Structure of Macromolecular Assemblies

Research

Towards extending the use of Electron Microscopy at different structural levels

The activity of the group aims to extend the use of improved methods in three-dimensional cryo-electron microscopy (3D-cryo-EM) in two main directions: Towards reaching subnanometer resolution using single particle reconstruction approaches, and towards the analysis of the subcellular domain using electron tomography

Molecular resolution

The model system we study is the assembly and maturation process of complex double stranded DNA viruses. We use the phages T7 and phi29 as model systems offering complementary aspects to cover the most interesting features shared by this virus family during their assembly, as the production of intermediates, the expansion of the shell by a complex subunit rearrangement, the transformation of inner capsid components (core, scaffolding), and the changes involved in the function of the DNA packaging machinery. We also improve the single particle reconstruction methods to get higher resolution data suitable to apply hybrid approaches, by a combination of 3D-cryo-EM and modelling methods, to compare quasi-atomic 3D maps of proheads and mature heads of T7.

 

Electron Tomography

The interactions of virus with the target cells are studied using electron tomography of infected cells. We are involved in the development of Tomographic methods using model large viruses (as Poxviruses) as test objects combining correlation with single particle reconstruction approaches.

 

Correlation of structural data and physical properties at the single particle level

The structural data obtained by 3D-cryo-EM from the different experimental systems under study in our group are currently combined with the study of the nano-mechanical properties of individual viral particles using atomic force microscopy and spectroscopy. We are also exploring the application of optical tweezers to test small forces involved in specific capsid transformations. The study of the deformation behaviour and material properties at the level of individual viral particles opens the possibility to perform systematic studies to correlate molecular structure, nanoscopic behaviour, and macroscopic properties of these macromolecular viral containers.

 

Cartography of a whole cell by cryo-X-ray nano-tomography

Cryo-electron tomography (cryo-ET) has proven to be a suitable tool in this context by combining improved preservation methods with tomographic three-dimensional reconstruction using electron microscope images at resolutions in the order of a few nanometers. One of the main limitations of cryo-ET is derived from the poor penetration of electrons in soft materials limited to 0.5 µm thick samples. This penetration level prevents the direct analysis of most cell types ultrastructure, where the only alternative is then to produce thin sections. Sectioning involves in classical approaches the use of chemical fixation and embedding and in cryo-methods the use fast freezing and cryo-processing. In both cases sectioning are prone to artifacts. In addition, the correlation of information from light microscopy to transmission electron microscopy has become an important issue to study biological events at different levels. The gap between these two microscopies in terms of magnification levels, thickness and resolution has complicated the microscopy information gateway.
X-ray microscopy then appears as a complementary approach to overcome these problems taking advance of thehigher penetration power of X-rays.

As in the case of cryo-ET, the combination of X-ray imaging with the preservation of the samples at cryogenic temperatures allows retrieving structural information close to physiological conditions. The tomographic reconstruction of X-ray projection images is a powerful tool that can also be combined with light microscopy.

 

Synthetic Biology

 

Physical characterization of the packaging machinery

Based on our previous structural knowledge on the packaging machinery of two viral model systems (T7 and phi29), we are developing a synthetic biology approach to insert phage connectors into nano-engineered lipidic bilayers, in collaboration with the group of Francisco Monroy (UCM). We are exploring the possibility of building up the entire packaging motor on these inserted connectors which will allow us to obtain efficient lipidic nanocontainers capable to carrying large amounts of DNA. On the other hand, we are also improving our optical tweezers set up to study, at the single molecule level, the physical properties of the membrane-inserted connectors and the molecular basis of their function.

 

Members

<

José López Carrascosa

Post-doctoral investigators

Fco Javier Chichón

Ana Cuervo
Rebeca Bocanegra
   

Predoctoral students

Jose Javier Conesa

   

Technician

Maria José Rodriguez

Encarna Cebrián

 

Publications

JOURNALS

2015

Sandra M. Ocampo, Vanessa Rodriguez, Leonor de la Cueva, Gorka Salas, José L. Carrascosa, María Josefa Rodríguez, Noemí García-Romero, Jose Luis F. Cuñado, Julio Camarero, Rodolfo Miranda, Cristobal Belda-Iniesta and Angel Ayuso-Sacido. g-force induced giant efficiency of nanoparticles internalization into living cells. doi: 10.1038/srep15160. Scientific Reports, 5:15160 (2015)

Veronica A. Gonzalez-Garcia, Rebeca Bocanegra, Mar Pulido-Cid, Jaime Martin-Benito, Ana Cuervo and Jose L. Carrascosa Characterization of the initial steps in the T7 DNA ejection process. doi: 10.1080/21597081.2015.1056904 Bacteriophage, vol. 5:3 (2015)

José M. Valpuesta and José L. Carrascosa Electron microscopy: the coming of age of a structural biology technique. doi: 10.1016/j.abb.2015.06.018 Archives of Biochemistry and Biophysics, 581 (2015)

Johann Mertens, Santiago Casado, Carlos P. Mata, Mercedes Hernando-Pérez, Pedro J. de Pablo, José L. Carrascosa and José R. Castón. A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability. doi: 10.1038/srep13486
Scientific Reports, 5 (2015)

Macarena Calero, Michele Chiappi, Ana Lazaro-Carrillo, Maria Jose Rodriguez, Francisco Javier Chichon, Kieran Crosbie-Staunton, Adriele Prina-Mello, Yuri Volkov, Angeles Villanueva and Jose L. Carrascosa Characterization of interaction of magnetic nanoparticles with breast cancer cells. doi: 10.1186/s12951-015-0073-9 Journal of Nanobiotechnology (2015)

Pérez-Berná, A. J., S. Marion, F. J. Chichón, J. J. Fernández, D. C. Winkler, J. L. Carrascosa, A. C. Steven, A. Šiber and C. San Martín (2015). "Distribution of DNA-condensing protein complexes in the adenovirus core." Nucleic Acids Research 43(8): 4274-4283.

Sorrentino, A., J. Nicolás, R. Valcárcela, F. J. Chichón, M. Rosanes, J. Avila, A. Tkachukc, J. Irwinc, S. Ferrer and E. Pereiro (2015). "MISTRAL: a transmission soft X-ray microscopy beamline for cryo nano-tomography of biological samples and magnetic domains imaging." Journal of Synchrotron Radiation(beamlines).

Vijayan, A., J. Garcia-Arriaza, C. R. S, J. J. Conesa, F. J. Chichon, C. Santiago, C. O. Sorzano, J. L. Carrascosa and M. Esteban (2015). "A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen." PLoS One 10(7): e0133595.

2014

Chagoyen, M., J. L. Carrascosa, F. Pazos and J. M. Valpuesta (2014). "Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex." Proteins-Structure Function and Bioinformatics 82(5): 703-707.

Cruz-Adalia, A., G. Ramirez-Santiago, C. Calabia-Linares, M. Torres-Torresano, L. Feo, M. Galan-Diez, E. Fernandez-Ruiz, E. Pereiro, P. Guttmann, M. Chiappi, G. Schneider, J. Lopez Carrascosa, F. Javier Chichon, G. Martinez del Hoyo, F. Sanchez-Madrid and E. Veiga (2014). "T Cells Kill Bacteria Captured by Transinfection from Dendritic Cells and Confer Protection in Mice." Cell Host & Microbe 15(5): 611-622.

Cuervo, A., P. D. Dans, J. L. Carrascosa, M. Orozco, G. Gomila and L. Fumagalli (2014). "Direct measurement of the dielectric polarization properties of DNA." Proceedings of the National Academy of Sciences of the United States of America 111(35): E3624-E3630.

Luque, D., A. de la Escosura, J. Snijder, M. Brasch, R. J. Burnley, M. S. T. Koay, J. L. Carrascosa, G. J. L. Wuite, W. H. Roos, A. J. R. Heck, J. J. L. M. Cornelissen, T. Torres and J. R. Caston (2014). "Self-assembly and characterization of small and monodisperse dye nanospheres in a protein cage." Chemical Science 5(2): 575-581.

Luque, D., J. Gomez-Blanco, D. Garriga, A. F. Brilot, J. M. Gonzalez, W. M. Havens, J. L. Carrascosa, B. L. Trus, N. Verdaguer, S. A. Ghabrial and J. R. Caston (2014). "Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage." Proceedings of the National Academy of Sciences of the United States of America 111(21): 7641-7646.

Morin, J. A., F. J. Cao, J. M. Lazaro, M. Salas, J. M. Valpuesta, J. L. Carrascosa and B. Ibarra (2014). "Identification of the Translocation Step of a Replicative DNA Polymerase." Biophysical Journal 106(2): 229A-229A. Rodriguez, J. M., F. J. Chichon, E. Martin-Forero, F. Gonzalez-Camacho, J. L. Carrascosa, J. R. Caston and D. Luque (2014). "New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage." Plos Pathogens 10(5).

2013

Ana Cuervo, Mar Pulido-Cid, Mónica Chagoyen, Rocío Arranz, Verónica A. González-García, Carmela García-Doval, José R. Castón, José M. Valpuesta, Mark J. van Raaij, Jaime Martín-Benito and José L. Carrascosa. Structural Characterización of the Bacteriophage T7 Tail Machinery. Journal of Biological Chemistry, 288, nº 36, pp., 26290-26299 (2013)

María I. Daudén, Jaime Martín-Benito, Juan C. Sanchez-Ferrero, Mar Pulido Cid, José M. Valpuesta and José L. Carrascosa.
Large Terminase Conformational Change Induced by Connector Binding in Bacteriphage T7.
Journal of Biological Chemistry, 288, nº 23, pp., 16998-17007 (2013)

Alexander Cartagena, Mercedes Hernando-Pérez, José L. Carrascosa, Pedro J. de Pablo and Arvind Raman.
Mapping in vitro local material properties of intact and disrupted virions at high resolution using multi-harmonic atomic force microscopy.
Nanoscale, 5, 4729-4736 (2013)

Elías Herrero-Galán, Maria Eugenia Fuentes-Perez, Carolina Carrasco, José M. Valpuesta, José L. Carrascosa, Fernando Moreno-Herrero, J.Ricardo Arias-González.
Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level.
J Am Chem Soc. 135(1), 122-131 (2013)

2012

Rocío Arranz, Rocío Coloma, F.Javier Chichón, José Javier Conesa, José L. Carrascosa, José Mª Valpuesta, Juan Ortín y Jaime Martín-Benito.
The structure of native influenza virion ribonucleoproteins.
Science, 338, 1635-1637 (2012)

Alejandro Peña, Inmaculada Matilla, Jaime Martín-Benito, José Mª Valpuesta, J. L. Carrascosa, Fernando de la Cruz, Elena Cabezón and Ignacio Arechaga.
The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases.
J Biol Chem. 287, 47pp, 39925-39932 (2012)

Jose A. Morin, Francisco J. Cao, José M. Valpuesta, José L.Carrascosa, Margarita Salas and Borja Ibarra.
Manipulation of single polymerase-DNA complexes. A mechanical view of DNA unwinding during replication.
Cell Cycle 11:16, 2967-2968 (2012)

Mercedes Hernando-Pérez, Roberto Miranda, María Aznar and José L.Carrascosa.
Direct Measurement of Phag phi29 Stiffness Provides Evidence of Internal Pressure.
Small, 8 (15), 2366-2370 (2012)

Laura Fumagalli, Daniel Esteban-Ferrer, Ana Cuervo, José L.Carrascosa and Gabriel Gomila.
Label-free identification of single dielectric nanoparticles and viruses with ultraweak polarization forces.
Nature Materials, 11 (9), 808-816 (2012)

Johann Mertens, María Ibarra, José L.Carrascosa, Javier Tamayo.
Interaction of viral ATPases with nucleotides measured with a microcantilever.
Sensor and Actuators B DOI:10.1016/j.snb.2012.03.062 (2012)

Ana Cuervo, José L.Carrascosa.
Bacteriophages: Structure.
eLS John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0024053 (2012)

Lara H. Molero, Iván López-Montero, Ileana Márquez, Sonia Moreno, Marisela Vélez, José L. Carrascosa and Francisco Monroy.
Efficient Orthogonal Integration of the Bacteriophage Ø29 DNA-Portal Connector Protein in Engineered Lipid Bilayers.
ACS Synth. Biol., 1, 414-424 (2012)

José A. Morín, Francisco J. Cao, José M. Lázaro, J. Ricardo Arias-González, José M. Valpuesta, José L. Carrascosa, Margarita Salas and Borja Ibarra.
Active DNA unwinding dynamics during processive DNA replication.
Proc. Natl. Acad. Sci. USA, PNAS, vol. 109, no. 21, 8115–8120 (2012)

J. Gómez-Blanco, D. Luque, J.M. González, J.L. Carrascosa, C. Alfonso, B.L. Trus, W.M. Havens, S.A. Ghabrial and J.R. Castón
Cryphonectria nitschkei Virus 1 Structure Shows that the Capsid  Protein of Chrysoviruses Is a Duplicated Helix-Rich Fold Conserved in Fungal Double-Stranded RNA Viruses.
J. Virol. Vol. 86 nº 15, 8314-8318 (2012)

Luque D, González JM, Gómez-Blanco J, Marabini R, Chichón J, Mena I, Angulo I, Carrascosa JL, Verdaguer N, Trus BL, Bárcena J, Castón JR.
Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T=3 capsid protein leads to larger T=4 capsids.
J Virol. Vol. 86, nº 12, 6470-6480 (2012).             

Ortega-Esteban A, Horcas I, Hernando-Pérez M, Ares P, Pérez-Berná AJ, San Martín C, Carrascosa JL, de Pablo PJ, Gómez-Herrero J.
Minimizing tip-sample forces in jumping mode atomic force microscopy in liquid.
Ultramicroscopy, 114, 56-61 (2012).

Peña A, Gewartowski K, Mroczek S, Cuéllar J, Szykowska A, Prokop A, Czarnocki-Cieciura M, Piwowarski J, Tous C, Aguilera A, Carrascosa JL, Valpuesta JM, Dziembowski A.
Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor.
EMBO J., 31 (6), 1605-1616 (2012).

Martinez-Martin D, Carrasco C, Hernando-Perez M, de Pablo PJ, Gomez-Herrero J, Perez R, Mateu MG, Carrascosa JL, Kiracofe D, Melcher J, Raman A.
Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy.
PLoS One. 7 (1): e30204 (2012).

Carrascosa JL, Glaeser RM.
Focused issue on X-ray microscopy of biological materials.
J Struct Biol. 177 (2): 177-8 (2012).

Chichón FJ, Rodríguez MJ, Pereiro E, Chiappi M, Perdiguero B, Guttmann P, Werner S, Rehbein S, Schneider G, Esteban M, Carrascosa JL.
Cryo X-ray nano-tomography of vaccinia virus infected cells.
J Struct Biol. 177 (2): 202-11 (2012).

Mertens J, Daudén MI, Carrascosa JL, Tamayo J.
Stepwise motion of a microcantilever driven by the hydrolysis of viral ATPases.
Nanotechnology. 23 (1):015501,  PMID: 22156040 (2012).

Cuervo A, Carrascosa JL.
Viral connectors for DNA encapsulation.
Current Opinion in Biotechnology, 23 (4), 529-536 (2012).

Silvia Hormeño, Borja Ibarra, José M. Valpuesta, José L. Carrascosa, J. Ricardo Arias-Gonzalez.
Mechanical Stability of low-humidity single DNA molecules.
Biopolymers, 97, nº4, 199-208 (2012).

2011

Irena L. Ivanovska, Roberto Miranda, J.L. Carrascosa, Gijs J.L. Wuite and Christoph F. Schmidt.
Discrete fracture patterns of virus shells reveal mechanical building blocks.
Proc. Natl. Acad. Sci. USA. 108 (31), 12611-12616 (2011).

Silvia Hormeño, Fernando Moreno-Herrero, Borja Ibarra, J.L. Carrascosa, José M. Valpuesta, and J. Ricardo Arias-Gonzalez.
Condensation Prevails over B-A Transition in the Structure of DNA at Low Humidity.
Biophysical Journal, 100, 2006-2015 (2011).

Silvia Hormeño, Borja Ibarra, J.L. Carrascosa, José M. Valpuesta, Fernando Moreno-Herrero and J. Ricardo Arias-Gonzalez.
Mechanical Properties of High-G•C Content DNA with A-Type Base-Stacking.
Biophysical Journal, 100, 1996-2005 (2011).

C. Carrasco, A. Luque, M. Hernando-Pérez, R. Miranda, J.L. Carrascosa, P.A. Serena, M. de Ridder, A. Raman, J. Gómez-Herrero, I.A.T. Schaap, D. Reguera and P.J. de Pablo.
Built-In Mechanical Stress in Viral Shells.
Biophysical Journal, 100, 1100-1108 (2011).

Alina Ionel, Javier A. Velazquez-Muriel, Daniel Luque, Ana Cuervo, José R. Caston, José M. Valpuesta, Jaime Martin-Benito and José L. Carrascosa.
Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7.
J Biol Chem., 286 (1) 234-242 (2011).

2010

Ignacio Arechaga, Oscar H. Martínez-Costa, Cristina Ferreras, José L. Carrascosa, and Juan J. Aragón.
Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function.
The FASEB Journal. 24, 4960-4968 (2010)

Luque D, González JM, Garriga D, Ghabrial SA, Havens WM, Trus B, Verdaguer N, Carrascosa JL, Castón JR.
The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication.
J Virol. 84 (14) 7256-7266 (2010)

Saugar I, Irigoyen N, Luque D, Carrascosa JL, Rodríguez JF, Castón JR.
Electrostatic interactions between capsid and scaffolding proteins mediate the structural polymorphism of a double-stranded RNA virus.
J Biol Chem. 285 (6) 3643-50, (2010)

Jason Pierson, José Jesús Fernández, Erik Bos, Shoaib Amini, Helmut Gnaegi, Matthijn Vos, Bennie Bel, Freek Adolfsen, José L. Carrascosa and Peter J. Peters.
Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box.
Journal of Structural Biology 169, 219-225, (2010)

BOOKS

Joaquin Oton, Carlos Oscar Sanchez Sorzano, Francisco Javier Chichón, José L Carrascosa, Jose M Carazo, Roberto Marabini
Soft X-ray Tomography Imaging for Biological Samples. Computational Methods for Three-Dimensional Microscopy Reconstruction, Chapter: 8, pp.260
Herman, Gabor T., Frank, Joachim, ed.
Springer New York Heidelberg Dordrecht London
ISBN 978-1-4614-9520-8

 

Ana Cuervo, María I. Daudén and José L. Carrascosa.
Nucleic Acid Packaging in Viruses.
Structure and Physics of Viruses. Series: Subcellular Biochemistry, Volumen: 68, 361-394, 2013
Mauricio G. Mateu, ed.
Dordrecht, Paises Bajos.
ISBN: 978-94-007-6551-1

 

José R. Castón and José L. Carrascosa.
The Basic Architecture of Viruses.
Structure and Physics of Viruses. Series: Subcellular Biochemistry, Volumen: 68, 53-75, 2013
Mauricio G. Mateu, ed.
Dordrecht, Paises Bajos.
ISBN: 978-94-007-6551-1

 

J.J. Fernández, J.I. Agulleiro, J.R. Bilbao-Castro, A. Martínez, I. García, F.J. Chichón, J. Martín-Benito, José L. Carrascosa.
Image processing in electron tomography.
Microscopy: Science, Technology, Applications and Education.
A.Méndez-Vilas and J.Díaz, eds
Vol. 1, pp: 19-28, 2010. Editorial: Formatex.
ISBN: 978-84-614-6189-9

 

José R. Castón, José F. Rodríguez and José L.Carrascosa.
Infectious Bursal Disease Virus (IBDV): A Segmented Double-Stranded RNA Virus With a T=13 Capsid That Lacks a T=1 Core.
Segmented Double-stranded RNA Viruses: Structure and Molecuar Biology, 133-144, 2008
J.T. Patton, eds. Caister Academic Press. Norfolk. UK.
ISBN: 978-1-904455-21-9

 

Funding

 

2015-2017.
Convocatoria 2014 Proyectos I+D - Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia
BFU2014-54181-P
Correlación entra la estructura, ensamblaje y propiedades de la máquinas virales involucradas en procesos clave del ciclo viral.
José L. Carrascosa.

2014-2018.
Nuevas fronteras del nanomagnetismo fundamental y aplicado.
S2013/MIT-2850 Convocatoria I+D(2+2) grupos de investigación de la Comunidad de Madrid.
José L. Carrascosa

2014-2017.
Retos Colaboración RTC-2014-1615-1
Desarrollo de una vacuna terapéutica frente al cáncer de cervix basada en la proteína de fusión entre el dominio extra a de la fibronectina y el antígeno E7 del HPV.
José L. Carrascosa.

2013-2017 
EXTREMA: Enhanced X-ray Tomographic Reconstruction: Experiment, Modeling and Algorithms. 
Acción COST MP1207
José L. Carrascosa. National coordinator

2011- 2014 
Financiación de la actividad del Comité Español de la IUPAB. 
ACI-COM-2011-1085. Subprograma de Fomento de la Cooperación Científica Internacional, Modalidad ACI-Comités.  CSIC
José L. Carrascosa

 

 

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