Transcript MACROMOLECULAR CRYSTALLOGRAPHY

Applications of Synchrotron Radiation
in Biology and Biotechnology
Zehra Sayers
Sabanci University, Turkey
Chair, SESAME Scientific Committee
UPHUK III
Bodrum, Turkey
Sept. 17-19, 2007
SYNCHROTRON RADIATION (SR)
Acceleration of charged
particles results in
emission of
electromagnetic radiation.
H. Winick
Initially thought as nuisance because of energy loss from
accelerated particles.
Importance recognized by early ’60s.
SR: Production
At low electron velocity
(non-relativistic case)
radiation is emitted in a
non-directional pattern.
When the electron velocity approaches
the velocity of light radiation is emitted in
the direction of motion and the radiated
power goes up dramatically.
SR: Basic properties
High flux and brightness
Pulsed time structure
Tunability
Polarized (linear,
elliptical, circular)
Small source size
Partial coherence
High stability
Flux = # of photons in given /
sec, mrad 
Brightness = # of photons in given /
sec, mrad , mrad , mm2
(a measure of concentration of the radiation)
SR:
Storage rings, bending magnets and insertion devices
Continuous spectrum
characterized by ec = critical
energy
bending magnet - a “sweeping searchlight”
ec(keV) = 0.665 B(T)E2(GeV)
eg: for B = 2T E = 3GeV ec =
12keV
wiggler - incoherent superposition
(bending magnet fields are
usually lower ~ 1 – 1.5T)
Quasi-monochromatic spectrum with
peaks at lower energy than a wiggler
1 =
u
2g2
(1 +
U
K2
)~
(fundamental)
2
g2
+ harmonics at higher energy
undulator - coherent interference
0.95 E2 (GeV)
2
u (cm) (1 + K )
2
K = g where  is the angle in each pole
e1 (keV) =
SR: Practical Production and Delivery to Users
the storage ring circulates
electrons and where they are
bent - synchrotron radiation is
produced
klystrons generate high power
radiowaves to sustain electron
acceleration, replenishing energy
lost to synchrotron radiation
electron gun
produces
electrons (at
e.g. 80 keV)
beam lines transport radiation
into “hutches” where
instrumentation is available for
experiments
special “wiggler”
insertion devices
used to generate
x-rays
linear accelerator/booster
accelerate e- which are
transported to storage ring (at
e.g. 7 GeV)
SR: Biological and Biotechnological Applications
“Biologists” are involved in 4 types of experiments at SR
sources:
Macromolecular Crystallography.
Spectroscopy.
X-ray Diffraction and Scattering from non-crystalline
systems.
Imaging.
WHAT ARE THE ADVANTAGES OF USING SR
TECHNIQUES IN BIOLOGY?
MACROMOLECULES OF LIVING SYSTEMS
•
Special architecture at molecular structure level;
Nucleic acids (DNA, RNA), Proteins, Lipids, Carbohydrates.
•
Examples:
DNA
Proteins
•
Hierarchical Organizational at larger scale:
Static and dynamic structures.
FUNCTIONAL ORGANIZATION
•
Examples:
Chromatin fibre dynamics
Cytoskeletal dynamics
SCHEME FOR FUNCTIONAL STUDIES
Structural Biology
Experimental Methods
Modelling
Bioinformaics
Conservation analysis
Cluster analysis
Molecular biology
Site directed mutagenesis
Activity measurements
Enzyme kinetics
Ligand interactins
Activity under perturbation
Test structural
models
Make functional
predictions
Test functional
predictions
Make structural
predictions
STRUCTURE AND FUNCTION RELATIONSHIP
• Experiments:
Static and Dynamic measurements
of structural parameters.
• Calculations:
Prediction of structure, structural
change where and how.
SR offers a wide selection of powerful experimental
tools for determination of structural parameters.
Time resolved data for establishment of correlation
between structural change and function.
MACROMOLECULAR CRYSTALLOGRAPHY
Determination of structure of macromolecules at atomic resolution.
Applications include:
Therapeutic drug design
Enzyme mechanisms
Supramolecular structure
Molecular recognition
Nucleic acids
Structural genomics
High-throughput crystallography
SR sources; high intensity, small beam size, and collimation.
The MAD (multi-wavelength anomalous ddiffraction) phasing
method readily applicable with tunable radiation at SR sources,
MACROMOLECULAR CRYSTALLOGRAPHY
SR offers possibility of using
Microcrystals
Large unit cell crystals
Cryo-crystallography
Minimizing radiation damage
Improvement of data quality
Automated crystal mounting robot
Crogenic robotic crystal transfer system
FedEx Crystallography!!!
SSRL, SAM
MACROMOLECULAR CRYSTALLOGRAPHY: Highlights
Nobel Prize 2003
Mechanism for the voltage dependent K-ion
channel.
Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene,
B.T. Chait, and R. MacKinnon, “X-ray structure
of a voltage-dependent K+ channel,” Nature
423, 33 (2003).
Nobel Prize 2007
Mechanism for RNA polymerase II.
Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, P.
Cramer, D.A. Bushnell, J. Fu, A.L. Gnatt, B.
Maier-Davis, N.E. Thompson, R.R. Burgess,
A.M. Edwards, P.R. David, and R.D. Kornberg,
“Architecture of RNA polymerase II and
implications for the transcription mechanism,”
Science 288, 640 (2000).
SPECTROSCOPY
Hard X-ray spectroscopy:
Extended x-ray absorption fine structure (EXAFS) spectroscopy,
X-ray absorption spectroscopy (XAS),
Near-edge x-ray absorption fine structure (NEXAFS) spectroscopy,
X-ray absorption near-edge structure (XANES) spectroscopy,
X-ray magnetic circular dichroism (XMCD)
Investigations of geometric and electronic structure.
Sensitive to element, oxidation state and symmetry of the molecules.
Tunability of SR is essential.
X-ray absorption spectroscopy
EXAFS; atomic arrangements, bond distance,
coordination no., symmtery,
XANES: valence,
Magnetic circular dichroism: spin-orbit magnetic
moments
X-ray fluorescence spectroscopy
Quantitative analysis of elemental distribution
Investigation of silent Zn in Metalloenzymes
Zn K-edge EXAFS as a function of time.
O. Kleinfeld, A. Frenkel, J.M.L. Martin, and I.
Sagi, “Active site electronic structure and
dynamics during metalloenzyme catalysis,”
Nat. Struct. Biol. 10, 98 (2003).
Imaging and Spectroscopy
Investigation of elemetal composition
of cancerous lung tissue can be
compared with that of healthy tissue
by X-ray
fluorescence mapping measurements.
An optical micrograph of lung tissue is
shown together with specific maps
showing Fe, Cu and Zn distributions in
the boxed
area of the tissue.
SSRL
X-RAY SCATTERING AND DIFFRACTION FROM
NONCRYSTALLINE SYSTEMS
Low resolution data on the size and shape of the molecule can be
obtained.
Time-resolved data in response to a perturbation on the system.
Protein solutions, fibers.
Biomaterials: membranes, lipid micelles.
Measurements can be made at small (SAXS) and/or wide angles (WAXS)
depending on the system.
Complementary data to crystallography, electron microscopy and
spectroscopic measurements.
Applications include:
Protein (DNA)-ligand interactions.
Drug delivery.
Material characterization.
Time-resolved changes instructure.
X-RAY SCATTERING AND DIFFRACTION FROM
NONCRYSTALLINE SYSTEMS
Examples:
Bacterial crystals
Rat tail tendon
IMAGING
Absorption contrast imaging
Phase contrast imaging
Fluorescence Imaging
Full field imaging
Diffraction enhanced imaging
Topography
Tomography
X-RAY THERAPY
Targeted and dose-controlled therapy.
CLOSER LOOK SMALL ANGLE X-RAY SCATTERING
(SAXS) FROM PROTEIN SOLUTIONS
SMALL ANGLE SOLUTION X-RAY SCATTERING
•
Small angle X-ray scattering results from inhomogeneities in the
electron density in a solution due to macromolecules dispersed in the
uniform electron density of the solvent (0).
A solution of macromolecules
Solute: protein, DNA,
polymer (p)
Solvent (0)
•
Scattering pattern is determined by the excess electron density of the
solute, (r)
(r) = (p-0)c(r) + s(r)
= av c (r) + s (r)
(1)
Where
p = the average electron density of the particle.
av = the average electron density of the particle above the level of the
solvent (contrast).
c (r) = dimensionless function describing the volume of the solute (with
the value 1 inside the particle and 0 elsewhere).
s (r) = fluctuations of the electron density above and below the mean
value (independent of the contrast).
•In an ideal solution all particles are identical and
randomly positioned and oriented in the solvent.
•Scattering pattern contains information about the
spherically averaged structure of the solute described by a
distance probability function p(r)
•p(r) is the spherically averaged autocorrelation function
of (r) and r2p(r) is the probability of finding a point inside
the particle at a distance between r and r+dr from any
other point inside the particle
Dmax
•
For a globular particle p(r) has two main regions
a. A region of sharp fluctuations due to neighbouring atom pairs (0.1
nmr 0.5 nm) and of damped oscillations due to structural domains
(i.e -helices in proteins)
b. A smooth region corresponding to intramolecular vectors.
•
Beyond Dmax p(r) vanishes
•
The scattering curve also contains two regions:
a. Small angle region; information on the long range organization
(shape) of the particle
b. Large (wide) angle region; internal structure of the particle
(deviations from p)
Large distances only
contribute at low angles.
Short distances contribute
over a large angular range
and at high angles their
contribution dominates the
scattering pattern.
SCATTERING PATTERN AND THE DISTANCE
DIFSTRIBUTION FUNCTION p(r)
Scattering intensity and the
distance distribution function
are related by a Henkel
transformation.
APPLICATIONS
•
Determination of radius gyration, radius gyration of the cross
section, molecular weight.
•
Shape determination; at low angle (2-3 nm) the scattering curve is
dominated by the shape of the particle.
•
Time-resolved measurements for determination of structural
changes during interactions or upon a perturbation on the system.
•
Modern methods allow domain structure analysis, possibility of
modeling loop domains, analysis of non-equilibrium systems
(Svergun and Koch 2002, Current Opinion in Structural Biology,
12:654-660).
METALLOTHIONEINS
6-8 kDa proteins that bind metals in a wide range of organisms.
High cysteine (cys) content (up to 30%) in the amino acid sequence
and bind metals through the thiol groups of cys residues.
Metal composition depends on the source and previous exposure to
metals. Human liver MT contains mainly Zn, that isolated from kidneys
contain Cd and Zn or Cu. In higher organisms MTs represent the only
protein that is a natural Cd ligand.
Precise physiological functions are not yet identified; MTs are involved
in transport and storage of essential metal ions (Cu and Zn) and
detoxification (Cd and Hg).
Durum wheat MT is expressed and synthesized at high levels during
exposure Cd.
durum WHEAT METALLOTHIONEIN
Amio acid sequence:
MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEV
AAGQSGEGCSCGDNCKCNPCNCHinge
N-terminal
C-terminal
region
Domain
Domain
b-domain
-domain
C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein
“Cystein motifs” (cys-motifs) are involved in metal binding.
Metal-binding domains are connected by a 42 residue hinge region.
Prepare recombinant proteins GSTdMT and dMT.
Balcali wheat can tolerate higher levels of Cd
in soil than C-1252.
Bacteria expressing recombinant dMT can
tolerate high levels Cd in growth medium.
MODELING THE STRUCTURE of dMT
Cys-motifs are clustered in the N- and C-termini of the protein forming the
metal-binding domains (b- and -domains).
The predicted 3D structure of dMT. Cadmium (blue
spheres)-binding metal centers at each pole of the
dumbbell-shaped molecule are depicted in ball and
stick representation with the extended hinge region
highlighted in ribbon representation.
Bilecen et al., 2005
PREPARATION AND CHARACTERIZATION of GSTdMT
Size-exclusion chromatography

GSTdMT
elutes
as dimer
UV Absorbance Measurements
Charge transfer band
between 250 and 260
nm due to Cd-thiol
interactions
SDS-PAGE Analysis
Native-PAGE Analysis

Dynamic Light Scattering
(DLS)
Measurements
PREPARATION AND CHARACTERIZATION of dMT
Size-exclusion chromatography
SDS-PAGE Analysis

Native-PAGE Analysis


UV Absorbance Measurements
Dynamic Light Scattering (DLS)
Measurements
EXPERIMENTAL SET-UP FOR SAXS MEASUREMENTS
THE PRINCIPLE OF A SMALL ANGLE X-RAY
SOLUTION SCATTERING EXPERIMENT
•
The optical system selects X-rays with a wavelength of 0.15 nm
and a narrow band-width
•
The beam is focused on a position sensitive detector with an
adequate cross section at the sample position
•
The incident beam intensity I0 is monitored.
•
IT is the intensity of the beam transmitted through the sample and
IT = I0 exp(-µt), where the factor (-µt) represents the absorbance of
a solution of thickness t
•
I(s) is the scattered intensity which depends on the scattering
vector s defined as
s = 2Sin/λ
where 2 is the scattering angle and λ is the wavelength
BASIC SAXS DATA REDUCTION
X33 camera of EMBL Hamburg Outstation on DORIS STORAGE ring of DESY,
Hamburg.
Data are collected and reduced using standard software
Reference measurements are made on solutions of bovine serum albumin.
Structural models can
be calculated ab initio
using software such as
GASBOR, SASHA etc
and rigid body
modelling using
MASSA, ASSA etc
(EMBL-Hamburg)
METALLOTHIONEINS
6-8 kDa proteins that bind metals in a wide range of organisms.
High cysteine (cys) content (up to 30%) in the amino acid sequence
and bind metals through the thiol groups of cys residues.
Metal composition depends on the source and previous exposure to
metals. Human liver MT contains mainly Zn, that isolated from kidneys
contain Cd and Zn or Cu. In higher organisms MTs represent the only
protein that is a natural Cd ligand.
Precise physiological functions are not yet identified; MTs are involved
in transport and storage of essential metal ions (Cu and Zn) and
detoxification (Cd and Hg).
Durum wheat MT is expressed and synthesized at high levels during
exposure Cd.
durum WHEAT METALLOTHIONEIN
Amio acid sequence:
MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEV
AAGQSGEGCSCGDNCKCNPCNCHinge
N-terminal
C-terminal
region
Domain
Domain
b-domain
-domain
C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein
“Cystein motifs” (cys-motifs) are involved in metal binding.
Metal-binding domains are connected by a 42 residue hinge region.
Prepare recombinant proteins GSTdMT and dMT.
Balcali wheat can tolerate higher levels of Cd
in soil than C-1252.
Bacteria expressing recombinant dMT can
tolerate high levels Cd in growth medium.
MODELING THE STRUCTURE of dMT
Cys-motifs are clustered in the N- and C-termini of the protein forming the
metal-binding domains (b- and -domains).
The predicted 3D structure of dMT. Cadmium (blue
spheres)-binding metal centers at each pole of the
dumbbell-shaped molecule are depicted in ball and
stick representation with the extended hinge region
highlighted in ribbon representation.
Bilecen et al., 2005
PREPARATION AND CHARACTERIZATION of GSTdMT
Size-exclusion chromatography

GSTdMT
elutes
as dimer
UV Absorbance Measurements
Charge transfer band
between 250 and 260
nm due to Cd-thiol
interactions
SDS-PAGE Analysis
Native-PAGE Analysis

Dynamic Light Scattering
(DLS)
Measurements
PREPARATION AND CHARACTERIZATION of dMT
Size-exclusion chromatography
SDS-PAGE Analysis

Native-PAGE Analysis


UV Absorbance Measurements
Dynamic Light Scattering (DLS)
Measurements
SAXS DATA from GSTdMT
Data collected from a1.5 mg/ml
GSTdMT solution at X33 camera on
DORIS storage ring. EMBL Hamburg
Outstation.
5.6
Guinier plot of GSTdMT
Linear Fit
5.5
GSTdMT
2
ln (I)
3
5.4
5.3
log (I)
1
5.2
0
5.1
-1
0.03
0.06
0.08
0.10
0.12
0.01
s2 (nm-2)
-2
0.02
0.00
-0.01
-0.02
-3
0.5
1.0
1.5
s (nm-1)
2.0
2.5
3.0
-0.03
0.06
GSTdMT exists as a dimer in solution.
The monomer has an extended structure.
0.08
0.10
s2 (nm-2)
0.12
ab initio SHAPE DETERMINATION of GSTdMT
Low-resolution GSTdMT structural model (GASBOR)
GST molecules are located in the center of the dimer and dMT
molecules extend from the center.
SAXS DATA from dMT
1.0 mg/ml dMT solution.
4.4
4.2
2.0
dMT
4.0
ln (I)
1.5
Log (I)
Guinier plot of dMT
linear fit
1.0
3.8
3.6
3.4
0.5
3.2
0.2
0.0
X Data
0.1
0.0
-0.1
-0.5
-0.2
0.5
1.0
1.5
s (nm-1)
2.0
2.5
3.0
-0.3
0.20
0.25
0.30
2
s (nm-2)
Experiments are possible only on SR source.
dMT exists as a dimer in solution with an
extended structure.
0.35
0.40
ab initio SHAPE DETERMINATION of dMT
Asymmetry in the structure of dMT?
Implications for Cd-binding?
Domain folding?
Functional implications.
FUTURE OUTLOOK
Macromolecular crystallography
High throughput crystal structure determination.
Automated remote screening and data collection.
Time-resolved crystallography.
Crystallography and SAXS.
X-ray Scattering
Cryo-SAXS.
Time-resolved SAXS.
High-resolution micro-beam SAXS.
Combination with SRCD.
Spectroscopy
Infrared microspectroscopy.
EXAFS and imaging.
Imaging
Imaging and spectroscopy.
3D tomography.
Imaging single particles…..
Useful information can be found at:
1. SSRL website: www-ssrl.slac.stanford.edu
2. www.lightsources.org
ACKNOWLEDEGEMENTS
Sabanci University
F.Dede
G. Dinler
F. Kisaayak
U. Sezerman
H. Budak
O.Gokce
I. Cakmak
SESAME
Z. Hussain
S. Hasnain
G. Vignola
H. Winick
EMBL Hamburg
M.H.J. Koch
D. Svergun
M. Roessle
A. Round
M. V. Petoukhov