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Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

Macromolecules are involved in myriads of interactions that regulate their cellular function. While years of structural biology progress was built by reducing this complexity, a molecular understanding of biological processes requires the characterization of ever larger and more dynamic molecular assemblies. Cryo-electron microscopy is rising to this challenge.

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3–6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystissp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP–PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

CRISPR–CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.

The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A number of CDK7 inhibitors have entered clinical trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have determined the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resolution using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the critical ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF–FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF–FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular β-sheet around a highly divergent β-strand of the BTB domain. Complex dissociation allows SCF–FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF–FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophilagenome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the DrosophilaP element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR–CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coliand human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Polycomb group proteins are transcriptional repressors controlling gene expression patterns and maintaining cell type identity. The chemical modifications of histones and DNA caused by the regulated activity of chromatin-modifying enzymes such as Polycomb help establish and maintain such expression patterns. Polycomb repressive complex 2 (PRC2) is the only known methyltransferase specific for histone H3 lysine 27 (H3K27) and catalyzes its trimethylation leading to the repressive H3K27me3 mark. Structural biology has made important contributions to our understanding of the molecular mechanisms that ensure the spatiotemporal regulation of PRC2 activity and the establishment of inactive chromatin domains marked by H3K27me3. In this review, we discuss the recent structural studies that have advanced our understanding of PRC2 function, in particular the roles of intersubunit interactions in complex assembly and the regulation of methyltransferase activity, as well as the mechanism of local H3K27me3 spreading leading to repressive domains.

The photosynthesis machinery in chloroplast thylakoid membranes is comprised of multiple protein complexes and supercomplexes1,2. Here, we show a novel supramolecular organization of photosystem I (PSI) in the moss Physcomitrella patensby single-particle cryo-electron microscopy. The moss-specific light-harvesting complex (LHC) protein Lhcb9 is involved in this PSI supercomplex, which has been shown to have a molecular density similar to that of the green alga Chlamydomonas reinhardtii3. Our results show that the structural organization is unexpectedly different—two rows of the LHCI belt exist as in C. reinhardtii4, but the outer one is shifted toward the PsaK side. Furthermore, one trimeric LHC protein and one monomeric LHC protein position alongside PsaL/K, filling the gap between these subunits and the outer LHCI belt. We provide evidence showing that Lhcb9 is a key factor, acting as a linkage between the PSI core and the outer LHCI belt to form the unique supramolecular organization of the PSI supercomplex in P. patens.

The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein–RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics. A cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme, which lengthens the protective caps on chromosomes.

Epigenetic regulation is mediated by protein complexes that couple recognition of chromatin marks to activity or recruitment of chromatin-modifying enzymes. Polycomb repressive complex 2 (PRC2), a gene silencer that methylates lysine 27 of histone H3, is stimulated upon recognition of its own catalytic product and has been shown to be more active on dinucleosomes than H3 tails or single nucleosomes. These properties probably facilitate local H3K27me2/3 spreading, causing heterochromatin formation and gene repression. Here, cryo-EM reconstructions of human PRC2 bound to bifunctional dinucleosomes show how a single PRC2, via interactions with nucleosomal DNA, positions the H3 tails of the activating and substrate nucleosome to interact with the EED subunit and the SET domain of EZH2, respectively. We show how the geometry of the PRC2–DNA interactions allows PRC2 to accommodate varying lengths of the linker DNA between nucleosomes. Our structures illustrate how an epigenetic regulator engages with a complex chromatin substrate. Cryo-EM analyses of human PRC2 bound to dinucleosomes with one unmodified (substrate) and one H3K27me3-containing (activating) nucleosome support a model for H3K27me3-based PRC2 activation and spreading.

ABSTRACTThe cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitrofrom these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.

Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIH subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Additionally, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.

ABSTRACTTFIID is a large protein complex required for the recognition and binding of eukaryotic gene core promoter sequences and for the recruitment of the rest of the general transcription factors involved in initiation of eukaryotic protein gene transcription. Cryo-electron microscopy studies have demonstrated the conformational complexity of human TFIID, where one-third of the mass of the complex can shift its position by well over 100 Å. This conformational plasticity appears to be linked to the capacity of TFIID to bind DNA, and suggests how it would allow both the recognition of different core promoter elements and the tuning of its binding affinity by regulatory factors.

This retrospective on the 25th anniversary of the publication of the first structure of tubulin is shaped by my own personal experiences rather than being a strict and complete historical account of the event. It is a reflection on how working in science felt many years ago, on the struggles and the joys of reaching for the high-hanging fruit, and, ultimately, on how relevant or not our personal scientific contributions are to the broader scientific community. Writing it brought back memories of my unique and sadly lost postdoctoral advisor Ken Downing, who dreamt of this structure and brought it to fruition against all odds.

In this Article, owing to issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections have been made to the Protein Data Bank (PDB) accessions in the ‘Data availability’ section, and this also affects Figs. 3, 4, Extended Data Fig. 6, Supplementary Table 1 and Supplementary Video 1. The original Article has been corrected online. See the accompanying Amendment for further details.

A long-standing conundrum is resolved about the underlying sequence determinants and molecular mechanism responsible for the recruitment of the protein kinase Hsl1 (an indispensable component of the so-called “morphogenesis checkpoint”) exclusively to the septin collar at the bud neck.

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

Next year will be the 50th anniversary of the discovery of tubulin. To celebrate this discovery, six leaders in the field of microtubule research reflect on key findings and technological breakthroughs over the past five decades, discuss implications for therapeutic applications and provide their thoughts on what questions need to be addressed in the near future.

The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1–13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP–TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

Single-particle cryo-electron microscopy (cryo-EM) has emerged over the last two decades as a technique capable of studying challenging systems that otherwise defy structural characterization. Recent technical advances have resulted in a 'quantum leap' in applicability, throughput and achievable resolution that has gained this technique worldwide attention. Here I discuss some of the major historical landmarks in the development of the cryo-EM field, ultimately leading to its present success.

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thalianausing atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.

Acetylation of α-tubulin is uniquely located on the microtubule lumen and away from most MAPs. It does not affect microtubule structure and must affect only proteins that bind directly to the lumen. αTAT1 can interact with the tubulin C-termini, which could facilitate access to its luminal site and make it responsive to other modifications.

Recent structural and biochemical studies of human TFIID have significantly increased our understanding of the mechanisms underlying the recruitment of TFIID to promoter DNA and its role in transcription initiation. Structural studies using cryo-EM revealed that modular interactions underlie TFIID’s ability to bind simultaneously multiple promoter motifs and to define a DNA state that will facilitate transcription initiation. Here we propose a general model of promoter binding by TFIID, where co-activators, activators, and histone modifications promote and/or stabilize a conformational state of TFIID that results in core promoter engagement. Within this high affinity conformation, we propose that TFIID’s extensive interaction with promoter DNA leads to topological changes in the DNA that facilitate the eventual loading of RNAP II. While more work is required to dissect the individual contributions of activators and repressors to TFIID’s DNA binding, the recent cryo-EM studies provide a physical framework to guide future structural, biophysical, and biochemical experiments.

The dynamic assembly and disassembly of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. Mathematical and computational modeling, especially coupled mechanochemical modeling, has contributed significantly to our understanding of microtubule dynamics. However, critical discrepancies exist between experimental observations and modeling results that need to be resolved before further progress toward a complete model can be made. Open sheet structures ranging in length from several hundred nanometers to one micron have often been observed at the growing ends of microtubules in in vitro studies. Existing modeling studies predict these sheet structures to be short and rare intermediates of microtubule disassembly rather than important components of the assembly process. Atomic force microscopy (AFM) studies also reveal interesting step-like gaps of the force-indentation curve that cannot yet be explained by existing theoretical models. We have carried out computational studies to compare the mechanical properties of two alternative models: a more conventional model where tubulin dimers are added directly into a microtubule lattice, and one that considers an additional type of tubulin lateral interaction proposed to exist in intermediate sheet structures during the microtubule assembly process. The first model involves a single type of lateral interactions between tubulin subunits, whereas the latter considers a second type that can convert to the canonical lateral contact during microtubule closure into a cylinder. Our analysis shows that only the second model can reproduce the AFM results over a broad parameter range. We propose additional studies using different sizes of AFM tips that would allow to unambiguously distinguish the relative validity of the two models.

Auf einen Klick:Kupferfreie Klick‐Chemie und eine Aldehydmarkierungsstrategie werden kombiniert, um heterodifunktionelle Proteinfusionen mit den orthogonalen Triazol‐ und Oxim‐Verknüpfungen herbeizuführen (siehe Schema). Durch die Einführung eines Formylglycins ermöglicht die Methode eine ortsspezifische Proteinkonjugation an humane Antikörper.

Clicking into place: Cu‐free click chemistry is combined with the aldehyde tag protein modification strategy to produce heterobifunctional protein fusions. This method relies on linkers that utilize orthogonal triazole and oxime chemistries (see scheme) to expand on conjugation with the fGly residue and enables site‐specific protein conjugation to full‐length human antibodies.

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P2-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo–electron tomography. We found networks of filaments both perpendicular and parallel to the mother–bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

Septins are essential for membrane compartmentalization and remodeling. Electron tomography of yeast bud necks shows filaments perpendicular and parallel to the mother-bud axis that resemble in vitro septin arrays. Filaments are still present, although disordered, in mutants lacking a single septin, underscoring the importance of septin assembly.

Chromosomes segregate by interaction of spindle microtubules with kinetochores. In budding yeast the Dam1 complex forms rings around microtubules and recapitulates the functionality of a kinetochore–microtubule attachment. Mapping of subunits within the complex and their organization within the ring has made it possible to generate a model of Dam1 ring assembly.

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore–microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.

Several models of Dam1 complex function at the kinetochore have been proposed. Here we show that removal of the E-hooks of tubulin reduces Dam1 binding 40-fold. We also report the first structure of the Dam1 ring around microtubules and evidence that this ring freely diffuses on microtubules based on imaging individual rings.

This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy.

Microtubule assembly and disassembly is a complex structural process that doesnot proceed by simple addition and subtraction of individual subunits to and from ahelical polymer, as would be the case for actin and other helical assemblies. Thedynamic process of microtubule growth and shrinking involves short-lasting polymerforms that differ substantially from the microtubule itself and constitute crucial assemblyand disassembly intermediates. Structural characterization thus depends on thestabilization of these brief intermediates and their preservation as polymericassemblies. This paper gives experimental details on the polymerization of GMPCPPtubulininto low-temperature stable polymers that we propose to correspond to the earlystages in microtubule assembly and includes new data on the effect of colchicine onGMPCPP-tubulin polymerization. Finally, we include our thoughts on the possiblebiological meaning of tubulin polymerization versatility.

Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

The multidrug efflux complex AcrAB–TolC confers intrinsic drug resistance in Escherichia coliby pumping antibiotics out of the cell. We determined a low-resolution (20 Å) structure of AcrA, the periplasmic component, by electron crystallography. Expressed with a His-tag at its carboxyl-terminus, the protein bound to lipid layers containing the nickel-chelating phospholipid DOGS-NTA. Under the lipid layers, AcrA crystallized in layer group P2122,with a unit cell size of 157 by 95 Å and a thickness of about 100 Å. The four asymmetric units in the unit cell are organized into what appears to be two rings, each with a central opening of 30 Å in diameter. Within each ring, the density can be interpreted as following a pseudo-helical path, approximately 210 Å long. This length matches that of monomeric AcrA in solution, previously estimated by light scattering and hydrodynamic measurements. On one side the density has a tubular shape, with a thickness of about 25 Å, while on the other side the densities of the upper and lower parts of the pseudo-helical path are fused into a shield.

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, γ-tubulin, gene of Aspergillus nidulansand have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of γ-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that γ-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of γ-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

αβ-tubulin heterodimers self-assemble to form microtubules nucleated by γ-tubulin in the cell. γ-tubulin is believed to recruit the αβ-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of δ-tubulin and ε-tubulin. δ-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and ε-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of αβ tubulin as a model, we have analyzed the sequences of γ-, δ- and ε-tubulin in regions corresponding to different polymerization interfaces in the tubulin αβ dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. δ-tubulin most likely interacts longitudinally with α-tubulin at the minus ends of microtubules, while ε-tubulin most likely binds to the plus end of β-tubulin. Conservation of key residues in γ-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.

Our understanding of the elaborate mechanism of gene transcription initiation in eukaryotes has been widened by recent structural information on some of the key components of the complex preinitiation transcriptional machinery. The high-resolution structures of both bacterial and eukaryotic polymerases are technical landmarks of great biological significance that have given us the first molecular insight into the mechanism of this large enzyme. While new atomic structures of different domains of general transcription factors, such as the double bromodomain of TAF250, have become available by means of X-ray crystallography and NMR studies, more global pictures of multisubunit transcription complexes, such as TFIID, TFIIH or the yeast mediator, have now been obtained by electron microscopy and image-reconstruction techniques. A combination of methodologies may prove essential for a complete structural description of the initial steps in the expression of eukaryotic genes.

The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (−1.8 °C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0 °C to ∼0.9 mg/ml (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985)Biochemistry24, 2790–2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5 °C) and high (25 °C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37 °C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 α-tubulins and 4 β-tubulins from the yellowbelly rockcod Notothenia coriicepsand 4 α-tubulins and 2 β-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the α- and β-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in α; S280G and A285S in β) were present in the S7-H9 interprotofilament “M” loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the α-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (α:S187A in helix H5 and β:Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitivetub1alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematictub1mutations were placed, along with the comparable set of tub2mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1alleles with the benomyl-resistant but otherwise entirely viable tub2-201allele.

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp)Pkl1plocalizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component.

Abstract: Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an “unconstrained” conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozenhydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules.