Plates-Formes de Recherche en Neurosciences

logo amu logo cnrs

Plates-formes PFRN

Accueil > Agenda > Les séminaires Jean Roche > Tomographie cellulaire et moléculaire.

Tomographie cellulaire et moléculaire.

Lundi 20 juin 2005, 11h, salle Lissitzky.


1 : Biochemistry (Mosc). 2004 Nov ;69(11):1219-25.

Electron tomography of biological samples.

Marco S, Boudier T, Messaoudi C, Rigaud JL.

Institut Curie, Section Recherche, UMR-CNRS 168 et LRC-CEA 34V 11, 75005 Paris, France. sergio.marco

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.

2 : Ultramicroscopy. 2004 Nov ;101(2-4):153-9.

Thermal AFM : a thermopile case study.

Fonseca L, Perez-Murano F, Calaza C, Rubio R, Santander J, Figueras E, Gracia I, Cane C, Moreno M, Marco S.

Centro Nacional de Microelectronica (IMB-CSIC) Campus Universidad Autonoma de Barcelona, 08193 Bellaterra, Spain. luis.fonseca

In this work, an atomic force microscope (AFM) with an integrated thermal sensor has been used to obtain the local spatial distribution of temperatures in a micromachined thermopile with submicron resolution. In this communication, we will show how the dimensional, structural and functional characteristics of a thermopile suits well with the requirements for AFM thermal imaging, and how a deeper insight of the thermopile operation can be gained with the aid of these advanced scanning probe-based tools.

3 : Biol Cell. 2003 Sep ;95(6):393-8.

Use of cryo-negative staining in tomographic reconstruction of biological objects : application to T4 bacteriophage.

Messaoudi C, Boudier T, Lechaire JP, Rigaud JL, Delacroix H, Gaill F, Marco S.

Institut Curie section recherche, UMR-CNRS 168 et LRC-CEA 34V. 11, rue Pierre et Marie Curie, 75231 Paris 05, France.

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks : the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.

4 : J Struct Biol. 2003 Jul ;143(1):33-44.

Revisiting the structure of Alvinella pompejana hemoglobin at 20A resolution by cryoelectron microscopy.

Jouan L, Marco S, Taveau JC.

Laboratoire des Proteines Complexes, J. E. 2320, Universite de Tours. 2, bis Boulevard Tonnelle, F-37032 Tours Cedex, France.

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs.

5 : Proc Natl Acad Sci U S A. 2003 Feb 18 ;100(4):1690-3. Epub 2003 Feb 6.

Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy.

Scheuring S, Seguin J, Marco S, Levy D, Robert B, Rigaud JL.

Institut Curie, Unite Mixte de Recherche-Centre National de la Recherche Scientifique 168 and Laboratoire de Recherche Correspondant-Commissariat a l’Energie Atomique 34V, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. simon.scheuring

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-A lateral and approximately 1-A vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.

6 : J Mol Biol. 2003 Jan 17 ;325(3):569-80.

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2).

Scheuring S, Seguin J, Marco S, Levy D, Breyton C, Robert B, Rigaud JL.

Institut Curie, UMR-CNRS 168 and LRC-CEA 34V, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. simon.scheuring

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A. Copyright 2003 Elsevier Science Ltd.

7 : J Mol Biol. 2002 Feb 1 ;315(5):1075-85.

Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis.

Chami M, Steinfels E, Orelle C, Jault JM, Di Pietro A, Rigaud JL, Marco S.

Institut Curie Section Recherche, UMR-CNRS 168 et LRC-CEA 8, 11 Rue Pierre et Marie Curie, Paris Cedex 05, 75231, France.

YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. >From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains : a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers. Copyright 2002 Elsevier Science Ltd.

    Ils nous font confiance

  • logo amu
  • logo cnrs
  • logo inserm
  • logo AP-HM
  • logo F�d�ration pour la Recherche sur le Cerveau
  • logo Fondation pour la Recherche Medical en France
  • logo IBiSA
  • logo Europe programme FEDER
  • logo Agence Nationale de la Recherche
  • logo Plateforme Technologique Aix-Marseille
  • logo Vect-Horus
  • logo Neuron Experts