Sharpening high resolution information in single particle electron ...

Journal of Structural Biology 164 (2008) 170–175

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Crystallization Notes

Sharpening high resolution information in single particle electron cryomicroscopy J.J. Fernández a,b,*, D. Luque a, J.R. Castón a, J.L. Carrascosa a a b

Centro Nacional de Biotecnologia, CSIC. Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain Department of Computer Architecture, University of Almeria, Almeria 04120, Spain

a r t i c l e

i n f o

Article history: Received 21 April 2008 Received in revised form 22 May 2008 Accepted 26 May 2008 Available online 3 June 2008 Keywords: Single particle Electron cryomicroscopy High resolution B-factor Temperature factor Sharpening Restoration Contrast loss Secondary structure elements

a b s t r a c t Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Single particle electron cryomicroscopy allows structure elucidation of unstained proteins and macromolecular assemblies at subnanometer resolution thanks to the technical and computational advances made during the last years (Henderson, 2004; Chiu et al., 2005; Frank, 2006). This experimental approach has made possible the structure determination of specimens with different symmetry levels in a resolution range of 4–9 Å, e.g. ribosome (Halic et al., 2006), groEL (Ranson et al., 2006; Ludtke et al., 2008), Bacteriophage T7 connector (Agirrezabala et al., 2005), and several icosahedral structures (e.g. Luque et al., 2007; Jiang et al., 2008; Zhang et al., 2008). There are exciting prospects that resolution levels adequate to build atomic models will soon be approachable with this technique (Henderson, 2004; Zhou, 2008). The main objective to reach high resolution is driven by the fact that density maps determined at subnanometer resolution allow identification of secondary structure elements (SSE) by their signature (Chiu et al., 2005; Baker et al., 2007). In the range 6–10 Å, a-helices become visible as straight rods of density and

* Corresponding author. Address: Department of Computer Architecture, University of Almeria, Almeria 04120, Spain. Fax: +34 950 015 486. E-mail addresses: [email protected], [email protected] (J.J. Fernández). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.05.010

b-sheets appear as continuous planes. Beyond 5–6 Å, loop densities may be discernable and the protein backbone may be traced. At resolutions higher than 4.5 Å, individual strands within the b-sheets may be observed. Eventually, resolutions better than 4 Å will allow the assignment of a sequence of aminoacid side chains into density features in the map (Henderson, 2004). Identification and interpretation of the structural features in a map are, however, hampered by the loss of contrast at high resolution. This degradation of the image contrast is caused, in a great extent, by factors related to the experimental imaging process (e.g. specimen movement and charging, radiation damage, partial microscope coherence, etc.) (Henderson, 1992; Wade, 1992), as well as by factors related to the computational procedures for structure determination (e.g. inaccurate determination of the orientation parameters, etc.) (Conway and Steven, 1999; Rosenthal and Henderson, 2003; Henderson, 2004). The combined effect of all these factors has been traditionally modeled by a Gaussian 2 amplitude decay of structure factors, given by eðBoverall =4d Þ with an overall temperature factor Boverall (also called B-factor) and where d denotes resolution (Å) (Glaeser and Downing, 1992; Conway and Steven, 1999; Rosenthal and Henderson, 2003). This amplitude fall-off significantly affects the high resolution components, thereby making the density map look apparently smooth and turning identification of SSE into a challenging task. Therefore, it is neces-

J.J. Fernández et al. / Journal of Structural Biology 164 (2008) 170–175

sary to restore (also called sharpen) these components in order to bring out the structural features concealed in the map. There have been several strategies to sharpen density maps in single particle electron cryomicroscopy. One common approach consists of correcting the amplitude fall-off by applying a B-factor 2 in the form eðBrestore =4d Þ , where Brestore is the negative of the estimated B-factor describing the contrast loss of the experimental data (e.g. Bottcher et al., 1997; Fotin et al., 2004). Other approaches use the X-ray solution scattering curve of the specimen (or a related homologue) to estimate the decay and correct the amplitudes accordingly (e.g. Gabashvili et al., 2000; Agirrezabala et al., 2005; Luque et al., 2007; Ludtke et al., 2008). Rosenthal and Henderson (2003) introduced a method for estimating the structure factor amplitude decay by comparison with an approximate theoretical scattering curve. This method allows objective sharpening of density maps, with neither further needs of ad hoc B-factors, nor the availability of an X-ray scattering reference. Moreover, that work also showed that a weighting function accounting for the noise in the map is essential to avoid its amplification, while at the same time increasing the robustness against oversharpening. Here, we introduce a program, EM-BFACTOR, that was developed to facilitate the general use of the method devised by Rosenthal and Henderson (2003). It allows automated determination of the B-factor for high resolution maps as well as noise-weighted sharpening of the density map to enhance features without noise amplification. Furthermore, the structure factor amplitudes of the map are also placed on an absolute scale. We have also further investigated on the sharpening method, and here, we describe its performance on several illustrative examples. 2. Methodology and implementation A Guinier plot shows the natural logarithm of the spherically averaged structure factor amplitudes of a map as a function of 2 the resolution ð1=d Þ. The low resolution region (to about 10 Å) is determined by the shape of the protein and the solvent contrast and shows a steep curve that gets the maximum scattering amplitude at zero, rapidly decaying afterwards. Beyond 10 Å, the amplitude depends on the specific structural features of the protein. In that range, the average scattering amplitude is determined by the random position of atoms in the protein and remains steadily constant with resolution, according to Wilson (1942). For an indepth theoretical background, refer to (Rosenthal and Henderson, 2003). The approach by Rosenthal and Henderson (2003) for B-factor determination relies on the fact that the high resolution contrast loss in an experimental map may be observed in a Guinier plot as the decay of its spherically averaged amplitude ðFÞ compared to the relatively flat theoretical scattering profile corresponding to the Wilson regime. In order to better estimate the B-factor, the noise-weighted amplitude C ref F is used as it represents more properly the average signal in the experimental map. The term C ref , which is computed by resolution shells from smoothed ver-ffi pa ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sion of the FSC (Fourier Shell Correlation) as 2FSC=ð1 þ FSCÞ, gives a measure of the average signal-to-noise ratio (SNR) in the reciprocal space and, hence, of the reliability in the structure factors. Our procedure for automated B-factor determination calculates the line that best fits the decay of the spherically averaged amplitude C ref F by a least squares procedure. For the fitting, a range of resolution is used, typically [10–15Å, Rmax ], where Rmax is the maximum resolution in the map as assessed by means of the FSC. The B-factor affecting the experimental map is then determined from the slope of the line fitted. The B-factor for sharpening ðBrestore Þ is thus set as the negative of the B-factor affecting the data.

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Contrast restoration is carried out by applying the B-factor in 2 the form eðBrestore =4d Þ . This type of restoration amplifies signal and noise regardless of the SNR, which may be especially dangerous at high resolution where the signal is weak. To attenuate the amplification of high resolution noise, a common solution is based on low-pass filtering. Rosenthal and Henderson (2003) devised an approach based on a down-weighting of resolution shells where noise is dominant by means of C ref . This noise-weighting has been shown to avoid noise intensification, increase the robustness against oversharpening and reduce the sensitivity to the resolution cutoff of the map. Therefore, the amplitudes F are restored in the 2 form C ref FeðBrestore =4d Þ . On the other hand, the experimental scattering amplitudes may be placed on an absolute scale by setting the zero angle scattering equal to 0:28 N atoms and the average amplitude in the high resolution region (Wilson regime) to pffiffiffiffiffiffiffiffiffiffiffiffiffiffi N atoms . N atoms denotes the number of carbon atom equivalents corresponding to the molecular mass of the protein, and 0.28 is the solvent contrast factor. The program EM-BFACTOR has been developed for automated Bfactor determination and sharpening of high resolution density maps as described above. Application of a specific value for B-factor is also possible. The program accepts 3D maps in any format common in EM (e.g. MRC, Spider, PIF, EM, Xmipp, etc.) by using the Bsoft library (Heymann and Belnap, 2007). EM-BFACTOR accepts FSC curves in the format of most common packages for single particle electron cryomicroscopy (Frealign, Spider, EMAN, Xmipp, Bsoft). The output sharpened map, placed on an absolute scale, can be written in any format common in EM. The program also generates a Guinier plot with the average structure factor amplitudes of the original map, the noise-weighted map and the sharpened map. The zero angle scattering and Wilson statistics used for scaling the map are also indicated. This plot, which is generated in Postscript file and in text format, can be used for assessment of the sharpening. An output report is shown on console with the progress of the program, the parameters for the least squares fitting and the B-factor that is finally found out. The command line user interface follows the Unixstyle and the options follows the conventions of Bsoft (Heymann and Belnap, 2007). Examples of commands and the output report are shown in Appendix. EM-BFACTOR and a comprehensive documentation will be available for public use at http:// www.ual.es/~jjfdez/SW/embfactor and through the wikipedia ‘‘Software tools for molecular microscopy”. 3. Illustrative examples EM-BFACTOR was tested on experimental 3D maps of several specimens to further investigate the method for automated B-factor determination and sharpening, and for illustrative purposes. First, we worked on two specimens whose structure has been solved at subnanometer resolution in our laboratory: Infectious bursal disease virus T = 1 subviral particle (IBDV T = 1 SVP) (Luque et al., 2007) and Bacteriophage T7 connector (Agirrezabala et al., 2005), solved at 7.2 and 8.0 Å resolution, respectively. Resolution was assessed by the standard technique based on the Fourier shell correlation (FSC) calculated between maps obtained from independent halves of the raw dataset at a 0.5 cutoff. IBDV T = 1 SVP was solved by X-ray crystallography and electron cryomicroscopy (Coulibaly et al., 2005; Luque et al., 2007), and it is thus adequate for objective assessment of the proposed contrast restoration methodology. The T7 connector map was sharpened according to the decay of the X-ray scattering amplitude of a homologous specimen, the Bacteriophage /29 connector (Agirrezabala et al., 2005). Therefore, this represents a complementary analysis to evaluate the performance of that sharpening strategy and whether it is possible further improvement. Second, we focused on several maps

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a

b

c

Guinier plot

d 12

Experimental amplitudes Cref-weighted amplitudes Sharpened amplitudes Wilson statistics: (Natoms)1/2

0.28xNatoms

11 10 9 8

lnF

7 6 5 4 3 2 0

0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 0.02

d−2 (Å−2) Fig. 1. Contrast restoration on IBDV T = 1 SVP at 7.2 Å. (a) trimer extracted from the unsharpened map; (b) trimer extracted from the sharpened map with EM-BFACTOR (Brestore ¼ 610 Å2); (c) X-ray structure of IBDV T = 1 SVP docked into a semi-transparent version of the sharpened trimer, rotated 70° around the X axis with respect to the view shown in (b); (d) Guinier plot provided by EM-BFACTOR showing the natural logarithm of the spherically average amplitude of the unsharpened map, the noiseweighted amplitude and the restored amplitudes after sharpening, as well as the zero scattering amplitude and the Wilson statistics. The pixel size was 1.4 Å.

from the EM depositions at the EBI Macromolecular Structure Database (http://www.ebi.ac.uk/msd/). The tests with these maps, which were already sharpened with different strategies, are helpful to confirm the potential role of this objective B-factor determination method.

Analyses with the IBDV T = 1 SVP were done on the raw structure (i.e. unsharpened) as solved at 7.2 Å resolution (Luque et al., 2007). The B-factor was determined with the program EM-BFACTOR as described above, obtaining Brestore ¼ 610 Å2. Fig. 1(a) and (b) show a trimer of the capsid protein before and after sharp-

Fig. 2. SSE identification on IBDV T = 1 SVP. (a) Ribbon diagram of IBDV T = 1 SVP subunit. Arrows indicate individual SSE, named as in Luque et al. (2007). (b and c) Results of the SSE identification for the map sharpened with the X-ray structure profile (b) and for the EM-BFACTOR sharpened map (c). a-helices and b-sheets are represented by green and cyan, respectively. (d) Comparison of the identified SSE, where the results for the map sharpened against the X-ray structure are shown in red and the ones for the EMBFACTOR sharpened map are shown in blue.

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map, the noise-weighted amplitude and the resulting curve after sharpening are shown. The raw map was also sharpened according to the decay of the X-ray scattering amplitude of the IBDV T = 1 SVP as solved previously (PDB entry 1wcd) (Coulibaly et al., 2005). Visually, the resulting map (not shown here) presented little difference with the map sharpened with EM-BFACTOR. For further assessment, SSE identified from each sharpened map were compared with the X-ray structure. The analysis and annotation of SSE is essential for extraction of significant functional and structural information, and at this intermediate resolution range (6–10 Å), a-helices and b-sheets are detectable. The IBDV T = 1 SVP map restored to follow the X-ray scattering profile and the one sharpened with EM-BFACTOR were analysed with SSEhunter (Baker et al., 2007). Both sharpening methods allow identification of the SSE of the IBDV T = 1 SVP subunit, and the annotated results can be basically superimposed (Fig. 2). The averaged scores for each individual SSE, reflecting their propensity for helix or sheet, are similar for both maps (Table 1). The fact that these scores are partly based upon local geometry features (Baker et al., 2007), and hence sensitive to amplitude sharpening, makes them appropriate for this quantitative comparison (see legend to Table 1 for description of the scoring algorithm). These results with real data demonstrate that the objective B-factor sharpening approach and

Table 1 Averaged scores of each single SSE of the IBDV T = 1 SVP subunit identified with SSEhunter SSE Sharpening method

a1

a2

a3

bS

bP

X-ray structure based EM-BFACTOR

1.15 1.18

0.84 0.82

0.71 0.60

1.79 1.72

1.62 1.54

Scores for the SSE in the density map sharpened following the X-ray structure profile (first row) and in the map corrected with EM-BFACTOR (second row) are shown. The SSE are named as illustrated in Fig. 2(a). SSEhunter (Baker et al., 2007) detects SSE based on density skeletonization, template-based search and local geometry analysis. Each of these three evaluation methods is scored (1) to (+1), summed and mapped in pseudoatoms that reflect the propensity for sheet-like and helix-like features. Each average score shown here is calculated using the X-ray scattering structure as a template so that the scores of the pseudoatoms located over each single SSE are averaged. Positive and negative values correspond to propensity for helix-like or sheet-like features, respectively. Maximum score for bsheet is 3; maximum score for a-helix is +3.

ening. The sharpening effect is apparent as significant structural features now become visible. The X-ray structure of IBDV T = 1 SVP fits well into the sharpened map (Fig. 1(c)). Fig. 1(d) shows the output Guinier plot provided by EM-BFACTOR, where the natural logarithm of the spherically average amplitude of the original

a

b

c

Guinier plot 10 Experimental amplitudes Cref-weighted amplitudes Sharpened amplitudes 1/2 Wilson statistics: (Natoms)

0.28xNatoms 9

lnF

8

7

6

5

4 0

0.002

0.004

0.006

d

0.008

−2

0.01

0.012

0.014

0.016

−2

(Å )

Fig. 3. Contrast restoration on T7 connector at 8.0 Å. (a) map with amplitudes enhanced as published in Agirrezabala et al. (2005); (b) map after additional sharpening with EM-BFACTOR (Brestore ¼ 343 Å2); (c) Guinier plot showing the residual scattering amplitude decay present in the initial map and the restored amplitude approaching Wilson statistics. The pixel size was 2.18 Å.

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Table 2 Summary of results from the application of EM-BFACTOR to a set of structures from the EM deposition at the EBI Macromolecular Structure Database EMD ID

Sample name

Resolution (Å)

B-factor (Å2)

1081 1125 1136 1180 1181 1202 1206 1285 1315 1334 1460 1461 5003

Native naked GroEL 80S wheat germ ribosome Poliovirus 135S particle GroEL-ATP7-GroES GroEL-ADP7-GroES GroEL-ADP-gp31 Bacteriophage phi6 nucleocapsid Yeast 80S ribosome in complex with the CrPV IRES RNA Complex of Thermus thermophilus 70S ribosomes and EF-G-GMPPNP Bacteriophages K1E and K1-5 Bovine rotavirus DLP Rotavirus VP6 protein Bacteriophage epsilon15

6.0 9.5 8.7 7.7 8.7 8.2 7.5 7.3 7.3 8.9 5.1 3.8 4.5

375 715 700 409 203 258 431 515 507 452 55 120 58

The columns correspond to the accession code, the name of the sample as appears in the database, the resolution of the map according to the database, and the B-factor found by EM-BFACTOR, respectively. The resolution of those maps was assessed by the standard technique based on the FSC at 0.3–0.5 cutoff. The cutoff used for the map with entry EMD-1460 was 0.143 (Zhang et al., 2008). The resolution of the map with entry EMD-1461 was assessed by comparison with the X-ray structure at 3.8 Å resolution (Zhang et al., 2008). Estimation of the B-factor was carried out from the experimental average scattering amplitudes, without noise-weighting.

the restoration based on the X-ray scattering amplitude of the specimen behave similarly. The structure of T7 connector was solved at 8.0 Å resolution by single particle electron cryomicroscopy (Agirrezabala et al., 2005). The amplitudes of the map were enhanced based on the X-ray scattering profile of the Bacteriophage /29 connector (Fig. 3(a)). EMBFACTOR has shown that further improvement was still possible, as after applying it to that amplitude-enhanced map, we obtained Brestore ¼ 343 Å2. The effect of this additional contrast restoration can be seen in Fig. 3(b). Fig. 3(c) shows the Guinier plot, where the remaining decay present in the X-ray corrected map is apparent. The B-factor sharpened map (Fig. 3(b)) clearly reveals more detailed structural features that previously appeared subtle, or even go unnoticed. The enhancement is visible in all the major domains of the structure: the crown at the top, the wings (corresponding to the outer 12 subunit ring), and the stalk (the narrower end at the bottom). At the crown, density masses that might correspond to helices are now clearly discernable. In the previous map, however, they appeared continuous with the surrounding densities. Similarly, the three subdomains that define certain vorticity of the structure look better detailed at the wing domain. Finally, new features at the distal stalk have been brought out after sharpening. Finally, we applied EM-BFACTOR to a set of structures solved at subnanometer resolution by electron cryomicroscopy that are deposited at the EBI MSD database. In these structures, the falloff of the experimental amplitudes was already corrected using different sharpening techniques. Our aim was to confirm if there were still chances for further amplitude enhancement, as happened with the T7 connector. A summary of the results for the tested specimens is shown in Table 2. When the B-factor is around or lighter than 200 Å2, little impact should be expected, as discussed by Rosenthal and Henderson (2003). However, stronger B-factors would involve significant enhancement of the density map. Note that the FSC curves are not available at the EBI MSD database and, as a result, the B-factor was estimated without noise-weighting. Therefore, some small deviations from the real B-factor value should be expected. 4. Conclusion The results obtained here confirm that the sharpening approach proposed by Rosenthal and Henderson (2003) can help to restore the contrast of a density map in an objective way, and that the availability of a reference is no longer crucial. Moreover, it has been shown that there may be still a chance to improve the density

maps that were sharpened with ad hoc B-factors or according to the decay of X-ray scattering profiles of related specimens. Sharpened maps facilitate annotation of SSE and other high resolution structural features may become discernable either by simple visualization or by means of computational tools for their identification and prediction. EM-BFACTOR is a program that has been developed to widely facilitate automated, objective B-factor determination and sharpening. The program, which is freely available, accepts the most common formats for 3D maps and for FSC curves in single particle electron cryomicroscopy. EM-BFACTOR helps to reveal the molecular features concealed in experimental density maps, thereby making sharpened maps better suited for interpretation. Acknowledgments The authors are deeply grateful to P.B. Rosenthal and R. Henderson for help and support and X. Agirrezabala for the T7 connector map. This work was initiated as part of a research visit (JJF) at the MRC Laboratory of Molecular Biology (Cambridge, UK). Work supported by EU-FP6-LSHG-CT-2004-502828, MEC-TIN2005-00447, MEC-BFU2005-06487 and JA-P06-TIC1426. Appendix A. EM-BFACTOR at work An example of a EM-BFACTOR command to sharpen a density map follows. This command allows automated B-factor determination, using the resolution range [8,12] Å. The input map is in Spider format. The input FSC curve is stored in a file called ‘‘myFSCcurve” which will be read and interpreted by the program after identification of its format. The pixel size in the map is 2.18 Å. The molecular mass of the specimen, 708 kDa, is specified for placing the map amplitudes on an absolute scale. As output, the program produces the sharpened map in MRC format, and the Guinier plot in text format is stored in the file ‘‘myguinier.plt” and also in Postscript format in the file ‘‘myguinier.plt.ps”. embfactor -molweight 708000 -FSC myFSCcurve -sampling 2.18 -resol 12,8 -guinier myguinier.plt inputmap.spi outputmap.mrc An example of the output report generated by the program follows (for brevity, only the most important lines are shown). This report shows the parameters for the absolute scaling, the format of the FSC curve, the parameters for the least squares fitting and the B-factor that was finally found out.


Reading FSC file: myFSCcurve FSC file: Spider Format Wilson Regime: 242.90 Zero Angle Scattering: 16520.00 Automatic determination of the B-factor affecting the data: Fitting y = a + b * x to Cref-weighted averaged structure factor curve: Resolution range for fitting: [12.00,8.00] Fitting parameters: a: 0.027726 b: 85.745102 B-factor found: 342.98 Restoring amplitudes by applying B-factor: 342.98

References Agirrezabala, X., Martin-Benito, J., Valle, M., Gonzalez, J.M., Valencia, A., Valpuesta, J.M., Carrascosa, J.L., 2005. Structure of the connector of bacteriophage T7 at 8 Å resolution: structural homologies of a basic component of a DNA translocating machinery. J. Mol. Biol. 347, 895–902. Baker, M.L., Ju, T., Chiu, W., 2007. Identification of secondary structure elements in intermediate-resolution density maps. Structure 15, 7–19. Bottcher, B., Wynne, S.A., Crowther, R.A., 1997. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386, 88– 91. Chiu, W., Baker, M.L., Jiang, W., Dougherty, M., Schmid, M.F., 2005. Electron cryomicroscopy of biological machines at subnanometer resolution. Structure 13, 363–372. Conway, J.F., Steven, A.C., 1999. Methods for reconstructing density maps of single particles from cryoelectron micrographs to subnanometer resolution. J. Struct. Biol. 128, 106–118. Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Navaza, J., Bressanelli, S., Delmas, B., Rey, F.A., 2005. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120, 761–772. Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573–579.

175

Frank, J., 2006. Three-dimensional electron microscopy of macromolecular assemblies. Visualization of Biological Molecules in Their Native State. Oxford University Press. Gabashvili, I.S., Agrawal, R.K., Spahn, C.M.T., Grassucci, R.A., Svergun, D.I., Frank, J., Penczek, P., 2000. Solution structure of the E. coli 70s ribosome at 11.5 Å resolution. Cell 100, 537–549. Glaeser, R.M., Downing, K.H., 1992. Assessment of resolution in biological electron crystallography. Ultramicroscopy 47, 256–265. Halic, M., Gartmann, M., Schlenker, O., Mielke, T., Pool, M.R., Sinning, I., Beckmann, R., 2006. Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312, 745–747. Henderson, R., 1992. Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice. Ultramicroscopy 46, 1–18. Henderson, R., 2004. Realising the potential of electron cryomicroscopy. Q. Rev. Biophys. 37, 3–13. Heymann, J.B., Belnap, D.M., 2007. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18. Jiang, W., Baker, M.L., Jakana, J., Weigele, P.R., King, J., Chiu, W., 2008. Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 451, 1130–1134. Ludtke, S.J., Baker, M.L., Chen, D., Song, J., Chuang, D.T., Chiu, W., 2008. De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure 16, 441–448. Luque, D., Saugar, I., Rodriguez, J.F., Verdaguer, N., Garriga, D., San Martin, C., Velazquez-Muriel, J.A., Trus, B.L., Carrascosa, J.L., Caston, J.R., 2007. Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch. J. Virol. 81, 6869–6878. Ranson, N.A., Clare, D.K., Farr, G.W., Houldershaw, D., Horwich, A.L., Saibil, H.R., 2006. Allosteric signalling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2, 147–152. Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745. Wade, R.H., 1992. A brief look at imaging and contrast transfer. Ultramicroscopy 46, 145–156. Wilson, A.J.C., 1942. Determination of absolute from relative X-ray intensity data. Nature 150, 151–152. Zhang, X., Settembre, E., Xu, C., Dormitzer, P.R., Bellamy, R., Harrison, S.C., Grigorieff, N., 2008. Near-atomic resolution using electron cryomicroscopy and singleparticle reconstruction. Proc. Natl. Acad. Sci. USA 105, 1867–1872. Zhou, Z.H., 2008. Towards atomic resolution structural determination by singleparticle cryo-electron microscopy. Curr. Opin. Struct. Biol. 18, 218–228.