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Advances in cryo-electron microscopy (cryo-EM) have made it possible to obtain structures of large biological macromolecules at near-atomic resolution. This "resolution revolution" has encouraged the use and development of modeling tools able to produce high-quality atomic models from cryo-EM density maps. Unfortunately, many practical problems appear when combining different packages in the same processing workflow, which make difficult the use of these tools by non-experts and, therefore, reduce their utility. We present here a major extension of the image processing framework Scipion that provides inter-package integration in the model building area and full tracking of the complete workflow, from image processing to structure validation.
The impact of structural biology on drug discovery is well documented, and the workhorse technique for the past 30 years or so has been X-ray crystallography. With the advent of several technological improvements, including direct electron detectors, automation, better microscope vacuums and lenses, phase plates and improvements in computing power enabled by GPUs, it is now possible to record and analyse images of protein structures containing high-resolution information. This review, from a pharmaceutical perspective, highlights some of the most relevant and interesting protein structures for the pharmaceutical industry and shows examples of how ligand-binding sites, membrane proteins, both big and small, pseudo symmetry and complexes are being addressed by this technique.
During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.
- MeSH
- aminoacyl-tRNA metabolismus MeSH
- elektronová kryomikroskopie * MeSH
- elongační faktor G chemie metabolismus MeSH
- Escherichia coli chemie metabolismus MeSH
- fosfáty metabolismus MeSH
- guanosintrifosfát chemie metabolismus MeSH
- malé podjednotky ribozomu bakteriální chemie metabolismus MeSH
- messenger RNA metabolismus MeSH
- proteosyntéza MeSH
- ribozomy chemie metabolismus MeSH
- RNA transferová metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Helix 38 (H38) of the large ribosomal subunit, with a length of 110 A, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 A from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.
- MeSH
- chlorid draselný chemie MeSH
- Deinococcus genetika MeSH
- elektronová kryomikroskopie MeSH
- Escherichia coli genetika MeSH
- Haloarcula marismortui genetika MeSH
- konformace nukleové kyseliny MeSH
- RNA ribozomální 23S chemie MeSH
- simulace molekulární dynamiky MeSH
- sodík chemie MeSH
- Thermus thermophilus genetika MeSH
- velké podjednotky ribozomu archebakteriální chemie MeSH
- velké podjednotky ribozomu bakteriální chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.
- MeSH
- chloroplasty chemie MeSH
- elektronová kryomikroskopie MeSH
- malé podjednotky ribozomu eukaryotické chemie MeSH
- molekulární modely MeSH
- proteiny chloroplastové chemie metabolismus MeSH
- ribozomální proteiny chemie metabolismus MeSH
- RNA ribozomální chemie MeSH
- Spinacia oleracea chemie MeSH
- stabilita RNA MeSH
- vazebná místa MeSH
- velké podjednotky ribozomu eukaryotické chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.
- MeSH
- amyotrofická laterální skleróza * genetika metabolismus MeSH
- dipeptidy metabolismus MeSH
- elektronová kryomikroskopie MeSH
- frontotemporální demence * genetika metabolismus MeSH
- lidé MeSH
- protein C9orf72 genetika metabolismus MeSH
- proteiny genetika metabolismus MeSH
- ribozomy metabolismus MeSH
- transferasy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Viruses from the family Iflaviridae are insect pathogens. Many of them, including slow bee paralysis virus (SBPV), cause lethal diseases in honeybees and bumblebees, resulting in agricultural losses. Iflaviruses have nonenveloped icosahedral virions containing single-stranded RNA genomes. However, their genome release mechanism is unknown. Here, we show that low pH promotes SBPV genome release, indicating that the virus may use endosomes to enter host cells. We used cryo-EM to study a heterogeneous population of SBPV virions at pH 5.5. We determined the structures of SBPV particles before and after genome release to resolutions of 3.3 and 3.4 Å, respectively. The capsids of SBPV virions in low pH are not expanded. Thus, SBPV does not appear to form "altered" particles with pores in their capsids before genome release, as is the case in many related picornaviruses. The egress of the genome from SBPV virions is associated with a loss of interpentamer contacts mediated by N-terminal arms of VP2 capsid proteins, which result in the expansion of the capsid. Pores that are 7 Å in diameter form around icosahedral threefold symmetry axes. We speculate that they serve as channels for the genome release. Our findings provide an atomic-level characterization of the genome release mechanism of iflaviruses.
- MeSH
- Dicistroviridae genetika fyziologie ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- genom virový MeSH
- kapsida ultrastruktura MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- Picornaviridae genetika fyziologie ultrastruktura MeSH
- statická elektřina MeSH
- svlékání virového obalu fyziologie MeSH
- včely virologie MeSH
- virové plášťové proteiny chemie ultrastruktura MeSH
- viry hmyzu genetika fyziologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.
- MeSH
- DNA viry genetika fyziologie ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- genom virový MeSH
- kapsida metabolismus ultrastruktura MeSH
- sestavení viru MeSH
- virion genetika fyziologie ultrastruktura MeSH
- virové proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy metabolismus MeSH
- ATPázy spojené s různými buněčnými aktivitami metabolismus MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- elektronová kryomikroskopie MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- replikace DNA * MeSH
- replikační protein C chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
