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Dive into the research topics where Ana Gonzalez is active.

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Featured researches published by Ana Gonzalez.


Nature Structural & Molecular Biology | 1999

Crystal structure of a DNA Holliday junction

Miguel Ortiz-Lombardía; Ana Gonzalez; Ramon Eritja; Joan Aymamí; Fernando Azorín; Miquel Coll

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 Å resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.


The EMBO Journal | 1998

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

F. Xavier Gomis-Rüth; Maria Solà; Paloma Acebo; Antonio Párraga; Alicia Guasch; Ramon Eritja; Ana Gonzalez; Manuel Espinosa; Gloria del Solar; Miquel Coll

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.


Acta Crystallographica Section D-biological Crystallography | 2008

New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

S. Michael Soltis; Aina E. Cohen; Ashley M. Deacon; Thomas Eriksson; Ana Gonzalez; Scott E. McPhillips; Hsui Chui; Pete W. Dunten; Michael Hollenbeck; Irimpan I. Mathews; Mitch Miller; Penjit Moorhead; R. Paul Phizackerley; Clyde A. Smith; Jinhu Song; Henry van dem Bedem; Paul J. Ellis; Peter Kuhn; Timothy M. McPhillips; Nicholas K. Sauter; Kenneth Sharp; Irina Tsyba; Guenter Wolf

Through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface, SSRL researchers have screened over 200u2005000 biological crystals for diffraction quality in an automated fashion. Three quarters of SSRL researchers are using these data-collection tools from remote locations.


Proceedings of the National Academy of Sciences of the United States of America | 2014

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Aina E. Cohen; S. Michael Soltis; Ana Gonzalez; Laura Aguila; Roberto Alonso-Mori; Christopher O. Barnes; Elizabeth L. Baxter; Winnie Brehmer; Aaron S. Brewster; Axel T. Brunger; Guillermo Calero; Joseph F. Chang; Matthieu Chollet; Paul Ehrensberger; Thomas Eriksson; Yiping Feng; Johan Hattne; Britt Hedman; Michael Hollenbeck; James M. Holton; Stephen Keable; Brian K. Kobilka; Elena G. Kovaleva; Andrew C. Kruse; Henrik T. Lemke; Guowu Lin; Artem Y. Lyubimov; Aashish Manglik; Irimpan I. Mathews; Scott E. McPhillips

Significance The extremely short and bright X-ray pulses produced by X-ray free-electron lasers unlock new opportunities in crystallography-based structural biology research. Efficient methods to deliver crystalline material are necessary due to damage or destruction of the crystal by the X-ray pulse. Crystals for the first experiments were 5 µm or smaller in size, delivered by a liquid injector. We describe a highly automated goniometer-based approach, compatible with crystals of larger and varied sizes, and accessible at cryogenic or ambient temperatures. These methods, coupled with improvements in data-processing algorithms, have resulted in high-resolution structures, unadulterated by the effects of radiation exposure, from only 100 to 1,000 diffraction images. 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.


eLife | 2015

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

D.A. Keedy; Lillian R. Kenner; Matthew Warkentin; Rahel A. Woldeyes; Jesse B. Hopkins; Michael C. Thompson; Aaron S. Brewster; Andrew H. Van Benschoten; Elizabeth L. Baxter; Monarin Uervirojnangkoorn; Scott E. McPhillips; Jinhu Song; Roberto Alonso-Mori; James M. Holton; William I. Weis; Axel T. Brunger; S. Michael Soltis; Henrik T. Lemke; Ana Gonzalez; Nicholas K. Sauter; Aina E. Cohen; Henry van den Bedem; Robert E. Thorne; J.S. Fraser

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function. DOI: http://dx.doi.org/10.7554/eLife.07574.001


Chemical Biology & Drug Design | 2014

Crystallographic Fragment‐Based Drug Discovery: Use of a Brominated Fragment Library Targeting HIV Protease

Theresa Tiefenbrunn; Stefano Forli; Meaghan Happer; Ana Gonzalez; Yingssu Tsai; Michael Soltis; John H. Elder; Arthur J. Olson; C.D. Stout

A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV‐1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with partial occupancy, resulting in weak, difficult‐to‐fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3‐bromo‐2,6‐dimethoxybenzoic acid (Br6) in the flap site and 1‐bromo‐2‐naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br‐fragments provides further insight into the nature of these surface pockets.


Acta Crystallographica Section D-biological Crystallography | 2001

Atomic resolution structure of Escherichia coli dUTPase determined ab initio

Ana Gonzalez; Gunilla Larsson; Rebecca Persson; Eila Cedergren-Zeppezauer

Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05 A resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45 A from a native crystal were also collected and the 100 K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor-substrate-analogue complexes of this protein at very high resolution.


Acta Crystallographica Section D-biological Crystallography | 2002

Advantages of high-resolution phasing: MAD to atomic resolution

Andrea Schmidt; Ana Gonzalez; Richard J. Morris; Marcelo D. Costabel; Pedro M. Alzari; Victor S. Lamzin

The structure of the endoglucanase A from Clostridium thermocellum CelA was re-solved by three-wavelength MAD. Experimental phases were obtained in the resolution range 25-1.0 A. Various structure-solution approaches were tested in order to quantify the contribution of each wavelength. Two-wavelength MAD phasing was sufficient to obtain excellent experimental phases. SAD at the remote wavelength also resulted in interpretable maps. The three-wavelength MAD electron-density map was of excellent quality: for parts of the structure, atom types and bond types could be easily assigned. Double bonds in peptide links and side chains could be located owing to their increased electron density indicating their pi character. Comparison with a previously determined structure of CelA at 1.65 A showed that, apart from a few additional multiple conformers and differently oriented side chains, major differences occur at the protein-solvent interface. A complete additional solvent shell could be observed and the inner shells have been completed. The high accuracy of the structure allowed unambiguous assignment of the protonation state for the active-site catalytic carboxylates.


Proceedings of the National Academy of Sciences of the United States of America | 2016

Measuring and modeling diffuse scattering in protein X-ray crystallography

Andrew H. Van Benschoten; Lin Liu; Ana Gonzalez; Aaron S. Brewster; Nicholas K. Sauter; J.S. Fraser; Michael E. Wall

Significance The structural details of protein motions are critical to understanding many biological processes, but they are often hidden to conventional biophysical techniques. Diffuse X-ray scattering can reveal details of the correlated movements between atoms; however, the data collection historically has required extra effort and dedicated experimental protocols. We have measured 3D diffuse intensities in X-ray diffraction from CypA and trypsin crystals using standard crystallographic data collection techniques. Analysis of the resulting data is consistent with the protein motions resembling diffusion in a liquid or vibrations of a soft solid. Our results show that using diffuse scattering to model protein motions can become a component of routine crystallographic analysis through the extension of commonplace methods. X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.


Acta Crystallographica Section D-biological Crystallography | 1999

Two-wavelength MAD phasing: in search of the optimal choice of wavelengths

Ana Gonzalez; J.-D. Pédelacq; Maria Solà; F.X. Gomis-Rüth; Miquel Coll; Jean-Pierre Samama; Stefano Benini

The multiwavelength anomalous dispersion (MAD) method is increasingly being used to determine protein crystal structures. In theory, data collection at two wavelengths is sufficient for the determination of MAD phases, but three or even more wavelengths are used most often. In this paper, the results of the phasing procedure using only two wavelengths for proteins containing different types of anomalous scatterers are analyzed. In these cases, it is shown that this approach leads to interpretable maps, similar in quality to those obtained with data collected at three wavelengths, provided that the wavelengths are chosen so as to give a large contrast in the real part of the anomalous scattering factor f. The consequences for a rational MAD data-collection strategy are discussed.

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Nicholas K. Sauter

Lawrence Berkeley National Laboratory

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Aaron S. Brewster

Lawrence Berkeley National Laboratory

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J.S. Fraser

University of California

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Jinhu Song

SLAC National Accelerator Laboratory

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D.A. Keedy

University of California

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Elizabeth L. Baxter

SLAC National Accelerator Laboratory

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Henrik T. Lemke

SLAC National Accelerator Laboratory

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