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X-ray crystallography. Electron microscopy. Direct methods. Acting as a tool of image processing. Dealing with pseudo symmetries. Two-stage image processing in Electron microscopy. Pseudo centro symmetry. Pseudo translational symmetries. Enantiomorphous phase ambiguity. - PowerPoint PPT Presentation
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Electron crystallographicmethods of solving
modulated structures
Electron microscopyElectron microscopyX-ray crystallographyX-ray crystallography
Direct methods
Pseudo translationalsymmetries
Dealing withpseudo symmetries
Two-stage image processing inElectron microscopy
Translationalphase ambiguity
Ab initio solutionof superstructures
Ab initio solution ofmodulated and
composite structures
Acting as a tool ofimage processing
Pseudo centro symmetry
Enantiomorphousphase ambiguity
Protein SAD/SIR phasing
Protein model completion
电子显微学电子显微学X - 射线晶体学X - 射线晶体学直接法
赝平移对称
处理赝对称性两步法电子
显微学图像处理
超结构从头求解
用于图像处理
赝中心对称
蛋白质 SAD/SIR相位推演
蛋白质模型完整化
电子晶体学方法求解调制结构
从头测定调制结构和组合结构
Initial phasing and Model completion of Proteins
Image processing inElectron Microscopy
SoftwareModulated and Composite structures
Enantiomorphous phase ambiguityresolved by the direct method
Sample: ZCW; Space group: P21
Acta Phys. Sin. 27, 169-174 (1978)
Enantiomorphous phase ambiguityresolved by the direct method
Direct method result Final map
a c
b
c
b
cK
Before treatment After treatment
X-ray Crystallography
Objectivelens
Back focal plane
Image plane
Object
Diffractionpattern Blurred
image
Fourier transform
Fourier transform
Electron microscopy
Object
Patterson map
Diffractionpattern
Diffractionwave front
Object
Patterson map
Diffractionwave front
Diffractionpattern
X-ray Crystallography
Multiplied with F*h
Objectivelens
Back focal plane
Image plane
Object
Diffractionpattern Blurred
image
Multiplied with acontrast transfer
function
Fourier transform
Fourier transform
Electron microscopy
Structure image blurred by
convoluting withthe Fourier
transform of acontrast transfer
function
Structure image blurred by
convoluting withIt’s inverse
Object
Patterson map
Diffractionwave front
Diffractionpattern
X-ray Crystallography
Multiplied with F*h
Objectivelens
Back focal plane
Image plane
Object
Diffractionpattern Blurred
image
Multiplied with acontrast transfer
function
Fourier transform
In the derivation of X-ray diffraction phases, direct methods are acting as a tool of image processing.
They convert the blurred image Patterson map to the deblurred image electron density map.
It is reasonable to expect that direct methods can be used in high-resolu-tion electron micro-scopy as a tool of image processing.
Fourier transform
Electron microscopy
<F >F
o
A
B
F”
F’”
’
F”
F *
Phase ambiguity intrinsic in SAD
F
A
B
N
FR
FD
FN
N
D
D
Phase ambiguity intrinsic in SIR
DR
R
D
N
N
1
( ) ( ' ")exp( 2 )N
oj j j j
j
F f f i f i
h h r
( ) '( ) "( )oF F F F F h h h h
* *( ) ( ) '( ) "( )oF F F F F h h h h
* 2 "F F F
exp , 2F F i F F F
"
, – SAD phase doublet
D – derivative, R – replacing atoms, N – native
D R NF F F
;N R N D R D
,I II – SIR phase doublet
, 3
1 1tanh sin
2 2
sin sinbest bes
c
t
P
m m
h h
h' h h' h h' h' h h' hh'
1 2
2
exp 2
1 1 2 1 cos2 cos2
2 2
m
P
2h h
h h
"
1tan( ) 2( )sin cos
2
best best
best P
h h h
h h h
Breaking SAD/SIR phase ambiguity by direct methods
' h h h
SAD/SIR data
P+(h) = 1/2
End
Calculate P+(h)
Eh ,’h , h
Calculate mh and h,best
LysozymeSpace group: P43212 Unit cell: a = 78.4, c = 37.0Å Number of residues in the ASU: 129Resolution limit: 2.01Å; Multiplicity: 47.2 Anomalous scatterer: S (10 )X-rays: Cr-K = 2.291Å, f ” = 1.14 Bijvoet ratio: <|F |>/<F > = 2.4% Data provided by Prof. Isao Tanaka and Dr. Nobuhisa Watanabe
AzurinSpace group: P4122 Unit cell: a = b = 52.65, c = 100.63ÅNumber of residues in the ASU: 129 Resolution limit: 1.9ÅMultiplicity: 10.0 Anomalous scatterer: Cu (1)X-rays: synchrotron radiation = 0.97Å f” = 2.206Bijvoet ratio: <|F|>/<F> = 1.44%Data provided by Prof. S. Hasnain
XylanaseSpace group: P21 Unit cell: a = 41.07, b = 67.14, c = 50.81Å = 113.5o Number of residues in the ASU: 303 Resolution limit: 1.75Å; Multiplicity: 15.9Anomalous scatterer: S (5 ) X-rays:synchrotron radiation= 1.488Å; f ” = 0.52Bijvoet ratio: <|F |>/<F > = 0.56% Data courtesy of Dr. Z. Dauter, National Cancer Institute, USA
Alanine racemaseSpace group: C 2 2 21 Unit cell: a = 72.68 b = 76.13, c = 136.27ÅNumber of residues in the ASU: 357 Resolution limit: 2.3ÅData collection range: 360o Anomalous scatterer: Se (8), S (6), P (1)X-rays: Cr-K, = 2.291ÅBijvoet ratio: <|F |>/<F > = 2.8% Data provided by Dr. Cheng Yang
Alanine racemaseSpace group: C 2 2 21 Unit cell: a = 72.68 b = 76.13, c = 136.27ÅNumber of residues in the ASU: 357 Resolution limit: 2.3ÅData collection range: 360o Anomalous scatterer: Se (8), S (6), P (1)X-rays: Cr-K, = 2.291ÅBijvoet ratio: <|F |>/<F > = 2.8% Data provided by Dr. Cheng Yang
SIR
SOLVE/RESOLVE + OASIS/DM
SOLVE/RESOLVE
SOLVE/RESOLVE
SAD
SOLVE/RESOLVE +OASIS/DM
SIR
SOLVE/RESOLVE SOLVE/RESOLVE+ OASIS/DM
SIR
SOLVE/RESOLVE
SAD
SOLVE/RESOLVE +OASIS/DM
SOLVE/RESOLVE SOLVE/RESOLVE+ OASIS/DM SOLVE/RESOLVE
SAD SOLVE/RESOLVE +OASIS/DM
2.1Å 3.0Å
3.5Å 4.0Å
