Low-dose phase retrieval of biological specimens using cryo-electron ptychography.

Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å2) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 µm), thus making it suitable for studies of many biologically important structures.

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution.

BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein. RESULTS: In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer. CONCLUSION: The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.

UNLABELLED: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE: In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.