Jesús G. Galaz-Montoya

Single Particle Tomography in EMAN2

Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally heterogeneous macromolecules. It can also provide direct observation (without labeling or staining) of complexes inside cells at nanometer resolution. The development of computational methods and tools for SPT remains an area of active research. Here we present the EMAN2.1 SPT toolbox, which offers a full SPT processing pipeline, from particle picking to post-alignment analysis of subtomogram averages, automating most steps. Different algorithm combinations can be applied at each step, providing versatility and allowing for procedural cross-testing and specimen-specific strategies. Alignment methods include all-vs-all, binary tree, iterative single-model refinement, multiple-model refinement, and self-symmetry alignment. An efficient angular search, Graphic Processing Unit (GPU) acceleration and both threaded and distributed parallelism are provided to speed up processing. Finally, automated simulations, per particle reconstruction of subtiltseries, and per-particle Contrast Transfer Function (CTF) correction have been implemented. Processing examples using both real and simulated data are shown for several structures.

TRiC’s tricks inhibit huntingtin aggregation

In Huntington’s disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC’s inhibition of mhttQ51 aggregation in vitro. DOI: http://dx.doi.org/10.7554/eLife.00710.001

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography.

Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.

Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography

Background: Huntington disease patients show an accumulation of oligomers and fibrillar species of mutant huntingtin (mHTT). Results: Cryoelectron tomography and subvolume averaging visualizes heterogeneous mHTT oligomeric species inside the chaperonin-like CCT5 cavity. Conclusion: The structural basis of mHTT aggregation inhibition by CCT5 is through capping of fibrils and encapsulation of oligomers. Significance: These structural mechanisms inspire the development of new strategies for inhibiting mHTT aggregation. Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its β-strand rich, DNA-binding domain (AML-(1–175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoproteins DNA-binding domain, which may be targeted for therapeutic benefit.

Chaperonin TRiC/CCT Recognizes Fusion Oncoprotein AML1-ETO through Subunit-Specific Interactions

AML1-ETO is the translational product of a chimeric gene created by the stable chromosome translocation t (8;21)(q22;q22). It causes acute myeloid leukemia (AML) by dysregulating the expression of genes critical for myeloid cell development and differentiation and recently has been reported to bind multiple subunits of the mammalian cytosolic chaperonin TRiC (or CCT), primarily through its DNA binding domain (AML1-175). Through these interactions, TRiC plays an important role in the synthesis, folding, and activity of AML1-ETO. Using single-particle cryo-electron microscopy, we demonstrate here that a folding intermediate of AML1-ETOs DNA-binding domain (AML1-175) forms a stable complex with apo-TRiC. Our structure reveals that AML1-175 associates directly with a specific subset of TRiC subunits in the open conformation.

EMAN2.1 - A New Generation of Software for Validated Single Particle Analysis and Single Particle Tomography

1. Graduate Program in Structural and Computational Biology & Molecular Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston TX 77025 2. National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza Houston TX 77025 3. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston TX 77025