Biomedical Engineering Reference
In-Depth Information
R
: coding sequences for two sticky tags are added, separated by a sequence coding
for a protease cleavage site. Upon introducing this engineered gene into a host cell,
the modified protein gets expressed and takes its place in its usual complexes—
assuming that there is no hindrance induced by the tags themselves. On lysing the
cell, the protein complexes containing protein
R
are retrieved thanks to two affinity
purification steps. Each purification step consists of capturing the complexes on an
affinity purification column thanks to one of the sticky tags. Between the first and
the second purification steps, the complexes hooked on the first column are released
thanks to addition of a protease which cuts the linker containing the first sticky
tag at the level of the cleavage site. This reveals the remaining sticky tag for the
second purification step. Upon completing these purification steps and dismantling
the complexes during electrophoresis, one obtains a gel with one band per protein
type. Mass spectrometry is then used to identify the protein types present.
This list of protein types obtained, also called a pullout, calls for two comments.
First, one does not know whether the list of interacting types corresponds a single
complex or to several complexes. For example, a list (
R,S,T
) obtained by tagging
R
may correspond to a single complex containing the three species, or to two
binary complexes respectively involving (
R,S
) and (
R,T
). Second, no information
on the stoichiometry of protein instances within a complex is available. Despite
these inherent combinatorial ambiguities, TAP data are of prime interest for the
reconstruction of large assemblies: knowing that protein instances participate in a
complex imposes distance restraints between them.
1.3.1.3
Reconstruction by Data Integration: Procedure and limitations
To deal with the ambiguities just discussed, the authors of [
4
] proposed a recon-
struction strategy based on three ingredients:
A model
M
for the protein instances of the NPC consisting of balls. Note that
a model involving
n
balls with prescribed radii defines a 3
n
dimensional space
corresponding to the
xyz
coordinates of all centers.
A scoring function measuring the coherence between a particular model and the
experimental data.
An optimization procedure aiming at finding the models which best comply with
the data.
Scoring function.
Consider a particular type of experimental data, such as those
described in Sect.
1.3.1.1
. In short, a restraint is a real-valued function measuring
the discrepancy between the model
M
and these data: a quadratic penalty is applied
the farther the model is from the data; if the model complies with the data, the
restraint is zero. Let us consider the following three examples:
•
A cryoEM envelope can be used to make sure that the model
M
does not protrude
from this envelope. That is, if the one-sided Hausdorff distance between the
model and the envelope is beyond some threshold, a penalty is applied.
Search WWH ::
Custom Search