W and Ju T (2010) Semi‐isometric registration of line features for flexible fitting of protein structures. Computer Graphics Forum (Proceedings of Pacific Graphics 2010) 29: 2243–2252.

Abeysinghe S, Ju T, Baker ML and Chiu W (2008) Shape modeling and matching in identifying 3D protein structures. Computer Aided Design 40: 708–720.

Baker ML, Abeysinghe SS, Schuh S et al. (2011) Modeling protein structure at near atomic resolutions with Gorgon. Journal of Structural Biology 174: 360–373.

Baker ML, Baker MR, Hryc CF and Dimaio F (2010a) Analyses of subnanometer resolution cryo‐EM density maps. Methods in Enzymology 483: 1–29.

Baker ML, Jiang W, Rixon FJ and Chiu W (2005) Common ancestry of herpesviruses and tailed DNA bacteriophages. Journal of Virology 79: 14967–14970.

Baker ML, Jiang W, Wedemeyer WJ et al. (2006a) Ab initio modeling of the herpesvirus VP26 core domain assessed by CryoEM density. PLoS Computational Biology 2: 1313–1324.

Baker ML, Ju T and Chiu W (2007) Identification of secondary structure elements in intermediate‐resolution density maps. Structure 15: 7–19.

Baker ML, Yu Z, Chiu W and Bajaj C (2006b) Automated segmentation of molecular subunits in electron cryomicroscopy density maps. Journal of Structural Biology 156: 432–441.

Baker ML, Zhang J, Ludtke SJ and Chiu W (2010b) Cryo‐EM of macromolecular assemblies at near‐atomic resolution. Nature Protocols 5: 1697–1708.

Böttcher B, Wynne SA and Crowther RA (1997) Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386: 88–91.

Chen DH, Baker ML, Hryc CF et al. (2011) Structural basis for scaffolding‐mediated assembly and maturation of a dsDNA virus. Proceedings of the National Academy of Sciences of the USA 108: 1355–1360.

Chiu W, Baker ML and Almo SC (2006) Structural biology of cellular machines. Trends in Cell Biology 16: 144–150.

Chiu W, Baker ML, Jiang W, Dougherty M and Schmid MF (2005) Electron cryomicroscopy of biological machines at subnanometer resolution. Structure 13: 363–372.

Cong Y, Baker ML, Jakana J et al. (2010) 4.0‐A resolution cryo‐EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proceedings of the National Academy of Sciences of the USA 107: 4967–4972.

Cong Y and Ludtke SJ (2010) Single particle analysis at high resolution. Methods in Enzymology 482: 211–235.

Conway JF, Cheng N, Zlotnick A et al. (1997) Visualization of a 4‐helix bundle in the hepatitis B virus capsid by cryo‐electron microscopy. Nature 386: 91–94.

DiMaio F, Tyka MD, Baker ML, Chiu W and Baker D (2009) Refinement of protein structures into low‐resolution density maps using rosetta. Journal of Molecular Biology 392: 181–190.

Emsley P, Lohkamp B, Scott WG and Cowtan K (2010) Features and development of Coot. Acta Crystallographica Section D: Biological Crystallography 66: 486–501.

Förster F, Pruggnaller S, Seybert A and Frangakis AS (2008) Classification of cryo‐electron sub‐tomograms using constrained correlation. Journal of Structural Biology 161: 276–286.

Frangakis AS, Böhm J, Förster F et al. (2002) Identification of macromolecular complexes in cryoelectron tomograms of phantom cells. Proceedings of the National Academy of Sciences of the USA 99: 14153–14158.

Grigorieff N and Harrison SC (2011) Near‐atomic resolution reconstructions of icosahedral viruses from electron cryo‐microscopy. Current Opinion in Structural Biology 21: 265–273.

Jiang W, Baker ML, Jakana J et al. (2008) Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 451: 1130–1134.

Jiang W, Baker ML, Ludtke SJ and Chiu W (2001) Bridging the information gap: computational tools for intermediate resolution structure interpretation. Journal of Molecular Biology 308: 1033–1044.

Jones TA, Zou JY, Cowan SW and Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallographica Section A 47(part 2): 110–119.

Ju T, Baker ML and Chiu W (2007) Computing a family of skeletons of volumetric models for shape description. Computer Aided Design 39: 352–360.

Kleywegt GJ and Jones TA (1997) Detecting folding motifs and similarities in protein structures. Methods in Enzymology 277: 525–545.

Kong Y and Ma J (2003) A structural‐informatics approach for mining beta‐sheets: locating sheets in intermediate‐resolution density maps. Journal of Molecular Biology 332: 399–413.

Kong Y, Zhang X, Baker TS and Ma J (2004) A structural‐informatics approach for tracing beta‐sheets: building pseudo‐C(alpha) traces for beta‐strands in intermediate‐resolution density maps. Journal of Molecular Biology 339: 117–130.

Li Z, Baker ML, Jiang W, Estes MK and Prasad BVV (2009) Rotavirus architecture at subnanometer resolution. Journal of Virology 83: 1754–1766.

Liu H, Jin L, Koh SBS et al. (2010) Atomic structure of human adenovirus by cryo‐EM reveals interactions among protein networks. Science 329: 1038–1043.

Ludtke SJ, Baker ML, Chen D‐HH et al. (2008) De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure 16: 441–448.

Ludtke SJ, Baldwin PR and Chiu W (1999) EMAN: semiautomated software for high‐resolution single‐particle reconstructions. Journal of Structural Biology 128: 82–97.

Nakagawa A, Miyazaki N, Taka J et al. (2003) The atomic structure of rice dwarf virus reveals the self‐assembly mechanism of component proteins. Structure 11: 1227–1238.

Pettersen EF, Goddard TD, Huang CC et al. (2004) UCSF Chimera – a visualization system for exploratory research and analysis. Journal of Computational Chemistry 25: 1605–1612.

Pintilie GD, Zhang J, Goddard TD, Chiu W and Gossard DC (2010) Quantitative analysis of cryo‐EM density map segmentation by watershed and scale‐space filtering, and fitting of structures by alignment to regions. Journal of Structural Biology 170: 427–438.

Rossmann MG, Bernal R and Pletnev SV (2001) Combining electron microscopic with X‐ray crystallographic structures. Journal of Structural Biology 136: 190–200.

Rossmann MG, Morais MC, Leiman PG and Zhang W (2005) Combining X‐ray crystallography and electron microscopy. Structure 13: 355–362.

Schröder GF, Brunger AT and Levitt M (2007) Combining efficient conformational sampling with a deformable elastic network model facilitates structure refinement at low resolution. Structure 15: 1630–1641.

Seidelt B, Innis CA, Wilson DN et al. (2009) Structural insight into nascent polypeptide chain‐mediated translational stalling. Science 326: 1412–1415.

Serysheva II, Hamilton SL, Chiu W and Ludtke SJ (2005) Structure of Ca2+ release channel at 14 A resolution. Journal of Molecular Biology 345: 427–431.

Siebert X and Navaza J (2009) UROX 2.0: an interactive tool for fitting atomic models into electron‐microscopy reconstructions. Acta Crystallographica Section D: Biological Crystallography 65: 651–658.

Suhre K, Navaza J and Sanejouand YH (2006) NORMA: a tool for flexible fitting of high‐resolution protein structures into low‐resolution electron‐microscopy‐derived density maps. Acta Crystallographica Section D: Biological Crystallography 62: 1098–1100.

Tama F, Miyashita O and Brooks CL (2004) Normal mode based flexible fitting of high‐resolution structure into low‐resolution experimental data from cryo‐EM. Journal of Structural Biology 147: 315–326.

Taylor DJ, Devkota B, Huang AD et al. (2009) Comprehensive molecular structure of the eukaryotic ribosome. Structure 17: 1591–1604.

Topf M, Baker ML, John B, Chiu W and Sali A (2005) Structural characterization of components of protein assemblies by comparative modeling and electron cryo‐microscopy. Journal of Structural Biology 149: 191–203.

Topf M, Baker ML, Marti‐Renom MA, Chiu W and Sali A (2006) Refinement of protein structures by iterative comparative modeling and CryoEM density fitting. Journal of Molecular Biology 357: 1655–1668.

Topf M, Lasker K, Webb B et al. (2008) Protein structure fitting and refinement guided by cryo‐EM density. Structure 16: 295–307.

Trabuco LG, Villa E, Schreiner E, Harrison CB and Schulten K (2009) Molecular dynamics flexible fitting: a practical guide to combine cryo‐electron microscopy and X‐ray crystallography. Methods 49: 174–180.

Volkmann N (2009) Confidence intervals for fitting of atomic models into low‐resolution densities. Acta Crystallographica Section D: Biological Crystallography 65: 679–689.

Volkmann N and Hanein D (1999) Quantitative fitting of atomic models into observed densities derived by electron microscopy. Journal of Structural Biology 125: 176–184.

Wriggers W, Milligan RA and McCammon JA (1999) Situs: a package for docking crystal structures into low‐resolution maps from electron microscopy. Journal of Structural Biology 125: 185–195.

Wynne SA, Crowther RA and Leslie AG (1999) The crystal structure of the human hepatitis B virus capsid. Molecular Cell 3: 771–780.

Yu X, Jin L and Zhou ZH (2008) 3.88 A structure of cytoplasmic polyhedrosis virus by cryo‐electron microscopy. Nature 453: 415–419.

Yuan S, Yu X, Topf M et al. (2010) Structure of an apoptosome–procaspase‐9 CARD complex. Structure 18: 571–583.

Zanetti G, Briggs JA, Grünewald K, Sattentau QJ and Fuller SD (2006) Cryo‐electron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathogens 2: 0790–0797.

Zhang J, Ma B, Dimaio F et al. (2011a) Cryo‐EM structure of a group II chaperonin in the prehydrolysis ATP‐bound state leading to lid closure. Structure 19: 633–639.

Zhang R, Hryc CF, Cong Y et al. (2011b) 4.4 Å Cryo‐EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus. EMBO Journal 30: 3854–3863.

Zhang X, Settembre E, Xu C et al. (2008) Near‐atomic resolution using electron cryomicroscopy and single‐particle reconstruction. Proceedings of the National Academy of Sciences of the USA 105: 1867–1872.

Zhou ZH, Baker ML, Jiang W et al. (2001) Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus. Nature Structural Biology 8: 868–873.

Zhu J, Cheng L, Fang Q, Zhou ZH and Honig B (2010) Building and refining protein models within cryo‐electron microscopy density maps based on homology modeling and multiscale structure refinement. Journal of Molecular Biology 397: 835–851.

Further Reading

Baumeister W and Steven AC (2000) Macromolecular electron microscopy in the era of structural genomics. Trends in Biochemical Sciences 25: 624–631.

Frank J (2002) Single‐particle imaging of macromolecules by cryo‐electron microscopy. Annual Review of Biophysics and Biomolecular Structure 31: 303–319.

Jiang W and Chiu W (2007) Cryoelectron microscopy of icosahedral virus particles. Methods in Molecular Biology 369: 345–363.

Zhou ZH (2008) Towards atomic resolution structural determination by single‐particle cryo‐electron microscopy. Current Opinion in Structural Biology 18: 218–228.

Zhou ZH, Dougherty M, Jakana J et al. (2000) Seeing the herpesvirus capsid at 8.5 A. Science 288: 877–880.

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Baker, Matthew L, Baker, Mariah R, and Cong, Yao(Jan 2012) Computational Methods for Interpretation of EM Maps at Subnanometer Resolution. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0023174]