A view of the three dimensional structure of globular proteins

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

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Three Dimensional structure and organization of diploid chromosomes by optical section microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127608 ◽

1987 ◽

Vol 45 ◽

pp. 633-635

Author(s):

David A. Agard ◽

Yasushi Hiraoka ◽

John W. Sedat

Keyword(s):

Dimensional Space ◽

Three Dimensional ◽

Developmental State ◽

Mitotic Cell ◽

Biological Properties ◽

Dimensional Structure ◽

Specific Gene ◽

Three Dimensional Structure ◽

Complementary Techniques ◽

Dynamic Structures

In an effort to understand the complex relationship between structure and biological function within the nucleus, we have embarked on a program to examine the three-dimensional structure and organization of Drosophila melanogaster embryonic chromosomes. Our overall goal is to determine how DNA and proteins are organized into complex and highly dynamic structures (chromosomes) and how these chromosomes are arranged in three dimensional space within the cell nucleus. Futher, we hope to be able to correlate structual data with such fundamental biological properties as stage in the mitotic cell cycle, developmental state and transcription at specific gene loci.Towards this end, we have been developing methodologies for the three-dimensional analysis of non-crystalline biological specimens using optical and electron microscopy. We feel that the combination of these two complementary techniques allows an unprecedented look at the structural organization of cellular components ranging in size from 100A to 100 microns.

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Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180161 ◽

1990 ◽

Vol 48 (1) ◽

pp. 282-283

Author(s):

José L. Carrascosa ◽

José M. Valpuesta ◽

Hisao Fujisawa

Keyword(s):

Dna Sequences ◽

Expression Vector ◽

Three Dimensional ◽

Deae Cellulose ◽

Dna Packaging ◽

Dimensional Structure ◽

Two Dimensional ◽

Freezing And Thawing ◽

Three Dimensional Structure ◽

Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

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