Compra de Maquinaria y Equipo

In order to obtain more detailed structural information about TatE complexes, the gel filtration-purified TatE complexes were analyzed by

negatively stained samples (

Electron microscopy micrographs of TatE particles were analyzed particle image processing techniques and

obtained using a random conical tilt strategy shows class averages which revealed

size classes. The complexes

diameter of ~ 8 nm and a dark central area indicating

Chapter 3. Purification and characterization of E. coli

PAGE of TatE and TatABC complexes

embranes were prepared from E. coli ΔtatAE cells expressing TatE or cells expressing TatABC proteins in which a Strep-II

terminus of TatE or TatC, respectively. The membranes were solubilised in digitonin and subjected to BN-PAGE. The gel was immunoblotted

Mobility’s of TatE and TatABC complexes are indicated on the Molecular mass markers are indicated on the left.

Electron microscopy of TatE complexes

more detailed structural information about TatE complexes, the gel purified TatE complexes were analyzed by electron microscop

negatively stained samples (carried out by Daniel Beck, University of

Electron microscopy micrographs of TatE particles were analyzed particle image processing techniques and a three-dimensional

obtained using a random conical tilt strategy. Figure 3.2.9 (courtesy of Daniel Beck) class averages which revealed that the TatE particles can be

complexes formed small ring-shaped structures with a maximum nm and a dark central area indicating the possibility of a pore or well

. coli TatE complexes

cells expressing TatE or E. coli IITMtag was present terminus of TatE or TatC, respectively. The membranes were solubilised in The gel was immunoblotted with antibodies to s are indicated on the

more detailed structural information about TatE complexes, the gel electron microscopy (EM) of , University of Warwick).

Electron microscopy micrographs of TatE particles were analyzed using single- reconstruction was (courtesy of Daniel Beck) can be separated in two shaped structures with a maximum the possibility of a pore or well.

Chapter

Figure 3.2.9 Micrographs of class averages of TatE complexes

Aligned class averages of the untilted particles and centered tilted particles shown. Class 1 consists

nm. (Image provided by Daniel Beck)

Figure 3.2.10 (courtesy

density map generated for both classes

was ~ 29 Å. As indicated by the original images,

structures. The dark central area observed in the micrographs was revealed to result from a central cleft or well within the ring, occluded on one side.

three-dimensional volume obtained

TatA complexes but much smaller in diameter (6 nm in the case of TatA

both three-dimensional volumes

Chapter 3. Purification and characterization of E. coli

Micrographs of class averages of TatE complexes

Aligned class averages of the untilted particles and centered tilted particles shown. Class 1 consists of 226 particles and class 2 contains 288. The scale bar is 10

vided by Daniel Beck)

courtesy of Daniel Beck) shows the three-dimensional electron generated for both classes. The estimated resolution of the

As indicated by the original images, these maps

structures. The dark central area observed in the micrographs was revealed to result from a central cleft or well within the ring, occluded on one side.

dimensional volume obtained was similar to previous structures

TatA complexes but much smaller in diameter (6–8 nm when compared with 9 m in the case of TatA (Gohlkeet al., 2005)). The diameter of the observed cleft for

dimensional volumes obtained was ~ 2.5 nm.

. coli TatE complexes

Aligned class averages of the untilted particles and centered tilted particles are of 226 particles and class 2 contains 288. The scale bar is 10

dimensional electron The estimated resolution of the density map these maps show ring-shaped structures. The dark central area observed in the micrographs was revealed to result from a central cleft or well within the ring, occluded on one side. The shape of the similar to previous structures shown for nm when compared with 9–13 diameter of the observed cleft for

Chapter

Figure 3.2.10 Three-

The small and large TatE

σ (S.D. above mean density). Dimensions shown from top to bottom diameter, complex height, and pore width. Molecular masses

on a protein density of 0.844 Da/Å

UCSF Chimera (Image provided by Daniel Beck

In addition to the single

contained multiple rings and appeared as poten ringed structures. Figure 3.2.11

the multi-ringed TatE complex structures. across a range of samples and grids and appeared the particles (~ 20% of the initial particle set

two rings with a small number showing three potential rings. 3.2.11, the class average of the most homogeneous set of structures consisted in a complex of 6 by 10.5 nm.

low resolution three-dimensional model of this class, contoured to the dimensions of Chapter 3. Purification and characterization of E. coli

-dimensional density maps of TatE complex assemblies

The small and large TatE complexes are shown filtered to 29 Å and contoured (S.D. above mean density). Dimensions shown from top to bottom

diameter, complex height, and pore width. Molecular masses were estimated based on a protein density of 0.844 Da/Å3. The figure was produced using the program

(Image provided by Daniel Beck).

to the single-ring structures described above, a number of particles contained multiple rings and appeared as potential dimers or trimers of the single ringed structures. Figure 3.2.11 (courtesy of Daniel Beck) shows

TatE complex structures. These larger structures were observed across a range of samples and grids and appeared to represent a significant subset of particles (~ 20% of the initial particle set). The majority of this set consisted of s with a small number showing three potential rings. As shown in Figure the class average of the most homogeneous set of particles

consisted in a complex of 6 by 10.5 nm. Crude size estimations based on a dimensional model of this class, contoured to the dimensions of . coli TatE complexes

dimensional density maps of TatE complex assemblies

complexes are shown filtered to 29 Å and contoured at ~ 5 (S.D. above mean density). Dimensions shown from top to bottom are: complex were estimated based produced using the program

number of particles tial dimers or trimers of the single- shows class averages of These larger structures were observed to represent a significant subset of ). The majority of this set consisted of As shown in Figure particles with two-ringed Crude size estimations based on a dimensional model of this class, contoured to the dimensions of

Chapter

the two-dimensional average, g with the uppermost band seen using twice the size of the single

The existence of multiringed forms of TatE may be relevant for the mechanism pore formation because they show that the purified TatE

associating to form larger structures structures still showed

Figure 3.2.11 Micrographs of

The most homogeneous particles

panel) and potential side view (Side panel by Daniel Beck).