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.

Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

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The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA–protein interactions and dissection of virus assembly on the basis of RNA nucleation.

Summarygp130 is a shared signal-transducing membrane-associated receptor for several hematopoietic cytokines. The 30 Å resolution cryo-electron microscopy (cryo-EM) structure of the Interleukin 11(IL-11)-IL-11 Receptor-gp130 extracellular complex reveals the architecture and dynamics of this gp130-containing signaling complex. Normal-mode analysis reveals a repertoire of conformational changes that could function in signal triggering. This suggests a concerted mechanism of signaling involving all the components of the complex. This could provide a general mechanism of signal transfer for cytokines utilizing the JAK-STAT signaling cascade.

The restriction factor Fv1 confers resistance to murine leukemia virus (MLV), blocking progression of the viral life cycle after reverse transcription, but before integration into the host chromosome. It is known that the specificity of restriction is determined by both the restriction factor and the viral capsid (CA), but a direct interaction between Fv1 and MLV CA has not yet been demonstrated. With the development of a previously unexplored method for in vitro polymerization of MLV CA, it has now been possible to display a binding interaction between Fv1 and MLV CA. C-terminally His-tagged CA molecules were assembled on Ni-chelating lipid nanotubes, and analysis by electron microscopy revealed the formation of a regular lattice. Comparison of binding data with existing restriction data confirmed the specificity of the binding interaction, with multiple positions of both Fv1 and CA shown to influence binding specificity.

Single-particle cryogenic electron microscopy (cryo-EM) can now yield nearatomic resolution structures of biological complexes. However, the referencebased alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional referencefree 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of $3.8 Å is presented as an example of the use of the ABC-4D procedure.