Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

DNA complexed with RECA protein

1992 ◽  
Vol 50 (1) ◽  
pp. 448-449
Author(s):  
Edward H. Egelman ◽  
Xiong Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Recent Work ◽  
Conformational Changes ◽  
Time Scale ◽  
Reca Protein ◽  
Structural Transitions ◽  
E Coli ◽  
Helical Polymer ◽  
Lines Of Evidence

We have been using electron microscopy and computed image analysis to understand the structure of the helical polymer that the RecA protein from E. coli forms on DNA. Recent work has adressed the following points:1) RecA binds an ATP analog, ATP-γ-S, and hydrolyzes this analog several thousand times more slowly than ATP is hydrolyzed by RecA. We have shown that structural transitions may be seen within RecA filaments on the same time scale (several hours) on which ATP-γ-S is being hydrolyzed, and several lines of evidence suggest that these conformational changes are due to the RecA ATPase. We have therefore been able to directly visualize these motions, using image analysis of bundles of RecA filaments.

RecA-DNA complexes: What we can learn about structure and dynamics from disordered filaments using computed image analysis

1986 ◽  
Vol 44 ◽  
pp. 144-145
Author(s):  
E.H. Egelman
Keyword(s):  
Image Analysis ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Strand Transfer ◽  
Homologous Pairing ◽  
Dna Complexes ◽  
Helical Polymers ◽  
E Coli ◽  
Structure And Dynamics ◽  
Static Images

The recA protein (38,000MW) of E. coli forms helical polymers which are able, in an ATP-dependent reaction, to mediate the entire genetic recombination process, including the search for homology, homologous pairing, and strand transfer. We have been using computed image analysis of electron micrographs of different recA complexes in an effort to understand the function of this protein in recombination. These filaments typically show poor helical order. We have studied the systematic deviations from helical order (the disorder) present in static images of recA complexes as a means of understanding the dynamics of recA filaments in solution.

scholarly journals Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

2012 ◽  
Vol 11 (11) ◽  
pp. 1430-1441 ◽  
Author(s):  
Esther van Duijn ◽  
Ioana M. Barbu ◽  
Arjan Barendregt ◽  
Matthijs M. Jore ◽  
Blake Wiedenheft ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Acquired Resistance ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Ribonucleoprotein Complexes ◽  
Specific Association

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

1998 ◽  
Vol 64 (2) ◽  
pp. 688-694 ◽  
Author(s):  
M. Loferer-Krößbacher ◽  
J. Klima ◽  
R. Psenner
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Image Analysis ◽  
Bacterial Biomass ◽  
Dry Weight ◽  
Dry Mass ◽  
Bacterial Cells ◽  
E Coli ◽  
Transmission Electron ◽  
Bacterial Assemblages

ABSTRACT We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW ofEscherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V 0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm3(n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm3) to 1,177 fg (V = 3.5 μm3). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to Vof individual cells varied from 466 fg of C μm−3 forVs of 0.001 to 0.01 μm3 to 397 fg of C μm−3 (0.01 to 0.1 μm3) and 288 fg of C μm−3 (0.1 to 1 μm3). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm−3 (0.1 to 1 μm3) and 211 fg of C μm−3 (1 to 4 μm3), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.

Structural analysis of the E. coli RUVB branch migration protein by EM and image analysis

1994 ◽  
Vol 52 ◽  
pp. 336-337
Author(s):  
X. Yu ◽  
K. Benson ◽  
A. Stasiak ◽  
I. Tsaneva ◽  
S. West ◽  
...  
Keyword(s):  
Image Analysis ◽  
Atp Hydrolysis ◽  
Genetic Recombination ◽  
Sos Response ◽  
Structure And Function ◽  
Holliday Junctions ◽  
Branch Migration ◽  
E Coli ◽  
Form Part ◽  
And Function

We have been interested in the structure and function of proteins involved in genetic recombinaton. The ruv locus on the E. coli chromosome contains three genes (ruvA, ruvB and ruvC) that are important for genetic recombination and DNA repair. The ruvA and ruvB genes form part of the SOS response to DNA damage and encode the RuvA and RuvB proteins. Together, RuvA and RuvB promote the branch migration of Holliday junctions in a reaction that requires ATP hydrolysis. Each protein plays a defined role, with RuvA responsible for DNA binding (and, in particular, junction recognition), whereas the RuvB ATPase provides the motor for branch migration. Sequence analysis has identified RuvB as a member of a superfamily of helicases, and experimentally it has been shown that RuvB, in the presence of RuvA, acts as an ATP-dependent helicase.When purified RuvB protein was incubated (in the presence of the ATP analog, ATP-γ-S) with covalently closed, relaxed dsDNA, double-ringed structures were observed on the DNA in the electron microscope (Fig. 1). The DNA must be passing through the center of these rings, since the rings are always aligned along a common axis.

scholarly journals Bacterial RecA Protein Promotes Adenoviral Recombination duringIn VitroInfection

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Jeong Yoon Lee ◽  
Ji Sun Lee ◽  
Emma C. Materne ◽  
Rahul Rajala ◽  
Ashrafali M. Ismail ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Human Adenovirus ◽  
Penton Base ◽  
Content Type ◽  
E Coli ◽  
Recombination Hot Spots ◽  
Loop 2

ABSTRACTAdenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of anEscherichia colilysate increased recombination; this was blocked in a RecA mutant strain,E. coliDH5α, or upon RecA depletion. Recombination increased in the presence ofE. colilysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiADsequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCEAdenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-Type proteins and proteasomes

1990 ◽  
Vol 103 (3) ◽  
pp. 197-203 ◽  
Author(s):  
Peter Zwickl ◽  
Günter Pfeifer ◽  
Friedrich Lottspeich ◽  
Friedrich Kopp ◽  
Burkhardt Dahlmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Protein Complexes ◽  
Large Protein

From Static Images To Dynamic Structures: The Use of Image Analysis and Video In Deriving Dynamical Information From Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 526-527
Author(s):  
Edward H. Egelman
Keyword(s):  
Image Analysis ◽  
Reca Protein ◽  
Helical Structures ◽  
Electron Microscopic ◽  
General Applicability ◽  
E Coli ◽  
Static Images ◽  
Helical Protein ◽  
Dynamic Structures ◽  
Dynamical Information

Advances in computer graphics and numerical processing, in video technology, and in image acquisition have enabled us to extend the power of the electron microscope in the analysis of macromolecular structures, particularly helical protein polymers. Three applications of this technology will be described:1) There are frequently times where the static images acquired from fixed, stained or frozen specimens leads to a loss of information about the dynamical properties of the molecules or structures being studied. We have been using computed image analysis, graphics and animation to recover the dynamical information that can be obtained from electron microscopic images.Using the RecA protein of E. coli , we have been able to capture different biochemical states as a function of time through the use of a slowly hydrolyzable ATP analog, ATP-γ-S. Threedimensional reconstruction of these helical structures, combined with computer-generated animation between different structures, have enabled us to directly visualize the motions within the protein polymer associated with the hydrolysis of the nucleotide analog. Modifications of the RecA protein, achieved through either proteolysis or mutation, have allowed us to use the same techniques to visualize domain-domain movements within the RecA filament which occur over a range of 5-10Å. The methods of analysis, graphics and animation which have been used will be discussed. The general applicability of these procedures to other systems will also be addressed.

An Ultrastructural Study of Chronic Pyelonephritis in Macaca Arctoides

1976 ◽  
Vol 34 ◽  
pp. 310-311
Author(s):  
T.W. Smith ◽  
J.A. Roberts ◽  
B.J. Martin
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ultrastructural Study ◽  
Effective Means ◽  
Chronic Pyelonephritis ◽  
Macaca Arctoides ◽  
Left Ureter ◽  
E Coli ◽  
Transmission Electron ◽  
Sizeable Number

Chronic pyelonephritis is one of the most common diseases of the kidney and accounts for a sizeable number of cases of renal insufficiency in man, however its pathogenesis requires further elucidation. Transmission electron microscopy may serve as a uniquely effective means of observing details of the nature of this disease. The present paper describes preliminary results of an ultrastructural study of chronic pyelonephritis in Macaca arctoides (stumptail monkey).The infection was induced in these experiments in a retrograde fashion by means of a unilateral catheterization of the left ureter whereby an innoculum of 10 cc of broth containing approximately 2 billion E. coli per cc and radio-opaque dye were injected under pressure (mimicing vesico-ureteric reflux).

Replication of Bacteriophage 186 DNA

1972 ◽  
Vol 30 ◽  
pp. 194-195
Author(s):  
Dhruba K. Chattoraj ◽  
Ross B. Inman
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Frame Of Reference ◽  
Dna Molecules ◽  
E Coli ◽  
Phage Dna ◽  
Partial Denaturation

Electron microscopy of replicating intermediates has been quite useful in understanding the mechanism of DNA replication in DNA molecules of bacteriophage, mitochondria and plasmids. The use of partial denaturation mapping has made the tool more powerful by providing a frame of reference by which the position of the replicating forks in bacteriophage DNA can be determined on the circular replicating molecules. This provided an easy means to find the origin and direction of replication in λ and P2 phage DNA molecules. DNA of temperate E. coli phage 186 was found to have an unique denaturation map and encouraged us to look into its mode of replication.

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