Single-particle electron cryo-microscopy: towards atomic resolution

Abstract

1. Introduction 3082. Electron microscopy 3112.1 Specimen preparation 3112.2 The electron microscope 3112.3 Acceleration voltage, defocus, and the electron gun 3122.4 Magnification and data collection 3133. Digitisation and CTF correction 3173.1 The patchwork densitometer 3183.2 Particle selection 3203.3 Position dependent CTF correction 3213.4 Precision of CTF determination 3214. Single particles and angular reconstitution 3234.1 Preliminary filtering and centring of data 3234.2 Alignments using correlation functions 3244.3 Choice of first reference images 3244.4 Multi-reference alignment of data 3254.5 MSA eigenvector/eigenvalue data compression 3284.6 MSA classification 3304.7 Euler angle determination (‘angular reconstitution’) 3324.8 Sinograms and sinogram correlation functions 3324.9 Exploiting symmetry 3354.10 Three-dimensional reconstruction 3374.11 Euler angles using anchor sets 3394.12 Iterative refinements 3395. Computational hardware/software aspects 3415.1 The (IMAGIC) image processing workstation 3425.2 Operating systems and GUIs 3425.3 Computational logistics 3445.4 Shared memory machines 3445.5 Farming on loosely coupled computers 3465.6 Implementation using MPI protocol 3475.7 Software is what it's all about 3476. Interpretation of results 3486.1 Assessing resolution: the Fourier Shell Correlation 3486.2 Influence of filtering 3516.3 Rendering 3516.4 Searching for known sub-structures 3526.5 Interpretation 3537. Examples 3537.1 Icosahedral symmetry: TBSV at 5·9 Å resolution 3547.2 The D6 symmetrical worm hemoglobin at 13 Å resolution 3567.3 Functional states of the 70S E. coli ribosome 3577.4 The 50S E. coli ribosomal subunit at 7·5 Å resolution 3598. Perspectives 3619. Acknowledgements 36410. References 364In the past few years, electron microscopy (EM) has established itself as an important – still upcoming – technique for studying the structures of large biological macromolecules. EM is a very direct method of structure determination that complements the well-established techniques of X-ray crystallography and NMR spectroscopy. Electron micrographs record images of the object and not just their diffraction patterns and thus the classical ‘phase’ problem of X-ray crystallography does not exist in EM. Modern microscopes may reach resolution levels better than ∼ 1·5 Å, which is more than sufficient to elucidate the polypeptide backbone in proteins directly. X-ray structures at such resolution levels are considered ‘excellent’. The fundamental problem in biological EM is not so much the instrumental resolution of the microscopes, but rather the radiation sensitivity of the biological material one wants to investigate. Information about the specimen is collected in the photographic emulsion with the arrival of individual electrons that have (elastically) interacted with the specimen. However, many electrons will damage the specimen by non-elastic interactions. By the time enough electrons have passed through the object to produce a single good signal-to-noise (SNR) image, the biological sample will have been reduced to ashes. In contrast, stable inorganic specimens in material science often show interpretable details down to the highest possible instrumental resolution.

Hierarchically Ordered Self‐Assembly of Amphiphilic Bifullerenes

“…They point to a preferential ultrastructural organisation of the aggregates and initiated a further treatment by a statistical method (multivariate statistical analysis, MSA),11, 12 which determines the structural differences of an image data set (considering several thousand individual particles) in terms of size, shape or inherent symmetry elements (details are given in the Supporting Information). The analysis allows for the identification (classification) of structurally identical particles, which are eventually aligned and summed up (Figure 3).…”

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

“…Images were normalized, band‐pass filtered and subjected to multireference alignment (MRA) using re‐projections (side views only) of the 3D reconstruction EMD‐2567 of the T4SS 3‐10 complex, followed by multivariate statistical analysis (MSA) (van Heel et al , 2000). The best classes representing characteristic views were used as new references for the next iteration of the alignment procedure.…”

GraFix: sample preparation for single-particle electron cryomicroscopy

“…Most molecular machines are dynamic, fragile multiprotein complexes 1 and are present at low copy number in the cell. Single-particle cryo-EM has been demonstrated to be a very powerful method for threedimensional structure analysis of large macromolecular complexes at increasing resolution 2 . For macromolecules with a high level of internal symmetry, structure determination by cryo-EM has become routine at a resolution of r10 Å , and there are examples where resolution better than 6 Å has been obtained 3 .…”