The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA.

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn2+, K+ and Mg2+, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.

Focused classifications and refinements in high-resolution single particle cryo-EM analysis.

Recent advances in cryo electron microscopy (cryo-EM) and image processing provide new opportunities to analyse drug targets at high resolution. However, structural heterogeneity limits resolution in many practical cases, hence restricting the level at which structural details can be analysed and drug design be performed. As structural disorder is not spread throughout the entire structure of a given macromolecular complex but instead is found in certain regions that move with respect to others and covering molecular scales from domain conformational changes up to the level of side chain conformations in ligand binding pockets, it is possible to focus the attention on those regions and the associated relative movements. Here we show how the usage of focused classifications and refinements provide insights into global conformational arrangements, exemplified on the human ribosome and on the cannabinoid G protein coupled receptor (GPCR), and how they can improve the local map resolution from an essentially disordered region to the 3-4 Å and finally to the 2 Å resolution range. A systematic analysis with variable spherical masks during focused refinement is presented showing that the choice of an optimal mask size helps refining to high resolution. This study covers several practical approaches on 4 examples illustrating how important mask size & shape and including neighbouring structural elements are for a focused analysis of a macromolecular complex. Such methods will be crucial for cryo-EM structure-based drug design of various medical targets and are applicable to single particle cryo-EM and electron tomography data.

High-resolution cryo-EM performance comparison of two latest-generation cryo electron microscopes on the human ribosome.

Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target. Astigmatism correction, CTF refinement and correction of higher order aberrations through refinement in separate optics groups helped to account for astigmatism/coma caused by beam tilting during multi-spot and multi-hole acquisition in neighbouring holes without stage movement. The obtained maps resolve Mg2+ ions, water molecules, inhibitors and side-chains including chemical modifications. The fact that both instruments can resolve such detailed features will greatly facilitate understanding molecular mechanisms of various targets and helps in cryo-EM structure based drug design. The methods and analysis tools used here will be useful also to characterize existing instruments and optimize data acquisition settings and are applicable broadly to other drug targets in structural biology.

Structural basis for safe and efficient energy conversion in a respiratory supercomplex.

Proton-translocating respiratory complexes assemble into supercomplexes that are proposed to increase the efficiency of energy conversion and limit the production of harmful reactive oxygen species during aerobic cellular respiration. Cytochrome bc complexes and cytochrome aa3 oxidases are major drivers of the proton motive force that fuels ATP generation via respiration, but how wasteful electron- and proton transfer is controlled to enhance safety and efficiency in the context of supercomplexes is not known. Here, we address this question with the 2.8 Å resolution cryo-EM structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from the actinobacterium Corynebacterium glutamicum. Menaquinone, substrate mimics, lycopene, an unexpected Qc site, dioxygen, proton transfer routes, and conformational states of key protonable residues are resolved. Our results show how safe and efficient energy conversion is achieved in a respiratory supercomplex through controlled electron and proton transfer. The structure may guide the rational design of drugs against actinobacteria that cause diphtheria and tuberculosis.

Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection.

Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.

Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms.

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Šresolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

Cryo-EM structure of the Ustilago maydis kinesin-5 motor domain bound to microtubules.

In many eukaryotes, kinesin-5 motors are essential for mitosis, and small molecules that inhibit human kinesin-5 disrupt cell division. To investigate whether fungal kinesin-5s could be targets for novel fungicides, we studied kinesin-5 from the pathogenic fungus Ustilago maydis. We used cryo-electron microscopy to determine the microtubule-bound structure of its motor domain with and without the N-terminal extension. The ATP-like conformations of the motor in the presence or absence of this N-terminus are very similar, suggesting this region is structurally disordered and does not directly influence the motor ATPase. The Ustilago maydis kinesin-5 motor domain adopts a canonical ATP-like conformation, thereby allowing the neck linker to bind along the motor domain towards the microtubule plus end. However, several insertions within this motor domain are structurally distinct. Loop2 forms a non-canonical interaction with α-tubulin, while loop8 may bridge between two adjacent protofilaments. Furthermore, loop5 - which in human kinesin-5 is involved in binding allosteric inhibitors - protrudes above the nucleotide binding site, revealing a distinct binding pocket for potential inhibitors. This work highlights fungal-specific elaborations of the kinesin-5 motor domain and provides the structural basis for future investigations of kinesins as targets for novel fungicides.

Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex.

Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5-Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance.

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor-microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor-microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

Volta phase plate data collection facilitates image processing and cryo-EM structure determination.

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.

Nucleotide- and Mal3-dependent changes in fission yeast microtubules suggest a structural plasticity view of dynamics.

Using cryo-electron microscopy, we characterize the architecture of microtubules assembled from Schizosaccharomyces pombe tubulin, in the presence and absence of their regulatory partner Mal3. Cryo-electron tomography reveals that microtubules assembled from S. pombe tubulin have predominantly B-lattice interprotofilament contacts, with protofilaments skewed around the microtubule axis. Copolymerization with Mal3 favors 13 protofilament microtubules with reduced protofilament skew, indicating that Mal3 adjusts interprotofilament interfaces. A 4.6-Å resolution structure of microtubule-bound Mal3 shows that Mal3 makes a distinctive footprint on the S. pombe microtubule lattice and that unlike mammalian microtubules, S. pombe microtubules do not show the longitudinal lattice compaction associated with EB protein binding and GTP hydrolysis. Our results firmly support a structural plasticity view of microtubule dynamics in which microtubule lattice conformation is sensitive to a variety of effectors and differently so for different tubulins.

Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes.

Cryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3Å. Such high-resolution features now enable the analysis of drug interactions with RNA and protein side-chains including even the visualization of chemical modifications of the ribosomal RNA. These advances represent a major breakthrough in structural biology and show the strong potential of cryo-EM beyond the ribosome field including for structure-based drug design.

Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.

Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.

Ribosome-SRP-FtsY cotranslational targeting complex in the closed state.

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP-RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

Structural basis of signal sequence surveillance and selection by the SRP-FtsY complex.

Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor.

OEP61 is a chaperone receptor at the plastid outer envelope.

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.