Jesus Gerardo Galaz Montoya

All Publications

• Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells. Structure (London, England : 1993) Wu, G. H., Mitchell, P. G., Galaz-Montoya, J. G., Hecksel, C. W., Sontag, E. M., Gangadharan, V. n., Marshman, J. n., Mankus, D. n., Bisher, M. E., Lytton-Jean, A. K., Frydman, J. n., Czymmek, K. n., Chiu, W. n. 2020

  Abstract

  Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

  View details for DOI 10.1016/j.str.2020.07.017

  View details for PubMedID 32814034

• Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane CELL HOST & MICROBE Jin, J., Galaz-Montoya, J. G., Sherman, M. B., Sun, S. Y., Goldsmith, C. S., O'Toole, E. T., Ackerman, L., Carlson, L., Weaver, S. C., Chiu, W., Simmons, G. 2018; 24 (3): 417-+

  Abstract

  Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.

  View details for PubMedID 30146390

  View details for PubMedCentralID PMC6137268

• Visualizing Adsorption of Cyanophage P-SSP7 onto Marine Prochlorococcus SCIENTIFIC REPORTS Murata, K., Zhang, Q., Galaz-Montoya, J. G., Fu, C., Coleman, M. L., Osburne, M. S., Schmid, M. F., Sullivan, M. B., Chisholm, S. W., Chiu, W. 2017; 7

  Abstract

  Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.

  View details for DOI 10.1038/srep44176

  View details for Web of Science ID 000395891400001

  View details for PubMedID 28281671

  View details for PubMedCentralID PMC5345008

• The advent of structural biology in situ by single particle cryo-electron tomography Biophysics Rreports Galaz-Montoya, J. G., Ludtke, S. J. 2017; 3 (1): 17-35
• Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO. The Journal of biological chemistry Roh, S. H., Kasembeli, M. n., Galaz-Montoya, J. G., Trnka, M. n., Lau, W. C., Burlingame, A. n., Chiu, W. n., Tweardy, D. J. 2016; 291 (9): 4732–41

  Abstract

  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 oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.

  View details for DOI 10.1074/jbc.M115.684878

  View details for PubMedID 26706127

  View details for PubMedCentralID PMC4813495

• Chaperonin TRiC/CCT Recognizes Fusion Oncoprotein AML1-ETO through Subunit-Specific Interactions. Biophysical journal Roh, S. H., Kasembeli, M. M., Galaz-Montoya, J. G., Chiu, W. n., Tweardy, D. J. 2016; 110 (11): 2377–85

  Abstract

  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-ETO's 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.

  View details for DOI 10.1016/j.bpj.2016.04.045

  View details for PubMedID 27276256

  View details for PubMedCentralID PMC4906440

• Quantifying Variability of Manual Annotation in Cryo-Electron Tomograms. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Hecksel, C. W., Darrow, M. C., Dai, W. n., Galaz-Montoya, J. G., Chin, J. A., Mitchell, P. G., Chen, S. n., Jakana, J. n., Schmid, M. F., Chiu, W. n. 2016; 22 (3): 487–96

  Abstract

  Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a "ground truth" for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.

  View details for DOI 10.1017/S1431927616000799

  View details for PubMedID 27225525

  View details for PubMedCentralID PMC5111626

• Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography. Journal of structural biology Galaz-Montoya, J. G., Hecksel, C. W., Baldwin, P. R., Wang, E. n., Weaver, S. C., Schmid, M. F., Ludtke, S. J., Chiu, W. n. 2016; 194 (3): 383–94

  Abstract

  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.

  View details for DOI 10.1016/j.jsb.2016.03.018

  View details for PubMedID 27016284

  View details for PubMedCentralID PMC4846534

• Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes SCIENTIFIC REPORTS Dalm, D., Galaz-Montoya, J. G., Miller, J. L., Grushin, K., Villalobos, A., Koyfman, A. Y., Schmid, M. F., Stoilova-McPhie, S. 2015; 5: 11212

  Abstract

  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.

  View details for DOI 10.1038/srep11212

  View details for Web of Science ID 000356519800001

  View details for PubMedID 26082135

  View details for PubMedCentralID PMC4469981

• Single particle tomography in EMAN2 JOURNAL OF STRUCTURAL BIOLOGY Galaz-Montoya, J. G., Flanagan, J., Schmid, M. F., Ludtke, S. J. 2015; 190 (3): 279–90

  Abstract

  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.

  View details for DOI 10.1016/j.jsb.2015.04.016

  View details for Web of Science ID 000356115600003

  View details for PubMedID 25956334

  View details for PubMedCentralID PMC4457691

• Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography. The Journal of biological chemistry Darrow, M. C., Sergeeva, O. A., Isas, J. M., Galaz-Montoya, J. G., King, J. A., Langen, R. n., Schmid, M. F., Chiu, W. n. 2015; 290 (28): 17451–61

  Abstract

  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.

  View details for DOI 10.1074/jbc.M115.655373

  View details for PubMedID 25995452

  View details for PubMedCentralID PMC4498080

• TRiC's tricks inhibit huntingtin aggregation ELIFE Shahmoradian, S. H., Galaz-Montoya, J. G., Schmid, M. F., Cong, Y., Ma, B., Spiess, C., Frydman, J., Ludtke, S. J., Chiu, W. 2013; 2

  Abstract

  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.

  View details for DOI 10.7554/eLife.00710

  View details for Web of Science ID 000328620500004

  View details for PubMedID 23853712

  View details for PubMedCentralID PMC3707056

• Interfacial behavior of N-nitrosodiethylamine/bovine serum albumin complexes at the air-water and the chloroform-water interfaces by axisymmetric drop tensiometry JOURNAL OF PHYSICAL CHEMISTRY B Juarez, J., Galaz, J. G., Machi, L., Burboa, M., Gutierrez-Millan, L. E., Goycoolea, F. M., Valdez, M. A. 2007; 111 (10): 2727–35

  Abstract

  Interfacial properties of N-nitrosodiethylamine/bovine serum albumin (NDA/BSA) complexes were investigated at the air-water interface. The interfacial behavior at the chloroform-water interface of the interaction product of phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dissolved in the chloroform phase, and NDA/BSA complex, in the aqueous phase, were also analyzed by using a drop tensiometer. The secondary structure changes of BSA with different NDA concentrations were monitored by circular dichroism spectroscopy at different pH and the NDA/BSA interaction was probed by fluorescence spectroscopy. Different NDA/BSA mixtures were prepared from 0, 7.5 x 10(-5), 2.2 x 10(-4), 3.7 x 10(-4), 5 x 10(-4), 1.6 x 10(-3), and 3.1 x 10(-3) M NDA solutions in order to afford 0, 300/1, 900/1, 1 500/1, 2 000/1, 6 000/1, and 12 500/1 NDA/BSA molar ratios, respectively, in the aqueous solutions. Increments of BSA alpha-helix contents were obtained up to the 2 000/1 NDA/BSA molar ratio, but at ratios beyond this value, the alpha-helix content practically disappeared. These BSA structure changes produced an increment of the surface pressure at the air-water interface, as the alpha-helix content increased with the concentration of NDA. On the contrary, when alpha-helix content decreased, the surface pressure also appeared lower than the one obtained with pure BSA solutions. The interaction of DPPC with NDA/BSA molecules at the chloroform-water interface produced also a small, but measurable, pressure increment with the addition of NDA molecules. Dynamic light scattering measurements of the molecular sizes of NDA/BSA complex at pH 4.6, 7.1, and 8.4 indicated that the size of extended BSA molecules at pH 4.6 increased in a greater proportion with the increment in NDA concentration than at the other studied pH values. Diffusion coefficients calculated from dynamic surface tension values, using a short-term solution of the general adsorption model of Ward and Tordai, also showed differences with pH and the NDA concentration. Both, the storage and loss dilatational elastic modulus were obtained at the air-water and at the chloroform-water interfaces. The interaction of NDA/BSA with DPPC at the chloroform-water produced a less rigid monolayer than the one obtained with pure DPPC (1 x 10(-5) M), indicating a significant penetration of NDA/BSA molecules at the interface. At short times and pH 4.6, the values of the storage elastic modulus were larger and more sensible to the NDA addition than the ones at pH 7.1 and 8.4, probably due to a gel-like network formation at the air-water interface.

  View details for DOI 10.1021/jp066061m

  View details for Web of Science ID 000244735300041

  View details for PubMedID 17315914