High-resolution cryo-electron microscopy study of structure and dynamics of yeast fatty acid synthase by single particle analysis [Elektronische Ressource] / von Preeti Kumari

Preeti Kumari

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 40 Mo

Extrait

High-resolution cryo-electron microscopy study of structure and
dynamics of yeast fatty acid synthase by single particle analysis.



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt im Fachbereich 14 Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe Universität
in Frankfurt am Main

von
Preeti Kumari
Aus
Munger
Indien


Frankfurt am Main (2009)


(D30)
1 CONTENTS
ABSTRACT
 8 

CHAPTER
1
 10

INTRODUCTION
 10

1.1
 FATTY
 A CIDS
 10

1.2
 FATTY
 A CIDS
 YNTHESIS
 12

1.3
 TYPE
I
FATTY
 A CIDS
 YNTHASE:
A
MEGASYNTHASE
MACH INE
 18

1.3.
1MAMMALIAN
FATTY
 ACID
SYNTHASE
 18

1.3.
2FUNGAL
FATTY
 ACID
SYNTHASE
 21

1.4
 FAS
INHIBITION
 24

1.5
 A CYLC
 ARRIER
PROTEIN
 26

CHAPTER
2
 28

ELECTRON
MICROSCOP&Y
M
 ETHODS 
 28

2.1
 INTRODUCTION
 28

2.2
 SINGLE
PARTICLE
A NALYSIS
 33 

2.2.

1 IMAGE
FORMATION
 33

2.2.
2NOISE
IN
EM‐IMAGES
 33

2.2.
3CONTRAST
TRANSFER
FUNCTION
 34

2.2.
42D 
IMAGE
ALIGNMENT
AND
CLASSIFICATION
 38

2.2.
53D ‐RECONSTRUCTION
METHODS
 41

2.2.
6RESOLUTION
ASSESSMENT
 46

2.2.
7MAP
 ANALYSIS
 47

2.3

 MATERIALS
AND
METHODS 
 49

2.3.

1 NEGATIVE
STAIN
 49

2.3.

2 CRYO ‐SAMPLE
PREPARATION
 49

2.3.
3ELECTRON
CRYO‐MICROSCOPY
 50

2.3.
4X‐RAY
SOLUTION
SCATTERING
 51

2.3.
5DATA
PROCESSING
 51

2.3.
6RESOLUTION
ASSESSMENT
 52

2.3.
7ESTIMATE
OF
3D ‐VARIANCE
 52

2.3.
8 MAP
VISUALIZATION
AND
ANALYSIS
 53

CHAPTER
3
 54

RESULTS
 54

3.1
 INITIAL
3D ­MAP
 54

3.2
 HIGH
RESOLUTION
3D ­MAP
 60

3.3 
 3D ­MAP
ANALYSIS
AT
 5.9
Å
 65

3.4
 STRUCTURE
OF
THEα
 
WHEEL
 69
6
3.5
 DIFFERENT
ORGANIZATIO N
OF
THE
REACTION
CHAMBER
WALL
 71

3.6
 MULTIPLE
POSITIONS
OF
THE
ACYL
CARRIER
PROTEIN
 75

3.7
 ACP
DOCKING
AND
RELATIV E
OCCUPANCIE
S 79

2 CHAPTER
4
 80

DISCUSSION
 80

PART
I:
ANALYSIS
OF
 YEAST
FAS
EM ­MAP
AT
5.9
Å
 80

4.1
 STRUCTURE
AND
DYNAMICS
OF
YEAST
FAS
 80

4.2
 STRUCTURE
OF
THEα
 
WHEEL
 80
6
4.3 
 REACTION
CHAMBER
CONOFRMATION
AND
BIOSYNT HETIC
ACTIVITY
 81

4.4
 SUBSTRATE
SHUTTLING
M ECHANISM
 83 

4.5
 LINKER
PEPTIDE
 83 

PART
II:
SINGLE
PARTICLE
ANALYSIS
TOWARD S
HIGHER
RESOLUTIONS
 86 

4.6
 FACTORS
LIMITING
HIGH­RESOLUTION
IN
EM 
 87 

4.6.
 1EM ‐DATA
COLLECTION
 87

4.6.
 2EM ‐DATA
PROCESSING
 91

4.7
 BREAKING
THE 
RESOLUTION
BARRIER
FOR
YEAST
FAS
STRUCTURE
 92

4.8
 NEAR­ATOMIC
&
SUB­NANOMETER
RESOLUTION
STRUCTURES
BYS
PA 
 93

4.9
 CONCLUSIONS
AND
OUTLOOK
 98

4.10
 A CCESSION
COD
E 99

BIBLIOGRAPHY:
 100

ZUSAMMENFASSUNG 
 109

PUBLICATIONS


3 LIST OF FIGURES
CHAPTER
1


Figure
1
Structural
representation
of
saturated
and
unsaturated 
fatty
acids
where
decanoic
acid
(C­10)

is
used
for
illustration
(Nelson
and
Cox
2000________________________________). ______________________________ 10

Figure
2.
Fatty
acid
synthesis
reaction
cycle
(Gipson
et
al.
2009).
Numbers
denote
the
steps
in
the
fatty

acid
chain
elongation
cycle
as
follows:
(1)(3)
acetyl/mylal
toransfer;
(2)
condensation;
(4)
ketoacyl

reduction;
(5)
dehydration;
(6)
enoyl
reduction. ____________________________ 12

Figure
3.
Structural
representation
of
acety­l
and
malonyl­CoA
used
as
substrates
in
fatty
acid

synthesis.________________________________ ________________________________ ______ 13

Figure
4.
Structural
representation
of
thio­lgroup
of
cysteine
in
KS
and
phoshpopantetheine
arm
of
ACP

found
in
both
Type
I
and
II
FAS
systems
(Wakil
et
al.). _____________________ 14

Figure
5a.
Reaction
step
1,
2
and
3
in
the
synthesis
of
fatty
acids
(Wakil
et
al.
198___________________3) 15

Figure
5b.
Reaction
stpe
4,
5
and
6
in
the
synthesis
of
fatty
acids
(Wakil
et
al.
1983) 16

Figure
5c.
Reaction
step
4,
5
and
6
in
the
synthesis
of
fatty
acid
(Wakil
et
al.
1983) 17

Figure
6a.
Diagrammatic
representation
of
one
α­polypeptide
chain
in
mammalian
FAS
showing
its

linear
domain
organization
(Maier
et
al.
2006).________________________________ ____________________________ 18

Figure
6b.
First
cryo­EM
map
of
mammalian
FAS
showing
different
views
(Brink
et
al.
200_________2). 20

Figure
6c.
X ­ray
structure
of
mammalian
FAS
at
4.5
Å
(Maier
et
al.
2006).______________________________ 20

Figure
6d.
3D­reconstructions
obtainde
by
EM
of
mammalian
FAS
showing
conformational
flexibility

(Brignole
et
al.
200________________________________9). _________________________ 20

Figure
7a.
Diagrammatic
representation
of
one
α­
and
 β­polypeptide
chain
in
fungal
FAS
showing
its

linear
domain
organization
(Gipson
et
al.
2009). ___________________________ 21

Figure
7b.
Early
tomographic
reconstruction
of
negatively
stained
yeast
FAS
(Hoppe
1976).__________ 22

Figure
7c.
Structure
of
yeast
FAS
by
early
single
particle
analysis
of
negatively
stained
specimen

(Kolodziej
et
al.
1996). ________________________________ ________________________ 22

Figure
7d.
X ­ray
structure
of
fungal
FAS
at
3.1
Å
(Jeni
et
al.
2007). _____ 22

Figure
8.
Structure
of
cerulenin
(Morisaki
et
al.
1993). ____________________ 25


CHAPTER
2


Figure
1.
Negative
staining
of
FAS
particles
showing
an
uneven
distribution
of
the
Uranyl
acetate
stain.
________________________________ ________________________________ _________________ 30

Figure
2a.
Holey­C
grid
showing
vitrified
water
in
thoe
lhes,
where
dark
colored
holes
show
thick
i32ce
.
Figure
2b.
Electron
micrograph
showing
FAS
particles
in
different
orientations
embedded
in
vitrified

water. ________________________________ __________ 32

Figure
3.
(a)
Contrast
transfer
function
for
a
defocus
series,
plots
for
defocus
values
at
2.5,

a2nd
3µm

are
shown
in
black,
blue
and
red
respectively;
(b)
dark
rings
seen
around
images
due
to
inaccurate
CTF

correction;
(c)
images
with
accurate
CTF
correction
show
no
dark
rings
around
them_________________. 36

Figure
4.
Different
methods
to
calculate
average
power
spectra
of
an
EM ­image:
on
left
is
shown
a

periodogram
generation
from
a
micrograph
using
the
small
overlapping
windows
method,
while
on

right
is
shown
usage
of
a
particle
stack
obtained
from
a
micrograph
for
generating
an
avraege
power

spectrum.________________________________ ________________________________ ______ 37

Figure
5.

Flow
chart
representing
an
outline
for
unsupervised
and
supervised
classification
in
single

particle
analysis. ______________________________ 40

Figure
6.
(a)
Pictorial
representation
of
angular
reconstitution
method
(vanHe
el
1987a)
showing
that

at
least
3
views
are
needed
for
3D ­reconstruction
of
any
asymmetric
structure.
(b)
Sinogram
correlation

function
for
two
different
views
of
FAS. ________________________________ ______ 42

Figure
7.
Principle
of
random
conical
tilt
methodw,i
snhg
how
many
rotated
images
within
a
cone

come
together
to
form
a
surface
(Radermacher
et
al.
1987). _______________ 43

Figure
8a.
Back
projection
reconstructs
an
image
by
taking
each
view
(shown
as
1D
view
1,
2
&
3)
and

“smearing”
it
along
its
direction
of
projection.
The
resulting
image
shown
as
a
2D
disc
in
(a)
is
a
blurry

4 version
of
the
original
image.
When
a
sufficient
number
of
views
are
used
during
back
projection
the

object
can
be
reconstructed
more
accurately
as
shown
as
a
2D
discn
i
(b)
(Smith
2002________________). 45

Figure
8b.
Forward
projection
of
the
reconstructed
image
(shown
as
a
2D­disc
here)
in
the
direction
of

the
original
views
(shown
as
1D
views
here)
produces
its
“r­eprojections”.
A
comparison
between
the

original
projection
and
its­
prerojection
can
be
used
to
assess
if
the
images
were
assigned
correct
angles

during
back­projection
(Smith
2002________________________________). ________________________________ _______ 45

Figure
9.
(a
)&
(b)
represent
the
mapping
of
Euler
angles
on
a
sphere
for
a
set
of
FAS
particles,
where

the
former
shows
an
uneven
distribution
of
views,
while
the
latter
represents
a
more
evenly
distributed

set
of
views________________________________. ____ 46 



CHAPTER
3 


Figure
1.
Flow
chart
for
ab
initio
model
building
using
the
angular
reconstitution
approach:
the

projections
assigned
with
wron
Eguler
angles
does
not
match
its
re­projection
(encircled
in
re______d). 56

Figure
2b.
Slices
(3.6
Å
thick)
of
the
initi­avlo
l3Dume
parallel
to
the
equator.__________________________ 57

Figure
2a.
Views
of
the
initial
­3mDodel
(D3
symmetry)
of
yeast
FAS
at
18
Å
showing
two
side
views

along
the
two­fold
axes
and
a
top
view
along
the
3­fold
axis
of
the
barrel________________________________. 57

Figure
2c:
Initial
model
showing
one
reaction
chamber
of
yeast
FAS
at
18
Å
as
seen
in
a
side
view
and

down
the
3 ­fold
axis.
The
color
scheme
represents
different
domains
contriebdu
bty
α
and
 β­chains
in

one
reaction
chamber
(Johansson
et
al.
2008)________________________________. ______________________________ 58

Figure
3:
Fitting
of
yeast
FAS­r
Xay
structure
(Leibundgut
et
al.
2007)
into
the
initial­
mEMap
(shown

for
one
dome).________________________________ _59

Figure
4.
Image
data.
(a)
Electron
micrograph
showing
differetn
orientations
of
FAS
particles
in

vitreous
ic