Guillermo Calero

University of Pittsburgh, Pittsburgh, Pennsylvania, United States

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Publications (22)247.82 Total impact

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    ABSTRACT: The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
    Proceedings of the National Academy of Sciences 10/2014; · 9.81 Impact Factor
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    ABSTRACT: The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation- sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals ofβ2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources
    Proceedings of the National Academy of Sciences 10/2014; 111(48):17122. · 9.81 Impact Factor
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    ABSTRACT: The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.
    Nature Chemical Biology 09/2014; in press. · 12.95 Impact Factor
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    ABSTRACT: Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.
    Acta Crystallographica Section F: Structural Biology Communications. 07/2014; 70(8).
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    ABSTRACT: Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.
    Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 07/2014; 369(1647).
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    ABSTRACT: The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
    Proceedings of the National Academy of Sciences 06/2014; 111(23):8470-8475. · 9.81 Impact Factor
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    ABSTRACT: Whereas individual RNA polymerase II (pol II) - general transcription factor (GTF) complexes are unstable, an assembly of pol II with six GTFs and promoter DNA could be isolated in abundant, homogeneous form. The resulting complete pol II transcription initiation complex (PIC) contained equimolar amounts of all 31 protein components. An intermediate in assembly, consisting of four GTFs and promoter DNA, could be isolated and supplemented with the remaining components for formation of the PIC. Nuclease digestion and psoralen cross-linking mapped the PIC between positions -70 and -9, centered on the TATA box. Addition of ATP to the PIC resulted in quantitative conversion to an open complex, which retained all 31 proteins, contrary to expectation from previous studies. Addition of the remaining NTPs resulted in runoff transcription, with an efficiency that was promoter-dependent and was as great as 17.5% with the promoters tested.
    Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
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    ABSTRACT: G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. β-Arrestins prolong G protein (G(S))-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gβγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G(S) activation and increases the steady-state levels of activated G(S), leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.
    Proceedings of the National Academy of Sciences 01/2013; · 9.81 Impact Factor
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    ABSTRACT: Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes, and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.
    Protein Expression and Purification 11/2012; · 1.43 Impact Factor
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    ABSTRACT: Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
    Structure 05/2012; 20(5):899-910. · 5.99 Impact Factor
  • Biophysical Journal 01/2012; 102(3):240-. · 3.67 Impact Factor
  • Biophysical Journal 01/2012; 102(3):244-. · 3.67 Impact Factor
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    ABSTRACT: Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.
    Nature 07/2011; 475(7355):240-3. · 38.60 Impact Factor
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    ABSTRACT: Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the "B finger," reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.
    Science 11/2009; 327(5962):206-9. · 31.20 Impact Factor
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    ABSTRACT: The second structure of a eukaryotic RNA polymerase II so far determined, that of the enzyme from the fission yeast Schizosaccharomyces pombe, is reported here. Comparison with the previous structure of the enzyme from the budding yeast Saccharomyces cerevisiae reveals differences in regions implicated in start site selection and transcription factor interaction. These aspects of the transcription mechanism differ between S. pombe and S. cerevisiae, but are conserved between S. pombe and humans. Amino acid changes apparently responsible for the structural differences are also conserved between S. pombe and humans, suggesting that the S. pombe structure may be a good surrogate for that of the human enzyme.
    Proceedings of the National Academy of Sciences 06/2009; 106(23):9185-90. · 9.81 Impact Factor
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    ABSTRACT: Structural information on nanometer-sized gold particles has been limited, due in part to the problem of preparing homogeneous material. Here we report the crystallization and x-ray structure determination of a p-mercaptobenzoic acid (p-MBA)-protected gold nanoparticle, which comprises 102 gold atoms and 44 p-MBAs. The central gold atoms are packed in a Marks decahedron, surrounded by additional layers of gold atoms in unanticipated geometries. The p-MBAs interact not only with the gold but also with one another, forming a rigid surface layer. The particles are chiral, with the two enantiomers alternating in the crystal lattice. The discrete nature of the particle may be explained by the closing of a 58-electron shell.
    Science 06/2009; 318(5849). · 31.03 Impact Factor
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    ABSTRACT: Synthesis, characterization, and functionalization of self-assembled, ligand-stabilized gold nanoparticles are long-standing issues in the chemistry of nanomaterials. Factors driving the thermodynamic stability of well documented discrete sizes are largely unknown. Herein, we provide a unified view of principles that underlie the stability of particles protected by thiolate (SR) or phosphine and halide (PR(3), X) ligands. The picture has emerged from analysis of large-scale density functional theory calculations of structurally characterized compounds, namely Au(102)(SR)(44), Au(39)(PR(3))(14)X(6)(-), Au(11)(PR(3))(7)X(3), and Au(13)(PR(3))(10)X(2)(3+), where X is either a halogen or a thiolate. Attributable to a compact, symmetric core and complete steric protection, each compound has a filled spherical electronic shell and a major energy gap to unoccupied states. Consequently, the exceptional stability is best described by a "noble-gas superatom" analogy. The explanatory power of this concept is shown by its application to many monomeric and oligomeric compounds of precisely known composition and structure, and its predictive power is indicated through suggestions offered for a series of anomalously stable cluster compositions which are still awaiting a precise structure determination.
    Proceedings of the National Academy of Sciences 08/2008; 105(27):9157-62. · 9.81 Impact Factor
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    ABSTRACT: GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTP-bound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl beta,gamma-methylene-diphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP- and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP- and GMP-PCP-bound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCP-bound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP- and GDP-bound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.
    Journal of Biological Chemistry 05/2008; 283(20):14153-64. · 4.65 Impact Factor
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    ABSTRACT: Structural information on nanometer-sized gold particles has been limited, due in part to the problem of preparing homogeneous material. Here we report the crystallization and x-ray structure determination of a p-mercaptobenzoic acid (p-MBA)-protected gold nanoparticle, which comprises 102 gold atoms and 44 p-MBAs. The central gold atoms are packed in a Marks decahedron, surrounded by additional layers of gold atoms in unanticipated geometries. The p-MBAs interact not only with the gold but also with one another, forming a rigid surface layer. The particles are chiral, with the two enantiomers alternating in the crystal lattice. The discrete nature of the particle may be explained by the closing of a 58-electron shell.
    Science 11/2007; 318(5849):430-3. · 31.20 Impact Factor
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    ABSTRACT: Yeast Mediator proteins interacting with Med17(Srb4) have been expressed at a high level with the use of recombinant baculoviruses and recovered in homogeneous form as a seven subunit, 223 kDa complex. Electron microscopy and single-particle analysis identify this complex as the Mediator head module. The recombinant head module complements "headless" Mediator for the initiation of transcription in vitro. The module interacts with an RNA polymerase II-TFIIF complex, but not with the polymerase or TFIIF alone. This interaction is lost in the presence of a DNA template and associated RNA transcript, recapitulating the release of Mediator that occurs upon the initiation of transcription. Disruption of the head module in a temperature-sensitive mutant in vivo leads to the release of middle and tail modules from a transcriptionally active promoter. The head module evidently controls Mediator-RNA polymerase II and Mediator-promoter interactions.
    Molecular Cell 09/2006; 23(3):355-64. · 15.28 Impact Factor

Publication Stats

895 Citations
247.82 Total Impact Points

Institutions

  • 2014
    • University of Pittsburgh
      • Department of Structural Biology
      Pittsburgh, Pennsylvania, United States
  • 2006–2013
    • Stanford University
      • Department of Structural Biology
      Palo Alto, CA, United States
  • 2008–2012
    • Stanford Medicine
      • Department of Structural Biology
      Stanford, California, United States
  • 2003
    • Cornell University
      • Department of Chemistry and Chemical Biology
      Ithaca, NY, United States