text:
Alternate pathways for folding in the flavodoxin fold family revealed by a nucleation-growth model.
ABSTRACT A recent study of experimental results for flavodoxin-like folds suggests that proteins from this family may exhibit a similar, signature pattern of folding intermediates. We study the folding landscapes of three proteins from the flavodoxin family (CheY, apoflavodoxin, and cutinase) using a simple nucleation and growth model that accurately describes both experimental and simulation results for the transition state structure, and the structure of on-pathway and misfolded intermediates for CheY. Although the landscape features of these proteins agree in basic ways with the results of the study, the simulations exhibit a range of folding behaviours consistent with two alternate folding routes corresponding to nucleation and growth from either side of the central beta-strand.
Article: The fuzzy oil drop model, based on hydrophobicity density distribution, generalizes the influence of water environment on protein structure and function.[Show abstract] [Hide abstract]
ABSTRACT: In this paper we show that the fuzzy oil drop model represents a general framework for describing the generation of hydrophobic cores in proteins and thus provides insight into the influence of the water environment upon protein structure and stability. The model has been successfully applied in the study of a wide range of proteins, however this paper focuses specifically on domains representing immunoglobulin-like folds. Here we provide evidence that immunoglobulin-like domains, despite being structurally similar, differ with respect to their participation in the generation of hydrophobic core. It is shown that β-structural fragments in β-barrels participate in hydrophobic core formation in a highly differentiated manner. Quantitatively measured participation in core formation helps explain the variable stability of proteins and is shown to be related to their biological properties. This also includes the known tendency of immunoglobulin domains to form amyloids, as shown using transthyretin to reveal the clear relation between amyloidogenic properties and structural characteristics based on the fuzzy oil drop model.Journal of Theoretical Biology 05/2014; · 2.35 Impact Factor
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ABSTRACT: Proteins are the molecular machines of the cell that fold into specific three-dimensional structures to fulfill their functions. To improve our understanding of how the structure and function of proteins arises, it is crucial to understand how evolution has generated the structural diversity we observe today. Classically, proteins that adopt different folds are considered to be nonhomologous. However, using state-of-the-art tools for homology detection, we found evidence of homology between proteins of two ancient and highly populated protein folds, the (βα)8-barrel and the flavodoxin-like fold. We detected a family of sequences that show intermediate features between both folds and determined what is to our knowledge the first representative crystal structure of one of its members, giving new insights into the evolutionary link of two of the earliest folds. Our findings contribute to an emergent vision where protein superfolds share common ancestry and encourage further approaches to complete the mapping of structure space onto sequence space.Nature Chemical Biology 07/2014; · 12.95 Impact Factor
Article: Topological frustration in beta alpha-repeat proteins: sequence diversity modulates the conserved folding mechanisms of alpha/beta/alpha sandwich proteins.[Show abstract] [Hide abstract]
ABSTRACT: The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of nonlocal interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of beta alpha-repeat proteins, which are dominated by local interactions, we performed a combined experimental and simulation analysis on two members of the flavodoxin-like, alpha/beta/alpha sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured submillisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine, and valine (ILV) side chains that form a large sequence-local cluster involving the central beta-sheet and helices alpha2, alpha 3, and alpha 4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely folded unproductive intermediates are a common property of the beta alpha-repeat motif.Journal of Molecular Biology 03/2010; 398(2):332-50. · 3.91 Impact Factor
Page 1
Alternate Pathways for Folding in the Flavodoxin Fold
Family Revealed by a Nucleation-growth Model
Erik D. Nelson* and Nick V. Grishin
Howard Hughes Medical
Institute, University of Texas
Southwestern Medical Center
6001 Forest Park Blvd., Room
ND10.124, Dallas, TX
A recent study of experimental results for flavodoxin-like folds suggests
that proteins from this family may exhibit a similar, signature pattern of
folding intermediates. We study the folding landscapes of three proteins
from the flavodoxin family (CheY, apoflavodoxin, and cutinase) using a
simple nucleation and growth model that accurately describes both
experimental and simulation results for the transition state structure, and
the structure of on-pathway and misfolded intermediates for CheY.
Although the landscape features of these proteins agree in basic ways
with the results of the study, the simulations exhibit a range of folding
behaviours consistent with two alternate folding routes corresponding to
nucleation and growth from either side of the central b-strand.
q 2006 Published by Elsevier Ltd.
Keywords: fold families; equilibrium intermediates; non-native interactions
*Corresponding author
From a folding perspective, the topology of a
protein is interpreted by the shape of its native
backbone which loosely determines the pattern of
atom-to-atom cross-links between its amino acid
residues. Over the past several years, simple
theoretical and computational models based essen-
tially on topology and minimal entropy loss1–3have
demonstrated that native topology is a "first order"
effect deciding the way a protein folds.4–12While
the data so far still provide a very incomplete
picture, it suggests that if we could provide any
consistent description of protein folding it would be
that evolutionary changes which, roughly speaking,
conserve topology13–15and act as perturbations
affecting mainly the depths of intermediates and the
heights offree energy barriers on a protein's folding
landscape rather than the basic mechanism16–18that
allows it to fold.
However, among these results have now
appeared a growing number of excursions away
from axiomatic correspondence between folding
and topology that must somehow find a place
within this picture.19–24For example, the small
proteins L and G share an almost identical,
symmetric topology, but both proteins nucleate
one of their two b-sheets preferentially, breaking
the symmetry of the native fold.20,21The small,
all-helical proteins Im7 and Im9 share essentially
the same topology, but Im7 folds through an on-
pathway intermediate in which a distorted arrange-
ment of its helices is stabilised by non-native
interactions.22,23Perhaps, it is not so surprising
that the folding mechanisms of these proteins are
varied. Their native shapes are not frustrated
mechanically7so they should have greater freedom
to respond to structural and energetic pertur-
bations, and their responses (the modulation of
intermediates and pathways by these pertur-
bations) may even be somewhat continuous.
On the other hand, even small perturbations
such as amino acid substitutions can sometimes
cause discrete interconversions of protein struc-
ture within a fold family (for instance, changing
b-strands to b-helices24,25). Moreover, the struc-
tural family of a protein (its fold type or fold
classification) often allows large loop insertions,
sometimes within secondary structure units, and
the substitution of one secondary structure type
for another, all of which can affect the entropy of
its folding units, the pattern of native contacts
between them, and the capacity of these units to
evolve more favourable contacts. Accordingly,
this more flexible interpretation of topology
(fold type) should permit more substantial
variations to occur among protein folding mech-
The landscape features that define the folding
pathways of larger proteins (w200 amino acid
residues) are more discrete, and should have more
capacity to accommodate perturbations. These
0022-2836/$ - see front matter q 2006 Published by Elsevier Ltd.
E-mail address of the corresponding author:
doi:10.1016/j.jmb.2006.02.026J. Mol. Biol. (2006) xx, 1–8
Page 2
features still appear to be guided by native
topology,5,26however, given the larger and less
predictable variations in structure that can be
admitted into the fold families of larger proteins, a
manifestly pathway-like protein could conceal, in
an evolutionary sense, alternate folding routes due
to multiple folding units that are responsive to
preferential stabilization by a suitable accumulation
of these perturbations. Therefore, as with proteins L
and G, a purely structural classification of protein
families can permit substantial variations among
the folding routes of a given fold type, but for larger
proteins this may start to define "discrete spectra"
of mechanical differences, or "modes" for folding
within a family.
If multiple routes do exist for a particular fold
type, when does nature choose from among them,
and when does it admit mixtures of the routes?
These types of problems are just now beginning to
be explored,19,27and they are of interest not simply
in terms of the physics of how proteins fold but
because they may provide information about low
lying conformational sub-states that decide how
proteins function. Because of the complexities
involved in obtaining this information experimen-
tally, simple, computationally efficient folding
models, such as those recently used to describe
protein transition state structures28–37could be very
useful to infer folding properties and thus direct the
process of these measurements more effectively.
Here, we use one of these models for a detailed
exploration of CheY and two other large proteins
from the flavodoxin fold family.27
The model is one of an extremely simple type in
which amino acid residues are allowed to exist in
just two states, either folded (frozen) or unfolded (a
discussionofthemodelisgivenintheAppendix). Its
energetics are heterogeneous and Go ¯-like, the
interaction between any two amino acid residues
being proportional to the number of atom-to-atom
contacts that would exist between them in the
native crystal structure of the protein. Each
collective state of the amino acid residues is
intended to represent a small micro-ensemble
consisting of the conformational states of unfolded
segments constrained by the frozen amino acid
residues and the cross-links that form between
them. The entropy of the micro-ensembles is
described using simple estimates from polymer
theory in which the unfolded segments are mod-
elled as random flight (gaussian) chains and only
the space occupied by frozen parts of the molecule
is excluded.
In current applications of this model,31–34the
micro-ensembles are limited to very simple objects
(for example, a nucleus or nuclei with two or fewer
loops) for the sake of simplifying the computations.
However, it is known that these approximations
begin to break down around ~ O100 amino acid
residues, precisely where the fine scale features of
folding start to matter less and where, due to its
speed, the model could be of most use. In a recent
paper,36we developed an approach to sample more
complex micro-ensemble topologies excluded in
previous work in order to investigate larger
proteins with multiple folding units. We found
that including these topologies often led to quali-
tative improvements in the calculation of transition
state structure, and that the dominantly occurring
micro-ensembles turned out to have a simple
scaling form (see the Appendix) for which an
explicit calculation of excluded volume effects38of
the type noted above would not be too forbidding.
Although we account for these effects in only an
order of magnitude sort of way, this approximation
seems to be enough to draw the kinds of
conclusions we need for this work.
The CheY topology studied here seems particu-
larly well suited to description by this model. The
transition state structure of CheY (3chy.pdb)
compares relatively well with available protein
engineering data39,40,43(correlation coefficient 0.62
or 0.94 if volume increasing mutations are
excluded) and the model detects the misfolded
and on-pathway intermediate states thought to
reflect topological frustration7,27between interior
(b-sheet) and exterior (a-helix) layers of the fold that
bridge two weakly interpenetrating domains43on
either side of the central b3strand. The level of
agreement is surprising since the misfolded inter-
mediate4,39,40is thought to result from the dynami-
cal connection between these layers and lead to a
non-native distortion of the helices, yet we observe
the intermediate in a model without explicit
dynamical constraints and native-only interactions
(Figures 1 and 2). On-pathway the agreement is
surprising as well. In crossing the transition state,
CheY nucleates from its N-terminal domain and
growth is thought to proceed by strands of the
b-sheet which frustrates the accretion of a-helices
onto the exterior. Again, this is exactly what we
observe in our simulations. In rough agreement
with the experimental results of Lopez-Hernandez
& Serrano,40,43the nuclear region includes b1-a1-b2
and part of a2(we refer to regions on either side of
the central b3strand as domains A and B below).
The minima in the free energy profile (Figure 2)
register with the formation of b-strands and the
helices start to form just before the maxima so that
the conflict in stability between interior and exterior
regions of the fold is periodically resolved in
crossing the barriers. The unusual unfolding and
refolding features of helices a4and a5in Figure 1(a)
and the accentuation of the intermediate barrier
after b4 in Figure 2 may signify non-native
interactions in the actual folding path as we explain
later below.
The flavodoxin study of Bollen & van Mierlo27
suggests that proteins from the same fold family
(CheY, cutinase and anabaena apoflavodoxin in this
instance) may exhibit a similar pattern of on and off-
pathway intermediates. These proteins have
lengths ranging from 128 to 197 amino acid residues
and very low sequence identity, and protein
engineering results exist only for the smallest
member, CheY.Interestingly,both cutinase
Alternate Pathways for Flavodoxin Folding
Page 3
(1agy.pdb) and apoflavodoxin (1ftg.pdb) contain a
number offlexible loop insertions (in cutinase these
include a-helical fragments) at points where
a-helices would connect to b-strands in the B (C-
terminal) domain of CheY. These insertions could
suggested by these authors and allow for greater
stability of the B domain which could lead to
variations among flavodoxin fold pathways.
Our results for cutinase and apoflavodoxin do
share many of the same features described for CheY.
Like CheY, the key folding event is growth of the
nucleus uptoandacrossthe b3stranddividingthe A
to varying degrees, the signal of a misfolded
intermediate in which helices but not strands or
loops (except in the nucleus) are folded, and minima
of b-strands (a-helices) consistent with frustration
between the interior and exterior regions of the
protein. However, at least for apoflavodoxin, the
structural mechanism for folding is quite different.
The nucleus of apoflavodoxin is on the opposite side
(C-terminal, or B-side) of the b3strand, including
connecting loops (see Figures 3 and 4) and growth
proceeds toward the N-terminal end of the b-sheet.
This result is at first difficult to accept given the
simplicity of the model and the fact that part of the
protein (the N-terminal strand b1) is confined in its
However, we note here that the number of atom-to-
and apoflavodoxin are also weighted in opposite
directions (see Figure 4) and this effect, together with
the results of the simulations.
The atom-to-atom contact profile for cutinase,
similar to its folding landscape, could best be
pictured as intermediate to CheY and apoflavo-
doxin. As in CheY, the formation of strands tends to
line up with minima in the free energy profile but
now the helices are included more within the
minima. Although we do not present folding plots
for cutinase, it is useful to summarize the results.
First, the CheY helix a4is unstructured in cutinase
0 64
Figure 1. Projection of the folding landscape onto (a) a-helices and (b) b-strands for CheY. Pn(q) is the probablity that
sub-structure n (helix or strand 1–5) is folded when there are q frozen amino acid residues. The folding process is
stepwise, nucleated by domain A (b1-a1-b2) at qw38 and proceeding to accrete each section, an–bnC1, in order along the
interior b-sheet. After the protein is nucleated, the addition of each new helix (strand) leads to a maxima (minima) in the
free energy profile (Figure 2). The misfolded "helical" intermediate observed by Clemente and co-workers is detected
near qw24. Across this region, the strands and loops in domain B remain unfolded totally, the number of nuclei jumps
(the probability of four nuclei reaching about 0.1 at qZ24) and the distribution of nuclear sizes changes abruptly from
bimodal (distributed about 2 and q amino acid residues) to unimodal (distributed about two amino acid residues, the
segment size used in the simulations) to bimodal before reaching the transition state. (c) Ribbon diagram of the CheY
crystal structure. Light blue regions indicate amino acid residues with native contacts defined by Nelson & Grishin7and
Shea et al.10
Alternate Pathways for Flavodoxin Folding
Page 4
so its C-terminal helix gets indexed as a4. In the
unfolded wing of the cutinase free energy profile,
part of its domain B is folded, including helices a3
and a4, and strands b4and b5. Although a3remains
frozen into the folded wing of the profile, most of
the segments unfold near qZL/2 (L is the length of
the protein) and are "simultaneously" replaced by
domain A, a2, and b3before the reaction proceeds.
The folding plots have an all or none character that
suggests the exchange of B-like for A-like nuclei is
part of the folding pathway27even though the
molecule begins this process from a partially
misfolded state.
Aside from structural processes, the results above
appear roughly consistent with the experimental
data. The sizes of free energy barriers are compar-
able in scale to the results reported by Bollen & van
Mierlo, and although it is difficult to establish the
topography of the landscape near the misfolded
intermediate, the profiles seem as if they could be
classified in a similar way. For example, the CheY
kinetics were analysed with both on and off-
pathway models by the Serrano group to indicate
that they lead to the same results.40This is
consistent with the fact that the main transition
state can be reached by a partial exchange of helical
structure in domain B for nuclear structure in
domain A as is indicated by our own results.
However, in apoflavodoxin and cutinase domain B
folds first, so the exchange should be qualitatively
different, and this may explain why an off-pathway
kinetic model27could describe these two experi-
ments better.
Does this over-simplified model predict the basic
signature of the folding landscapes?
The model appears to be operating as intended.
(i) The transition state structure of the CheY
topology agrees well with experiment. (ii) Complex
diagrams (nested, inter-linked loop, etc.36) are very
infrequent in simulations for this fold type. (iii)
There are very few contacts between domain A and
domain B (after strand b3) so the nuclei in these two
regionsarefreetofoldinparallel (seetheAppendix).
Figure 2. Structural events along the free energy profile,
F(q), of CheY. The misfolded helical intermediate is
centered at qZ24 (u), and the nucleus, b1-a1-b2, is formed
at qZ38 (t). Each major basin in the profile corresponds to
the completion of a helix an(left side of basin), formation
of a strand bnC1(middle of basin), and partial formation
of the following helix anC1 (right side of basin). The
structure of the misfolded intermediate, and the registry
of helices with maxima in F(q) indicates topological
frustration between the b-interior and a-exterior of the
protein as suggested by Bollen & van Mierlo.27The depth
of minima (height of maxima) in this region reflect loop
closure events that are sensitive to the entropy approxi-
mations used in these types of models. The basic structure
of the profile is in agreement with that in Clementi et al.4
except for the placement of the transition state.
050100 150
050100 150
Figure 3. Projection of the folding landscape onto (a) a-helices and (b) b-strands for apoflavodoxin. The helix indices
follow the crystal structure data in which a2-a3corresponds to the CheY helix a2. The strand indices are the same in all
three of the flavodoxin proteins. The nucleus of apoflavodoxin includes part of the C-terminal helix a6, all of b5, most of a
large loop l6preceeding, or inserted into b5, a small part of helix a5and the loop preceeding it (see Figure 4). As the
transition state is crossed, the rest of a6forms, and folding continues to alternate from a to b moving from C to
N-terminal ends until the protein is folded. Again, there are two minima (basins) in folded wing of the free energy
profile, comparable in size to CheY, that register with the formation of bn-anK1layers. The signature of an intermediate
with helical structure is visible near qZ36.
Alternate Pathways for Flavodoxin Folding
Page 5
(iv) The patterns of atom-to-atom contacts are
consistent with the way each protein folds, and
although the entropy cost to freeze unfolded
segments of proteins depends on amino acid
composition,25it seems unlikely that including
this dependence could lead to something concerted
enough to reverse the effect in Figure 4. Finally, (v)
in mechanical unfolding7of apoflavodoxin, both
domain A (the CheY nuclear region) and the helix-
strand combination a6-b5in domain B (the apo-
flavodoxin nucleus) are dynamically confined by
their local environments, moving essentially as
fixed units while the protein unfolds and remaining
so long after the core of the protein is exposed to
As we noted above, non-native interactions can
have a substantial impact on, or even control the
folding of certain proteins, and some of our results
seem to suggest these effects. Although the model
does not include non-native interactions directly,
proteins do, and the results may reflect their
absence in the model at certain points along the
folding profiles. The effects of non-native inter-
actions have never been looked at using this type of
model and hence it is difficult to decide when they
could be present, or what signature they would
leave on the model kinetics. Consequently, we
decided to look at the Im7 folding landscape
where these effects have been mapped out.22,23
Im7 folds through a single intermediate in which
three of its fourhelices (a1, a2, and a4) are structured
but distorted non-natively, maximizing the burial of
hydrophobic side-chains that would be exposed
had the helices adopted their native positions. In
crossing the transition state into the native fold, the
helices acquire their native orientations, and the
binding site for helix a3is exposed allowing it to
fold and ultimately lock the whole protein into its
native structure. Our results for Im7 are shown
in Figure 5. Its sister protein, Im9, folds across
a smooth free energy barrier but still shows some
indications of an intermediate perhaps suggesting
the results seen at low pH.23Both proteins condense
into relatively large, partially unfolded ensembles
(Figure 5(b)) due to exposed side-chains in the turn
regions of the folds. This situation can be improved
a bit by extending the contact radii or by including
the dependence of the entropy on amino acid type,
however, the results here are still very instructive.
Again, in the intermediate parts of the protein are
stabilized by non-native interactions. When the
transition state is crossed, these stabilizing contacts
are exchanged for native contacts and the energetics
of the protein and the model converge. Any
qualitative differences that exist between the
model and the protein due to the missing non-
native interactions should be evident before the
transition region where these interactions are lost
and the differences between the two pathways are
reversed. Regions of the protein that are stabilized
by non-native interactions in the intermediate
should be less stable in the model and may tend
to fold late, while regions that are not stabilized by
these interactions would tend to fold early. Because
this behaviour is reversed on crossing the transition
state, it should be evident (if the effect is strong
enough) as some type of "wrinkle" in the time order
for folding the sub-structures involved in the
intermediate, and this is exactly what we observe.
The folding order for sub-structures in the
protein and the model converge on the right side
of the transition barrier just after the major
intermediate (we refer to this point as q* in
Figure 5(b)). Within the model intermediate, helices
a1 and a2 are structured, and as the transition
barrier is crossed helix a3folds, unfolds, and then
refolds after helix a4converging with the experi-
ments. The barrier is a residue of the competition
between (i) the free energy of freezing helix a4
leaving the loop including helix a3unfolded and
2 (a)(b)
Figure 4. Profile of atom-to-atom contacts, ci, for (a) CheY and (b) apoflavodoxin. ciis the number of atom-to-atom
contacts with amino acid i divided by the mean (a contact is registered when atoms from non-nearest neighbor amino
acids are less than 5 A˚apart; the Figure is coarse grained in blocks of two amino acids). Shaded bars in the lower part of
the Figures indicate the nuclear regions. For each fold, the number of contacts between domain A and the "nuclear part"
of domain B (after the dividing b3strand) is about the same as that for two amino acids. The local accumulations of
contacts and the opposing slopes of the profiles coincide with the location of nuclei and the direction of their growth.
Alternate Pathways for Flavodoxin Folding


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The Role of Non-Governmental Organisations in Public Interest Litigation
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Budget Cut of 66% imposed on CE Schemes
Sponsor.ie reduces fees!
Hi there, I am in the process of setting up a charity and need all the help and Advice I can get.
We are in the process of setting up a community based cancer support centre. Any advice on wording re job description for centre manager and part time...
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New Firmware for Canon EOS C500/C500 PL Cinema EOS Cameras (ver. 188.8.131.52.00)
Canon released a new firmware for the EOS C500/C500 PL Cinema EOS Cameras, version 18.104.22.168.00. Hey, it's the camera used to capture comet ISON. The links:
Download Firmware Version 126.96.36.199.00 for EOS C500.
Download Firmware Version 18.104.22.168.00 for EOS C500 PL
Download the new version of the Instruction Manual for the EOS C500.
Download the new version of the Instruction Manual for the EOS C500 PL.
Canon product advisory:
Thank you for using Canon products.
Canon has released a firmware update for the EOS C500/C500 PL Cinema EOS Cameras.
EOS C500 Cinema EOS Camera & EOS C500PL Cinema EOS Camera
1. When using the Magnify focus assist function, the ability to move the magnified viewing area around the LCD has been added.
2. Maximum ISO setting has been increased to ISO 80,000.
3. Cinema Gamut mode and DCI-P3+ mode have been added to provide expanded color gamut options in RAW capture.
4. 4096×1080-pixel RAW format resolution has been added.
5. A Key Lock menu setting has been added which now makes it possible to lock all operations, including the START/STOP button.
6. Using the optional Canon WFT-E6 Wireless File Transmitter, the camera's remote-control application allows up to two users to access the same unit via a Wi-Fi® link providing simultaneous and independent control of camera operation and metadata input.
7. Canon Log LUT support has been made possible for HD/SD SDI terminal output.
8. ACESproxy output from monitor terminal has been added.
9. [ND]/[ND-] have been added as functions that can be allocated to any assignable button.
EOS C500 Digital Cinema Camera only
10. Ability to assign the two control dials (body and grip) to operate either Iris or ISO sensitivity independently has been added.
11. Peripheral Illumination Correction Data has been added for seven (7) Canon Cinema lenses (EF mount) and eleven (11) Canon EF Lenses.


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I was the DP and cinematographer on all four parts of this little mini-series of videos we did for our weekend services at church. The actors were fantastic, and I think these turned out pretty well!
Directed and Written by Mike Hadley
DP - Tim Johnson
Edited by Travis Wermedal
Dude - Matt Houchin
Girl - Danielle Anderson
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Featured Business
Dr. Jerry H. Grant
1320 N Highland
El Dorado, AR 71730
(870) 862-2285
Read more about this business
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Featured Business
Larry's Rexall
102 Elm St.
El Dorado, AR 71730
(870) 863-8111
Read more about this business
/ View Map
Reddies rip Boll Weevils 71-0
ARKADELPHIA - Henderson State's defense forced a season-high six turnovers, while the offense totaled 655 yards as the 14th-ranked Reddies shutout Arkansas-Monticello by a score of 71-0 on Saturday afternoon at Carpenter-Haygood Stadium in Arkadelphia, Ark.
Henderson State, 6-0 o...
Published: 10/07/2012
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We recommend
Product Info
Marquez Shoe - GQYS700001
The nuts and bolts
- The three-hole Marquez Shoe doesn't like to brag. But he knows he's your favorite. Great looks, supreme comfort, extreme durability—why be humble?
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- 100% Suede Leather, Outsole: 100% Rubber
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Items purchased online or by phone may be returned or exchanged by mail or at any DC Shoes/Quiksilver/Roxy retail store.
For a full refund, returns must be made with the original receipt and merchandise must be in its original condition.
Click here for our entire return policy


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|Casual Dining
|Love Seafood Cafe
Soul food, seafood and BBQ, something for every taste.
|Save 10% on food.
|Casual Dining
|Rusty Bellies Waterfront Grill
Seafood restaurant. Open for lunch and dinner.
|Save 10% on food and non-alcoholic beverages.
|Casual Dining
|Santorini Taverna & Grill
Santorini Taverna in Eden Prairie is legendary for its American cuisine, Greek classics and its amazing outdoor summer deck. Santorini offers the savory tastes of the Mediterranean, as well as numerous seafood and vegetarian selections.
|Save 10% on lunch, dinner or brunch.
|Casual Dining
Little French bistro located in the heart of downtown Tampa.
|Save 15% on food, non-alcoholic beverages, and catering services.
|Fast Food
Ice, Custard and Happiness equals Rita's
|Save 20% on your total order.
|Casual Dining
|Liquid Ginger Asian Grille & Teahouse
Enjoy tea from all over the world then snack on great Asian cuisine.
|Save 10% on food and non-alcoholic beverages.
|Casual Dining
|Christopher's Family Pizzeria
Great old fashioned pizza, ribs, kid friendly, delivery
|Save 15% on all food and non alcoholic beverages.
|Casual Dining
|Rocky's Cafe and Pizza
Offering wings, pizza, and more Italian cuisine for the whole family to enjoy.
|Save 10% on food and non-alcoholic beverages.
|Casual Dining
|Port of Madison Restaurant
Variety is the key at this popular restaurant, located in the Holiday Inn Huntsville-West. It offers a breakfast buffet as well as menu selections Monday to Friday from 6am to 10 am, 5pm to 11pm, and Saturday and Sunday from 7am to 10am, 5pm to 11pm.
|Save 15% on food.
|Casual Dining
|Wine Cellar
Elegant dark wood walls and large windows enhance the restaurant's warm, inviting atmosphere. Its well-rounded menu offers some unique selections. The four-course prix fixe menu changes every other week.
|Save 15% on food and non-alcoholic beverages.


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"Disconnect between Brooklyn and Ireland"
I enjoyed the Irish voices and the experience of coming to America, as seen through a Foreigner's eyes. I was disappointed not so much that the main character had little character. The author was unconvincing in setting the reader up to understand eillis' behavior toward her marriage and American Husband. The ending also left me wondering.
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If you find this review inappropriate and think it should be removed from our site, let us know. This report will be reviewed by Audible and we will take appropriate action.


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No Reservations kicked off its very first episode in the City of Light 6 seasons ago. For the 100th episode, Tony and the crew return to Paris, the city where it all began.Go to Episode
Hugo Desnoyer Boucherie
Tony and Eric Ripert visit one of the most well-known boucheries in Paris. Hugo Desnoyer supplies some of the best restaurants in Paris.
45 Rue Boulard, 75014 Paris, France
Marx Food Lab
Tony and Eric visit the labs of food wizard Thierry Marx. While wildly experimental --there's a reason his recent book is called 'Planet Marx' -- his take on modern (or molecular) cuisine has a lightness of both touch and spirit.
4 Rue du Bouloi, 75001 Paris, France
More Paris Travel Guides
Trip Flip: Paris, France Travel Guide
Bert takes newlyweds Craig and Heather Bacon to France for an ultimate Parisian experience.
The Layover: 40 Hours in Paris
Tony has 40 hours to eat his way through Paris.
description comes here
Other Anthony Bourdain Travel Guides
Anthony Bourdain ends his No Reservations ...
Dominican Republic
Get a complete list of the restaurants, ...
No Reservations in Burgundy
Get Tony Bourdain's Travel Guide for Burgundy ...
Anthony Bourdain's Rio Travel Guide
Check out all the places Anthony Bourdain ...
More Travel Inspirations
GAC America's Amusement Parks
Buckle up and take a tour of the most thrilling amusement parks in America.
3-Ingredient Apps for Thanksgiving
Try Food.com's quick and easy apps for a stress free turkey day.
Food Network's Top Turkey Day Tips
Plan Your Menu with easy, how-to videos from Bobby, Sunny and more.


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Brothers jailed for conspiring to attend terror training camp in Syria
Two brothers have been jailed for conspiring to attend a terror training camp in Syria.
A London clinic is launching Britain's first "talking therapy" programme for obese patients.
22 November 2012
09 November 2012
Sickness benefit will be stripped from claimants who refuse to get treatment for their problems under a crackdown to be tested in a £25 million Government trial.
06 November 2012
A former event organiser for MP Zac Goldsmith is threatening to sue a hospital after receiving electric shock therapy allegedly without giving permission.
30 October 2012
A new national service to treat patients with blood cancer that affects the spine was launched today in London.
12 October 2012
Many healthcare workers do not know how to use inhalers properly so are not in a position to teach asthma patients to care for themselves, according to an article in a medical journal.
04 October 2012
Paul Merton tells of his spell in a psychiatric hospital in his first 'more fascinating than funny' tour show since 1998
03 October 2012
Want to smooth away the stress? Our writers test five of the best treatments in town
04 July 2012
An exhibition which features surgeons sawing through a living person's skull had to be altered after more than a dozen visitors fainted from shock.
15 June 2012
Children who witness violence at home are more likely to seriously harm someone else, carry a weapon or be excluded from school, according to a survey.
31 May 2012


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The Hill: GOP lawmaker: I saw TSA pat down 'little old lady,' child, but not Arab manSubmitted by legalizeliberty on Thu, 05/26/2011 - 10:40
The Transportation Security Administration is too worried about "political correctness," according to a Republican lawmaker.
Rep. Paul Broun (R-Ga.), a member of the House Homeland Security Committee, said TSA is too politically correct when it chooses which passengers to search at airport security checkpoints.
During a recent trip, Broun said he saw TSA pat down an elderly person and a child, but not a man he himself deemed suspicious.
"I walked through … right behind me there was a grandmother — little old lady, and she was was patted down," Broun said on C-SPAN's "Washington Journal."
"Right behind her was a little kid who was patted down. And then right behind him was a guy in Arabian dress who just walked right through. Why are we patting down grandma and kids?"
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Assembly Minority Leader Brian Kolb: $850,000 "Golden Parachute" For New MTA Boss Is An Affront To Taxpayers
July 31, 2009
"News that the incoming head of the Metropolitan Transportation Authority (MTA) could receive an $850,000 'golden parachute' is an affront to hard-working New York taxpayers and small businesses that, earlier this year, picked up the multi-billion dollar tab for the MTA's bailout. While I am uncertain as to what possessed Governor Paterson to offer such an exorbitant compensation package, I am certain the MTA is a public entity that has been hemorrhaging public monies and is sorely in need of stricter internal fiscal controls and accountability. Coming at a time when 854,200 New Yorkers are unemployed – the most since 1976 – this latest MTA revelation clearly illustrates why a fiscal oversight control board is needed to ensure a distressing pattern of fiscal and administrative irresponsibility at the authority does not continue. I call on the MTA Board to send a message by rejecting the proposed compensation package."
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- Assembly Minority Leader Brian Kolb Statement In Recognition Of Veterans Day
- Getting Back-To-School: Dealing With Rising Costs And Common Core
- Assembly Minority Leader Brian Kolb Receives Top Award From NFIB
- Assembly Minority Leader Brian Kolb Receives Highest Scores From Unshackle Upstate And NFIB
- Assembly Minority Conference Scores New York's Highest Marks From Unshackle Upstate And NFIB


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West Coast rap newcomer Kreayshawn recently opened up on her rags to riches rise in hip-hop and how she nearly ended up having a career doing blue collar work.
Admitting she survived a difficult childhood, Kreay credited a stint with free education and training program Job Corps for getting the wheels rolling.
"I went through so many hard, crazy, random things, even in my childhood," says the 22-year-old Natassia Zolot (Kreayshawn), "that for a second I thought 'What is my life going to be?' It was insane — I could've wound up dead." She says she turned her life around through Job Corps. "I really think it's a good place to go," Kreayshawn says of the free education/training program that helps low-income youth earn a diploma or GED, then learn career-defining work skills. "Right before I went, I was living at (Mob partner) V. Nasty's house, and I just didn't really see myself doing much of anything. Then I heard of Job Corps, I went, and it helped me get my life on track. And if you graduate, they even help you get an apartment." (SF Examiner)
She also said Job Corps landed her a j-o-b followed by a one-time purchase which sparked a rap career.
Zolot lived in Job Corps' dorms on Treasure Island, regularly rode the 108 bus and studied facility maintenance. "So I learned how to power-wash cement, which was actually kind of scary — the water is so hot, we'd have to wear these big overall outfits," she says. "I also know how to paint curbs and fire hydrants — it was actually some pretty interesting stuff." Soon, she had her own apartment, a job at IKEA, and enough money for a MacBook, on which she made her first music and videos. (SF Examiner)
Following the recent passing of Apple co-founder Steve Jobs, Kreay said his MacBook laptop line is responsible for helping fuel a viral hip-hop takeover.
"RIP Steve Jobs," said Kreayshawn. "If it wasn't for him me and alot of artists wouldn't have the digital proficiency that we do today. Buying my first MacBook was a huge change in my life for me and it also helped me make sense of all the arts that I can accomplish on one piece of technology created a man named Steve Jobs." (Hip Hop DX)
In September, Kreay revealed she has other career backup plans if hip-hop does not pan out to her liking.
"I'm a rapper, I'm a graphic designer director, model, deejay and a professional b*tch snatcher," Kreayshawn said. "There's a lot of people that want to push you down. One, for being a female and one for being white. It's hard but I don't see it like that. In Oakland, that's how you grew up…The best about everything that's going on is I have my head on my shoulders, I know what I want, I know my vision and it's not like I signed this record deal blindly — whatever happens, happens. I know that I'm a good person and that I'm smart and funny. If the music thing doesn't work out, I'm talented at a million other things." ("Carson Daly")
Check out a recent Kreayshawn interview below:


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Real Estate Technology & Tools
Who do you typically turn to first when considering the purchase of a new piece of technology for your real estate business? If you're like a lot of agents, you'll pick the brains of other agents to find out what works and what's not worth your money.
But the choices – there are so many of them! When you find yourself feeling overwhelmed, grab a cup of coffee and dig into the following pages. Let your colleagues walk you through those choices and help you decide on how and where to spend your money.
Feel free to check back daily because we add hundreds of new posts every day. This is the place to find everything an agent needs to know about tech and tools to help you run a lean, mean real estate machine.
Trending blogs on Real Estate Technology & Tools
(The Ballen Companies, TBG, TBM, ASAN)
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The online marketer today is not the same as the marketer even 5 years ago. We must be fast to implement, integrate, and innovate. Today, we must be quick to adapt. We must be ready to make a sudden change. We must accept that what worked yesterday may not work today. We must realize competition...
(Floor Coverings International - Westchester NY & Stamford CT)
Comments 16
One of my end of year projects is to get some videos done. Over the weekend, I finally made my first "slide" video. I'm hoping to make 4 more for the end of the year, and I'm really hoping this will help my website and web presence. So, yesterday I was doing some research and found this very ...
(Re/Max Sunset Realty)
Comments 8
Traveling For Thanksgiving-Check Your Flight Traveling for Thanksgiving? With all the crazy weather we have had and are still having, you may want to check flight cancellations and delays before heading out the door. You can do that on your iPad or iPhone. Flightaware for iPad will help ...
Comments 16
A recent inquiry by someone who wants to break into the luxury real estate market in 2015 spurred this question, "Is it time to update your existing website or create an additional website for the luxury market?" This morning, as we were reading our roundup of luxury news, this headline pop...
(RE/MAX Properties SW, Inc.)
Comments 19
We've found that a short video works as well as a bio for a lot of today's younger clients. You might take a look at this one (warning: full volume audio) and give me your comments: If enough people are interested, I'll post a second blog detailing the steps to take to do it yourself. Namaste! Eric
(Homesmart International)
Comments 0
How to sell more homes in 2015 How to Find MORE TIME in Every Day Today we will share with you the TOOL that will assist you in making a HUGE change in your life and in your business. Please understand, this won't happen overnight and it may not happen within a couple of weeks, I know becau...
(RE/MAX Properties SW, Inc.)
Comments 3
There is a ton of competition in the real estate industry today. In our market alone, the MLS roster has 24,637 agents. Tha's today; there's undoubtedly more coming into the business tomorrow. So, how do you stand out? I've been in this business long enough to know that you can spend tens of tho...
(Somers & Company)
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Seacrest FL Home for Sale | 82 Clareon Dr Whites Gulfview Estates Comfort has no boundaries in this Seacrest FL 4-bedroom beach home for sale. Boasting of 2,394 square feet of heated and cooled living space, this home graciously features 3 baths, 2-car attached garage, large windows and high cei...
(High Country Realty - Prescott - AZ)
Comments 5
Is there a Way to Search for most Recent Posts on a Particular Topic? I have been recently been searching for a new CRM. I entered "CRM Wordpress" in the searchbox. The results gave me a few posts in Active Rain mostly from 2010. Things change fast in our business. I am NOT interested in what wa...
(IXACT Contact Real Estate CRM)
Comments 0
As social media explodes in popularity and mobile use expands, you might be re-strategizing how to spend your marketing dollars. You might also be cringing at the idea of spending an inordinate amount of money on relatively new, untested waters, like social media ads. But, you have to go where th...
(The Berkshire Group)
Comments 1
My Iphone screen is barely visible....why? All of a sudden this morning, the screen on my Iphone has turned very dark so I can hardly read the phone numbers of incoming calls or see the rectangle at the bottom to swipe it open. For those of you that have an IPhone, has this ever happened to yo...
(The Jordon Wheeler Group)
Comments 9
Another Phone Upgrade - Samsung Note 4 I upgraded my cell phone over the weekend from the Samsung Note 2 to the Samsung Note 4. Two generations later and I can tell the differences between the two phones. I got the Samsung Note 2 earlier this year, and I have enjoyed it a lot. I rarely bite t...
(Coldwell Banker Lake Country)
Comments 125
Posting this from New Orleans, where I'm attending my first National Association of Realtors national convention. One of the highlights of today was a seminar on the use of drones for aerial photography for real estate use. We had two industry experts with the best intel available - the good new...
(Sharon Tara Transformations)
Comments 32
Today I'm going to share a tutorial video showing how I use Ribbet to colorize old black and white photographs. Ribbet is very user friendly and the technique I use is like coloring or painting when you were a kid. It's really just a matter of choosing a color, applying it, and then adjusting the...
(Homesmart International)
Comments 0
STOP – LOOK – LISTEN When we realize according to NAR (where we pay millions of dollars to) that the #1 complaint against real estate agents is: non-communications and if those of us who want to increase our income, make it a point to communicate more frequently not only with our clients and cus...
(BPO Automation Group & BPO University)
Comments 2
AutoFill Your BPO's in 5 Minutes 11/24/2014 BPO's Leaving You Stressed? There is a Better Way! Let us help! Sign Up Today for Half Price! AutoFill Your BPO's in "5 Minutes" Guaranteed Contact...
(Tech Training for You!)
Comments 21
In this post, we will take a look at the difference between the high resolution photos that we take with our cameras as compared to the optimized copy that we should be using when we are working with web presentations. Having high resolution images is great for certain types of presentations such...
(RealSupport Inc.)
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Blink and you'll miss it! It's that time of year again once more, and it seems as though 2014 has flown by. Our Real Estate Marketing group wanted to take the opportunity to give thanks to our clients, our colleagues and the team members that make up our innovative, professional Real Estate Virtu...
(Woodland Management Service)
Comments 2
Every week I pick 10 posts that I think that could use a second look, a little more exposure, or that are so good that I want all my friends to have a chance to experience it. This week is no exception and it has been a challenge to narrow the list down to 10, I hope that you enjoy these along wi...
(Real Living | At Home)
Comments 2
I read a good article this week : http://www.inman.com/next/drone-technology-helps-agents-sell-houses/ about the use of drones for real estate photography. Currently in the US; the FAA does not allow the use of drones for commercial purposes. Our neighbor to the north; Canada - does allow t...


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by Sheldon Rosner • November 19, 2014 • Astrology, Humor, News, Philosophy, Politics, Psychology/Sociology, Spirituality • 0 Comments
Our journey through Sagittarius begins on the New Moon in that sign on the 22nd, anniversary of the JFK assassination, 51 years ago, which means Chiron has returned for that event. I wish we could solve the mystery of who actually shot him. As for this first day of Sag, Mercury square Jupiter goes out […]Read more →


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Online dating brings singles together who may never otherwise meet. It's a big world and the SeniorPeopleMeet.com community wants to help you connect with singles in your area. Whether you are seeking just a date, a pen pal, a casual or a serious relationship, you can meet singles in Muscatine today! Iowa is known as the "The Hawkeye State" and SeniorPeopleMeet.com is here to bring their Senior Singles together.Search Single Senior Men in Muscatine | Search Single Senior Women in Muscatine
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