S Ludtke

The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT

TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

Visualizing virus assembly intermediates inside marine cyanobacteria

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Structure of the voltage-gated L-type Ca2+ channel by electron cryomicroscopy.

Voltage-dependent L-type Ca2+ channels play important functional roles in many excitable cells. We present a three-dimensional structure of an L-type Ca2+ channel. Electron cryomicroscopy in conjunction with single-particle processing was used to determine a 30-Å resolution structure of the channel protein. The asymmetrical channel structure consists of two major regions: a heart-shaped region connected at its widest end with a handle-shaped region. A molecular model is proposed for the arrangement of this skeletal muscle L-type Ca2+ channel structure with respect to the sarcoplasmic reticulum Ca2+-release channel, the physical partner of the L-type channel for signal transduction during the excitation-contraction coupling in muscle.

EMAN2.1 - A New Generation of Software for Validated Single Particle Analysis and Single Particle Tomography

1. Graduate Program in Structural and Computational Biology & Molecular Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston TX 77025 2. National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza Houston TX 77025 3. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston TX 77025

Visualizing virus assembly intermediates inside marine cyanobacteria by zernike phase contrast electron cryo-tomography

1. National Center for Macromolecular Imaging, Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. 2. Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, USA. 3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 4. Department of Biology, Northeastern University, Boston, MA, USA. 5. National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Japan † Present address: FEI, 5350 Dawson Creek Drive, Hillsboro, OR, USA

3-D TEM Reconstructions with EMAN2

Transmission Electron Microscopy (TEM), in both negative stain and cryo, is a powerful technique for determining 3-D structural information about biological molecules/ macromolecules. EMAN was originally developed a decade ago to ease the task of performing high resolution single particle reconstructions, by automating portions of the reconstruction process which were considered robust and providing an easy to use graphical user interface (GUI) for tasks not considered ready for automation[1]. The techniques used for reconstruction were a hybrid of some methods not then in common use, such as direct Fourier inversion for reconstruction and full amplitude and phase CTF correction, as well as other established techniques pioneered in earlier software packages such as SPIDER[2] and IMAGIC[3]. EMAN evolved to offer additional techniques for 2-D analysis, 2-D and 3-D population dynamics studies, and various post-processing operations such as secondary structure localization, skeletonization and crystal-structure docking. The resolution capabilities of single particle reconstruction have advanced rapidly over the last decade. In the last year, several single particle reconstructions have been solved at ~4 Å resolution, and subnanometer resolutions have been achieved in numerous labs around the world.