Using Scipion for stream image processing at Cryo-EM facilities.

Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

Structural Study of Heterogeneous Biological Samples by Cryoelectron Microscopy and Image Processing.

In living organisms, biological macromolecules are intrinsically flexible and naturally exist in multiple conformations. Modern electron microscopy, especially at liquid nitrogen temperatures (cryo-EM), is able to visualise biocomplexes in nearly native conditions and in multiple conformational states. The advances made during the last decade in electronic technology and software development have led to the revelation of structural variations in complexes and also improved the resolution of EM structures. Nowadays, structural studies based on single particle analysis (SPA) suggests several approaches for the separation of different conformational states and therefore disclosure of the mechanisms for functioning of complexes. The task of resolving different states requires the examination of large datasets, sophisticated programs, and significant computing power. Some methods are based on analysis of two-dimensional images, while others are based on three-dimensional studies. In this review, we describe the basic principles implemented in the various techniques that are currently used in the analysis of structural conformations and provide some examples of successful applications of these methods in structural studies of biologically significant complexes.

Chaperonin complex with a newly folded protein encapsulated in the folding chamber.

A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.