Small, solubilized platinum nanocrystals consist of an ordered core surrounded by mobile surface atoms.

In situ structures of Platinum (Pt) nanoparticles (NPs) can be determined with graphene liquid cell transmission electron microscopy. Atomic-scale three-dimensional structural information about their physiochemical properties in solution is critical for understanding their chemical function. We here analyze eight atomic-resolution maps of small (<3 nm) colloidal Pt NPs. Their structures are composed of an ordered crystalline core surrounded by surface atoms with comparatively high mobility. 3D reconstructions calculated from cumulative doses of 8500 and 17,000 electrons/pixel, respectively, are characterized in terms of loss of atomic densities and atomic displacements. Less than 5% of the total number of atoms are lost due to dissolution or knock-on damage in five of the structures analyzed, whereas 10-16% are lost in the remaining three. Less than 5% of the atomic positions are displaced due to the increased electron irradiation in all structures. The surface dynamics will play a critical role in the diverse catalytic function of Pt NPs and must be considered in efforts to model Pt NP function computationally.

Structure and assembly of the NOT10:11 module of the CCR4-NOT complex.

NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.

Method for 3D atomic structure determination of multi-element nanoparticles with graphene liquid-cell TEM.

Determining the 3D atomic structures of multi-element nanoparticles in their native liquid environment is crucial to understanding their physicochemical properties. Graphene liquid cell (GLC) TEM offers a platform to directly investigate nanoparticles in their solution phase. Moreover, exploiting high-resolution TEM images of single rotating nanoparticles in GLCs, 3D atomic structures of nanoparticles are reconstructed by a method called "Brownian one-particle reconstruction". We here introduce a 3D atomic structure determination method for multi-element nanoparticle systems. The method, which is based on low-pass filtration and initial 3D model generation customized for different types of multi-element systems, enables reconstruction of high-resolution 3D Coulomb density maps for ordered and disordered multi-element systems and classification of the heteroatom type. Using high-resolution image datasets obtained from TEM simulations of PbSe, CdSe, and FePt nanoparticles that are structurally relaxed with first-principles calculations in the graphene liquid cell, we show that the types and positions of the constituent atoms are precisely determined with root mean square displacement values less than 24 pm. Our study suggests that it is possible to investigate the 3D atomic structures of synthesized multi-element nanoparticles in liquid phase.

Structural analysis of the PTEN:P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1.

The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gßγ subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gßγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.

SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent.

The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear. To investigate, we assayed the chromatin modifying functions of SAGA and SLIK, revealing identical kinetics on minimal substrates in vitro. We also examined the binding of SAGA and SLIK to TBP and concluded that interestingly, both protein complexes have similar affinity for TBP. Additionally, despite the loss of Spt8 and C-terminus of Spt7 in SLIK, TBP prebound to SLIK is not released in the presence of TATA-box DNA, just like TBP prebound to SAGA. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (KD = 10-17 nM), and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of the Spt8 subunit in SLIK neither drive any major conformational differences in its structure compared with SAGA, nor significantly affect HAT, DUB, or DNA-binding activities in vitro.

Correlating 3D Surface Atomic Structure and Catalytic Activities of Pt Nanocrystals.

Active sites and catalytic activity of heterogeneous catalysts is determined by their surface atomic structures. However, probing the surface structure at an atomic resolution is difficult, especially for solution ensembles of catalytic nanocrystals, which consist of heterogeneous particles with irregular shapes and surfaces. Here, we constructed 3D maps of the coordination number (CN) and generalized CN (CN_) for individual surface atoms of sub-3 nm Pt nanocrystals. Our results reveal that the synthesized Pt nanocrystals are enclosed by islands of atoms with nonuniform shapes that lead to complex surface structures, including a high ratio of low-coordination surface atoms, reduced domain size of low-index facets, and various types of exposed high-index facets. 3D maps of CN_ are directly correlated to catalytic activities assigned to individual surface atoms with distinct local coordination structures, which explains the origin of high catalytic performance of small Pt nanocrystals in important reactions such as oxygen reduction reactions and CO electro-oxidation.

SINGLE: Atomic-resolution structure identification of nanocrystals by graphene liquid cell EM.

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a "one-particle Brownian 3D reconstruction method" based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo-electron microscopy. Our developments are made available through the open-source software package SINGLE.

SIMPLE 3.0. Stream single-particle cryo-EM analysis in real time.

We here introduce the third major release of the SIMPLE (Single-particle IMage Processing Linux Engine) open-source software package for analysis of cryogenic transmission electron microscopy (cryo-EM) movies of single-particles (Single-Particle Analysis, SPA). Development of SIMPLE 3.0 has been focused on real-time data processing using minimal CPU computing resources to allow easy and cost-efficient scaling of processing as data rates escalate. Our stream SPA tool implements the steps of anisotropic motion correction and CTF estimation, rapid template-based particle identification and 2D clustering with automatic class rejection. SIMPLE 3.0 additionally features an easy-to-use web-based graphical user interface (GUI) that can be run on any device (workstation, laptop, tablet or phone) and supports a remote multi-user environment over the network. The new project-based execution model automatically records the executed workflow and represents it as a flow diagram in the GUI. This facilitates meta-data handling and greatly simplifies usage. Using SIMPLE 3.0, it is possible to automatically obtain a clean SP data set amenable to high-resolution 3D reconstruction directly upon completion of the data acquisition, without the need for extensive image processing post collection. Only minimal standard CPU computing resources are required to keep up with a rate of ∼300 Gatan K3 direct electron detector movies per hour. SIMPLE 3.0 is available for download from simplecryoem.com.

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.

Critical differences in 3D atomic structure of individual ligand-protected nanocrystals in solution.

Precise three-dimensional (3D) atomic structure determination of individual nanocrystals is a prerequisite for understanding and predicting their physical properties. Nanocrystals from the same synthesis batch display what are often presumed to be small but possibly important differences in size, lattice distortions, and defects, which can only be understood by structural characterization with high spatial 3D resolution. We solved the structures of individual colloidal platinum nanocrystals by developing atomic-resolution 3D liquid-cell electron microscopy to reveal critical intrinsic heterogeneity of ligand-protected platinum nanocrystals in solution, including structural degeneracies, lattice parameter deviations, internal defects, and strain. These differences in structure lead to substantial contributions to free energies, consequential enough that they must be considered in any discussion of fundamental nanocrystal properties or applications.

Point-group symmetry detection in three-dimensional charge density of biomolecules.

MOTIVATION: No rigorous statistical tests for detecting point-group symmetry in three-dimensional (3D) charge density maps obtained by electron microscopy (EM) and related techniques have been developed. RESULTS: We propose a method for determining the point-group symmetry of 3D charge density maps obtained by EM and related techniques. Our ab initio algorithm does not depend on atomic coordinates but utilizes the density map directly. We validate the approach for a range of publicly available single-particle cryo-EM datasets. In straightforward cases, our method enables fully automated single-particle 3D reconstruction without having to input an arbitrarily selected point-group symmetry. When pseudo-symmetry is present, our method provides statistics quantifying the degree to which the 3D density agrees with the different point-groups tested. AVAILABILITY AND IMPLEMENTATION: The software is freely available at https://github.com/hael/SIMPLE3.0.

AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar.

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.

The TAFs of TFIID Bind and Rearrange the Topology of the TATA-Less RPS5 Promoter.

The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS).

Rapid near-atomic resolution single-particle 3D reconstruction with SIMPLE.

Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.

Single-particle cryo-EM-Improved ab initio 3D reconstruction with SIMPLE/PRIME.

Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here, we extend our previous work on PRIME and introduce new stochastic optimization algorithms that improve the robustness of the approach. Our refined method for identification of homogeneous subsets of images in accurate register substantially improves the resolution of the cluster centers and of the ab initio 3D reconstructions derived from them. We now obtain maps with a resolution better than 10 Å by exclusively processing cluster centers. Excellent parallel code performance on over-the-counter laptops and CPU workstations is demonstrated.

Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome.

An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site.IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821-832, 2015, https://doi.org/10.1038/nrd4675). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

High-resolution cryo-EM: the nuts and bolts.

Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. Successful high-resolution structure determination by cryo-EM always depends on the quality of the protein sample. While structural heterogeneity remains a key challenge for cryo-EM, it also represents a rare opportunity to study the intrinsic conformational flexibility of macromolecular assemblies. Here, we review the key technological advancements that have made this 'resolution revolution' possible and give a concise overview of the technical challenges that needed to be overcome to allow high-resolution structure determination.

A Stochastic Hill Climbing Approach for Simultaneous 2D Alignment and Clustering of Cryogenic Electron Microscopy Images.

A critical step in the analysis of novel cryogenic electron microscopy (cryo-EM) single-particle datasets is the identification of homogeneous subsets of images. Methods for solving this problem are important for data quality assessment, ab initio 3D reconstruction, and analysis of population diversity due to the heterogeneous nature of macromolecules. Here we formulate a stochastic algorithm for identification of homogeneous subsets of images. The purpose of the method is to generate improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion. We show that our method overcomes inherent limitations of widely used clustering approaches and proceed to test the approach on six publicly available experimental cryo-EM datasets. We conclude that, in each instance, ab initio 3D reconstructions of quality suitable for initialization of high-resolution refinement are produced from the cluster centers.