Download BCM 6200 - Protein crystallography - I Crystal symmetry X-ray

BCM 6200 - Protein crystallography - I
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Crystal symmetry
X-ray diffraction
Protein crystallization
X-ray sources
SAXS
Université de Montréal
Faculté de Médecine
Département de biochimie
X-ray sources
• The hardware needed for the collection of X-ray diffraction data are
1) X-ray source
2) X-ray detector
3) Crystal rotation instrument to collect the diffracted data
• X-ray sources breakdown into three kinds:
1) home sources
o Microfocus sealed x-ray tube beam
4 x 108 ph/sec (200m dia)
o Rotating Anode Generator ~
2 x 1010 ph/sec (200m dia)
o MetalJet D2
5 x 1010 ph/sec (10m dia)
2) synchrotron X-ray sources – 2nd/3rd generation
o CLS CMCF 081D-1 beamline (3rd)
2 x 1012 ph/sec (100 m)2
o APS 14-BM-D beamline (3rd)
2 x 1010 ph/sec (200 m dia)
o NSLS X26 beamline (2nd)
5 x 1011 ph/sec (100 um)2
3) X-ray free electron laser (XFEL )
> 4 x 1021 ph/sec in 5–10 fs pulses
- improvement in peak brightness ~ ten orders of magnitude over 3rd generation Xray light sources
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
2
Home X-ray sources
• X-ray tubes consist of a filament that acts as a cathode.
• Electrons are emitted by the glowing cathode and
accelerated by several tens of kVs across the vacuum
towards the anode, which consists of a metal target made of
a characteristic material, usually copper or chromium, for
protein crystallography
• As the electron beam impacts the anode, the high kinetic
energy of the electrons is converted during deceleration
into X-rays producing
• a continuous spectrum consisting of bremsstrahlung
("braking radiation") and
MetalJet (Ga rich alloy
• emission lines characteristic for electronic transitions
9.2keV – molten at RT)
caused in the anode material.
• The characteristic X-ray emissions, which are important for crystallography, have an
intensity that is several orders of magnitude higher than the bremsstrahlung.
• The specific components of the X-rays emission are cut out from the bremsstrahlung and
other emission lines by filters, monochromators or X-ray mirrors, and the resulting
monochromatic X-rays are collimated and focused onto the crystals.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
3
Home X-ray sources
• The electron energy = e x V (accelerating
voltage), where e is the electron charge.
• The photon energy is hv = h(c/), where h is
Planck's constant, v is the frequency of the
radiation, c is the speed of light, and  is the
wavelength.
• Therefore
• The sharp peaks in the spectrum are due to
electron transitions between inner orbitals in
the atoms of the anode material.
• The high-energy electrons reaching the
anode kick electrons out of low-lying orbitals
in the anode atoms.
• Electrons from higher orbitals occupy the
empty positions and the energy released in
this process is emitted as X-ray radiation of
specific wavelengths as shown
The spectrum from an X-ray tube with a copper
anode (8.0 keV). It shows a continuous spectrum
and two sharp peaks due to quantized electrons
in the copper. I is the energy of the emitted
radiation on an arbitrary scale.
The wavelength of an in-house source is fixed by the choice of anode target material
and is not tunable, as is the case at a synchrotron.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
4
Home X-ray sources
• For emission of the characteristic lines
in the spectrum, a minimum excitation
voltage is required.
• For example, for the emission at Cu K
line, V should be at least 8 kV.
• If a higher voltage is applied, the
intensity emitted is stronger with
respect to the continuous radiation, up
to V / Vmin  4.
• The intensity is also proportional to the
tube current, as long as the anode is
not overloaded.
• The heating of the anode caused by the
electron beam at the focal spot limits
the maximum power of the tube.
• This limit can be moved to a higher
power loading if anode target rotates
at high speed to efficiently distribute
and dissipate heat.
Université de Montréal
Faculté de Médecine
Département de biochimie
Schematic representation of the atomic energy levels and
transitions causing characteristic X-ray wavelengths. K
radiation from a transition of the L-shell to the K -shell and
K for a transition from the M-shell to the K-shell. Because of
the fine structure in the L-shell, K is split into K(1) and
K(2). The weighted average value for K(1) and K(2)
wavelengths when Cu is the anode material is taken as
1.54178 Å, while K is 1.39217 Å.
MetalJet technology replaces the solid anode
with a jet of liquid metal that is molten and
continuously regenerated. MetalJet sources
thus generate more x-rays from a small spot
with performance closer to synchrotron-lightsource
BCM 6200
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Synchrotron radiation
Insertion device
Synchrotron radiation is emitted when a beam of
electrons moving close to the speed of light is bent by
a powerful magnetic field
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Highly intense X-ray beam
Wavelength tunable
X-ray bunches – picosecond intervals
Polarized
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
6
Synchrotron devices
Intensity
• At a synchrotron facility, bunches of electrons, several GeV
in energy, move in a large, carefully steered, closed electron
beam loop containing bending elements and linear
segments, collectively called the storage ring.
• In each section, magnetic devices are inserted - bending
magnets in the curved sections, insertion devices called
wigglers and undulators in the straight sections - to bend,
wiggle or undulate the path of the electrons while they
pass around the ring.
• Due to the acceleration experienced in the bending
magnets or insertion devices, the electrons emit a narrow
fan of intense white (polychromatic) radiation ranging from
soft UV to hard X-rays over a very tightly defined angle
tangential to the ring.
• The radiation is 'tunable' by cutting out fine bands (few eV
or 10-5 Å wide) of wavelengths appropriate for particular
experiments with monochromator crystals that selectively
pass the wavelength of choice.
From http://internal.physics.uwa.edu.au/~hammond/SyncRes/making-synchrotron-light.htm
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
7
Properties of Synchrotron Radiation
 Intensity
• The main advantage of synchrotron radiation for X-ray diffraction is
its high intensity, which can be at least several orders of magnitude
greater than for a conventional X-ray tube.
• A further advantage is the very low angular divergence of the beam
resulting in sharper diffraction spots reducing overlap between
diffracted intensities.
 Tunability
• Synchrotron radiation is tunable. Any suitable wavelength in the
spectral range can be selected with a monochromator.
• This property is essential for routinely solving protein structures by
anomalous dispersion.
• Wavelengths tuned to 1 A or less reduces absorption and minimizes
radiation damage to the protein crystal even at 100 K.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
8
Properties of Synchrotron Radiation
 Lifetime
• The lifetime of a storage ring filling is limited. When the intensity of the radiation has
fallen to a certain minimum value, a new injection is required.
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Top-off operations is a quasi-continuous injection mode that increases the flux and brightness of
a synchrotron source and improves thermal stability of optical components by maintaining a
constant current in the storage ring.
• Positive charged ions that form accumulate in the electron beam, leading to beam losses.
• A beam composed of positrons has a longer lifetime, because positrons repel positive ions
created in the residual gas, reducing losses.
 Time Structure
• Synchrotron radiation is produced in flashes by the circulating bunches of charged
particles.
• Synchrotrons operate in a single or multi bunch mode with a bunch length in the
picosecond range.
• This allows structural changes in the nanosecond timescale to be observed.
 Polarization
• Synchrotron radiation is highly polarized - fully polarized with the electric vector in the
orbit plane (parallel polarization) and a small perpendicular component out of the plane.
• Important when applying the correct polarization factor and affects anomalous X-ray
scattering of atoms.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
9
Monochromators
• Following the Bragg law (2d sin θ = n ), each
component wavelength of a polychromatic
beam of radiation directed at a single crystal
of known orientation and d-spacing will be
diffracted at a discrete angle.
• Monochromators make use of this fact to
selectively remove radiation outside of a
tunable energy range, and pass only the
radiation of interest
• Mirrors reflect polychromatic X-rays at their
respective grazing angle.
A double monochromator.
The rotation axis for
changing the wavelength is
perpendicular to the plane
of the page. This gives a
small vertical shift to the exit
beam after rotation, but the
incoming and exit beams are
parallel.
Université de Montréal
Faculté de Médecine
Département de biochimie
The white synchrotron light beam impinges onto
a polychromator bent crystal, at several different
incident angles, reflecting a polychromatic beam.
The reflected beam is selected with a specific
bandwidth of hundreds of eV and it is horizontally
focused at the sample position. Vertical focusing
is accomplished by a bendable mirror before the
crystal.
BCM 6200
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Detectors
• Photographic film is a classical detector for X-ray radiation, but not used much because of
the availability of far more sensitive image plates and area detectors. The single
advantage of film over area detectors was its superior resolution resulting from its fine
grain  single photon counters  image plates.
• CCD (charge coupled device) detectors can collect Xray data at high frequency (seconds exposure times).
Their light-sensitive integrated circuit stores and
displays the number of X-ray photons collected at
each pixel as an electrical charge that can be readout
rapidly. They have fast response, low noise, a
reasonable dynamic range, and good linearity. A
disadvantage is that they operate at very low
temperatures (-70° C) to minimize internal noise.
• Pilatus - silicon pixel detectors developed at Swiss Light Source.
X-rays are converted to an electrical signal by photoelectric
effect in silicon and counted directly by a series of cells bonded
to the silicon detector, rather than relying on a phosphor. Each
pixel has its own amplifier, discriminator (for distinguishing Xrays of the desired energy from noise) and counter circuit; fast
response (7 ms readout), no readout noise, superior dynamic
range - count rate > 6 × 106 ph/s/pixel, high resolution (pixel)
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
11
Rotation (Oscillation) Instrument
• Goniometers are used to rotate crystals. The crystal (and the reciprocal lattice) must
rotated through the Ewald sphere to collect all diffracted intensities.
• An efficient strategy uses small oscillation (rotation) steps to collect diffracted intensities
to avoid overlaps. Data collection strategies should minimize systematic errors such as
spot overlaps and maximize data completeness; high redundancy is also an advantage.
• Modern oscillation cameras allow rotation of the
crystal around one or more axes. The reflections
appear in a seemingly disordered arrangement
on the detector.
• Their position is determined from the following:
1. The crystal orientation.
2. The unit cell parameters in the crystal.
3. The crystal-to-film distance and the wavelength.
4. The film center.
• Intelligent data reduction software mentioned
previously can recognize (index) the intensity spots, Modern KAPPA Goniometer showing the
apply correction factors, and supply the protein
incident X-ray beam collimator with beam stop,
the CCD detector face, the cold nitrogen jet
crystallographer with a final dataset.
nozzle, and the protein crystal mounted on the
goniometer head
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
12
Radiation Damage
• The problem of radiation damage suffered by protein crystals during x-ray structure
determination has plagued crystallographers since the early days of the field
• Often multiple crystals were required to obtain a complete room temperature
data set.
• The introduction of macromolecular crystallography cryocooling techniques in the
1990s reduced the rate of radiation damage to the extent that researchers could
largely disregard it.
• One could normally obtain all of the required data from a single crystal.
• Increased lifetime at 100 K relative to room temperature is  70.
• However, with the advent of 3rd generation synchrotrons and the provision of
undulator fed beamlines, the observation of radiation damage has become
commonplace:
• Manifested as a reduction in diffracting power and a loss of high-resolution
reflections even at cryotemperatures of 100 K.
• In addition, the incident x-rays induce specific structural changes in the protein in a
well defined sequence, some amino acids being more susceptible than others.
• This phenomenon can change the biological information inferred from an x-ray
structure, the active sites of enzymes and metalloproteins being particularly affected.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
13
Radiation Damage
• X-ray photons cause radical formation, which leads to chemical reactions that
gradually destroy crystalline order.
• Radicals are produced in the biological macromolecules and the solvent.
• Some radicals (e.g., oxygen or hydroxy radicals) diffuse away and exercise their
damaging effect at other sites in the crystal.
• Radiation damage can be reduced:
a. with sensitive X-ray detectors that allow relatively short exposure times, and
b. strongly reduced by cooling crystals to cryogenic temperatures (100 and 120 K).
• At these temperatures, radicals are still created by the X -ray photons but their
diffusion through the crystal is eliminated.
• Even at cryotemperatures, specific groups in the protein are nevertheless damaged.
a. Disulfide bonds are especially prone to be damaged, leading to bond cleavage.
b. Carboxylic acids can be decarboxylated; cysteine, methionine and tyrosine can
also suffer.
• Data treatment: Extrapolate to zero dose improves value of the measured intensity.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
14
Cryocrystallography and radiation damage
(a) A 100K cryocooled protein crystal held in a vitrified cryobuffer loop. (b) A 100K
cryocooled protein crystal that has been subjected to data collection at three positions
on a synchrotron undulator X-ray beamline and then allowed to warm up in cryobuffer.
The release of secondary radiation products on warming has blackened the crystal at the
beam spots. Additionally, gas evolved on warming.
Garman E (2003) ‘Cool’ crystals: macromolecular cryocrystallography and
radiation damage. Curr Op Struct Biol 13(5), 545–551
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
15
Cryoprotectants
How to cryocool : Garman E.
(1999) Cool data: quantity AND
quality. Acta Crystallogr D Biol
Crystallogr. 55(10):1641-53.
Garman E (2003) ‘Cool’ crystals:
macromolecular cryocrystallography and
radiation damage. Curr Op Struct Biol
13(5), 545–551
Bar chart showing the most commonly used cryoprotectant agents. Co-crystallisation
refers to conditions that do not require the addition of cryoprotectant agent. Cryobuffers
that contain more than one cryoprotectant were grouped into the ‘mixed’ category. Data
were compiled from a survey of all papers published in Acta Crystallographica D: 2000,
146; 2001, 175; 2002, 210; Jan–May 2003, 122. MPD, 2-methyl-2,4-pentanediol.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
16
Radiation Damage at 100K
The program RADDOSE is
widely used to compute
the dose absorbed by a
macromolecular crystal
during an X-ray diffraction
experiment. A number of
factors affect the absorbed
dose, including the
incident X-ray flux density,
the photon energy and the
composition of the
macromolecule and of the
buffer in the crystal.
Radiation damage induces systematic decay in the diffraction pattern. This is evident
from the monotonic decrease in the numbers of diffraction spots and resolution
estimate. It is clear that the sample decayed significantly after image 300. The blue and
red circles show the total number and the number of ‘good’ spots respectively, while
the green triangles show the resolution limit.
Winter G, McAuley KE. Automated data collection for macromolecular
crystallography (2011) Methods Struct. Proteo. 55(1), 81–93
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
17
Site Specific Radiation Damage at 100K
Owen RL, Rudiño-Piñera E, Garman EF. Experimental determination of the
radiation dose limit for cryocooled protein crystals. Proc Natl Acad Sci USA.
2006;103(13):4912-7.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
Electron density of the ferritin
structures (contoured at 0.2
electrons per Å3) refined from the
first eight data sets (Top and
Middle), showing the specific
structural damage suffered by Glu63, Arg-52 (from a symmetryrelated molecule), and a water
molecule. Between each data set,
crystals were exposed to controlled
periods of unattenuated beam.
Bottom shows residue Thr-29 from
every other data set. It shows no
degradation in the quality of the
electron density, similar to the
sulfur-containing residue (Cys-48)
visible in the top eight maps. The
resilience of these residues to high
dose illustrates the highly specific
nature of radiation damage in
proteins.
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Radiation Safety
• High-energy photons of X-rays have a harmful effect on living tissue. Therefore,
they must be used with great care, taking all necessary precautions.
• Local regulations for the protection of personnel should be obeyed and
unauthorized use of X -ray equipment should be forbidden.
• A somewhat confusing number of radiation units are in use.
• A definition of the important ones is as follows.
• The radiation absorbed dose (rad) is a measure for the amount of
radiation that corresponds to the energy absorption in a certain medium,
such as a tissue.
 One rad is the dose of radiation corresponding to an energy
absorption of 0.01 J/kg medium.
• It has been found that the same absorbed dose from different types of
radiation does not always have the same harmful effect in biological
systems.
 To convert absorbed dose to dose equivalent, or "rem," where we
now consider the biological effects in man, one modifies with a
quality factor. For practical scenarios, with low "linear energy
transfer" (LET) radiation such as gamma or x rays, 1 rad = 1 rem.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
19
X-ray free electron laser (XFEL)
• An unique capability of XFELs is their peak flux or brightness (flux per source
size and per emission angle)
• An XFEL source emits the same number of X-rays that we can get today in 1
second from a 3rd generation synchrotron source (1012-1013 ph/s) in a single
ultra-fast single burst of about 100 femtoseconds.
• Hence XFELs allow snapshots to be taken of the motion of atoms, in effect
almost freezing their motion, and of chemical processes such as bond
breakage.
• A second feature of XFELs is the elimination of the need of growing crystals of
sufficient size for structure determination.
• With 3rd generation synchrotrons, the necessary increase in X-ray dose to
record data from crystals that are too small leads to extensive damage before
a diffraction signal can be recorded.
• Using femtosecond pulses from a X-ray free-electron laser, structure
determination can be undertaken from single-crystal X-ray diffraction
‘snapshots’ collected from a fully hydrated stream of nanocrystals.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
20
How it works: X-ray free electron laser
• The key to understanding of how an XFEL works is the free-electron laser instability
or self-organization of electrons in a relativistic beam.
• This instability takes an electron beam with a random electron position distribution,
and changes it into a distribution with electrons regularly spaced at about the x-ray
wavelength, producing what could be called a 1-dimensional electron crystal. This
creates spikes of intensity.
Basic ingredients of a free-electron laser, including an electron gun, an accelerator and an
'undulator' or 'wiggler' that produces a periodic B-field. Bottom: as the electron bunch travels
along the undulator or wiggler, microbunches are progressively built. The microbunching is due to
the interaction between the travelling electrons and the previously emitted waves.
Ribic PR, Margaritondo G (2012) Status and prospects of x-ray free-electron lasers (X-FELs): a simple
presentation. J. Phys. D: Appl. Phys. 45 213001 doi:10.1088/0022-3727/45/21/213001
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
21
Data Collection: X-ray free electron laser
Layout of the diffraction experiment. The XFEL beam is focused on the liquid jet, which
contains fully hydrated crystals. The
diffracted x-rays are collected with two CCD
detectors. The inset in the upper-left corner
shows a scanning electron microscope (SEM)
image of the nozzle, the flowing jet and
focusing gas.
Technical details from Chapman H N & 87 others (2011). Femtosecond X-ray protein
nanocrystallography. Nature, 470(7332), 73-77.
• Nanocrystals flow in their buffer solution in a gas-focused, 4-μm-diameter jet at a
velocity of 10 m s−1 perpendicular to the pulsed XFEL beam that is focused on the jet.
• Two pairs of high-frame-rate pnCCD detectors record low- and high-angle diffraction
from single X-ray FEL pulses, at the FEL repetition rate of 30 Hz.
• Crystals arrive at random times and orientations in the beam, and the probability of
hitting one is proportional to the crystal concentration.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
22
Proof of Concept
• Proof of concept with photosystem I, one of
the largest membrane protein complexes.
• More than 3,000,000 diffraction patterns
were collected and a three-dimensional data
set was assembled from individual
photosystem I nanocrystals (200 nm to
2 μm in size).
• The problem of radiation damage mitigated
by using pulses briefer than the timescale of
most damage processes.
• A new approach to structure determination
of macromolecules that 1) do not yield
crystals of sufficient size for studies using
conventional radiation sources or 2) are
(a) Diffraction 'snapshot' of a single nanocrystal of the protein
particularly sensitive to radiation damage.
photosystem I. Some peaks labeled with their Miller indices
(hkl). The resolution in the lower detector corner is 8.5 Å.
(b) Diffraction data from 15000 nanocrystal patterns merged into a single dataset, displayed on the linear colour
scale shown on the right. (c) Region of the 2mFo − DFc electron density map at 1.0σ (purple mesh), calculated from
the 70-fs data and (d) obtained from conventional synchrotron diffraction experiments on photosystem I single
crystals (for comparison). From Chapman H N & 87 others (2011). Femtosecond X-ray protein nanocrystallography.
Nature, 470(7332), 73-77
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
23
Outrunning radiation damage
(a) Lyosozyme exposed to an X-ray pulse with
an FWHM of 2 fs, and disintegration followed
in time. Atomic positions in the first two
structures (before and after the pulse) are
practically identical at this pulse length
because of an inertial delay in the explosion.
(c) Behavior of lyosozyme during an Xray pulse with an FWHM of 50 fs
(b) Lysozyme exposed to the
same number of photons as in
a, but the FWHM of the pulse
was 10 fs. Images show the
structure at the beginning, in
the middle and near the end of
the X-ray pulse
Université de Montréal
Faculté de Médecine
Département de biochimie
Using a computer
simulation, by the end of
a XFEL 100-fs pulse each
atom of lysozyme was
ionized once, on average,
and motion of nuclei had
begun.
Neutze R, Wouts R, van der Spoel D,
Weckert E, Hajdu J. (2000) Potential for
biomolecular imaging with femtosecond
X-ray pulses. Nature. 406(6797):752-7.
BCM 6200
24
Experimental setup: Time resolved
nanocrystallography
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•
•
•
•
Experimental setup and intersection
of the 3 interacting beams: X-rays,
pump laser, and the liquid jet
containing the co-crystals.
The upper left insert shows the
thermal glow of the 2 keV X-rays
interacting with the liquid jet.
The lower insert shows the scatter
from the frequency-doubled Nd:YAG
pump laser from the liquid jet.
The overlap of the two beams (liquid
jet and X-rays) can be seen, with the
pump laser intersecting the liquid jet
of cocrystals and extending upstream
towards the nozzle.
The pump laser is extended
upstream of the X-ray pulse to
compensate for the approximately
130 μm travel of the crystals
between the pump and probe pulses
for the 10 μs delay time.
Aquila & 79 et al. (2012) Time-resolved protein nanocrystallography using an X-ray free electron laser. Opt Express. 20(3): 2706–2716
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
25
Direct observation of ultrafast collective motions
in CO myoglobin upon ligand dissociation
(A) Stereo figure, showing the structure of myoglobin
as well as the F(light) – F(dark) difference electron
density for the 0.5 ps time delay data, contoured at
+3σ (green) and –3σ (red). (B to D) Difference
electron density maps (F(light) – F(dark), showing the
bound and photodissociated CO, the doming of the
heme, the out-of-plane movement of the iron, and
the concomitant movement of His93 away from the
heme as well as the rotation of His64 and the
movement of Phe43, which is displaced in different
directions at the two time delays shown (0.5 and 10
ps). The orientation in panel (B) is rotated by 90° with
respect to the orientation in panels (C) and (D).
Barends TR, Foucar L, Ardevol A, Nass K, Aquila A, Botha S, Doak RB, Falahati K, Hartmann E, Hilpert M, Heinz M, Hoffmann MC, Köfinger J, Koglin JE,
Kovacsova G, Liang M, Milathianaki D, Lemke H, Reinstein J, Roome CM, Shoeman RL, Williams GJ, Burghardt I, Hummer G, Boutet S, Schlichting I. Direct
observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science. 2015 Sep 10.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
26
Coupling of ultrafast local modes to slower,
global motions in CO myoglobin
Ultrafast heme modes (0.5 - 3 ps),
such as heme doming (①), are
coupled to the F-helix through
residues contacting the heme such as
His93 and His97 (②). These motions
are transmitted to other parts of the
protein ( ~ 10 ps) such as the CD
corner (through the Lys98-Lys42
interaction), ultimately resulting in a
coupling with slower normal modes
(100 ps) of the protein such as the
motions of the E- and F-helices (③).
Barends TR, Foucar L, Ardevol A, Nass K, Aquila A, Botha S, Doak RB, Falahati K, Hartmann E, Hilpert M, Heinz M, Hoffmann MC, Köfinger J, Koglin JE,
Kovacsova G, Liang M, Milathianaki D, Lemke H, Reinstein J, Roome CM, Shoeman RL, Williams GJ, Burghardt I, Hummer G, Boutet S, Schlichting I. Direct
observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science. 2015 Sep 10.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
27
Future enhancements
• Data are collected on fully hydrated nanocrystals without cryogenic cooling.
• Use of X-ray laser pulses that are so short that only negligible X-ray-induced radiation
damage occurs during data collection.
• Significant improvements in sample utilization by exploiting higher X-ray repetition rates
(greater number of crystal hits) or by slowing the liquid flow.
• A viscous LCP microjet consuming approximately two orders of magnitude less
sample (membrane proteins) developed
• An injection system using microcrystals suspended in a grease matrix, makes it
possible to collect complete SFX diffraction data with 100 μl or less of microcrystals.
• Further efficiency gains would result from indexing and merging a greater proportion of
patterns into the 3D data set, which may be achieved by applying methods for merging
continuous diffraction patterns of single molecules or by using ‘post-refinement’ to
obtain accurate structure factor estimates from fewer diffraction patterns.
• These methods will also remove the twinning ambiguity that exists in our current
indexing scheme.
• Our method also has potential application to the study of chemical reactions, such as the
processes in photosynthesis or enzymatic reactions.
1. Chapman H N & 87 others (2011). Femtosecond X-ray protein nanocrystallography. Nature, 470(7332), 73-77.
2. Neutze et al. Membrane protein structural biology using X-ray free electron lasers. Curr Opin Struct Biol. 2015 Sep 2;33:115-125. Review.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
28
Linac Coherent Light Source
(LCLS) at SLAC
• LCLS - 38,000 megawatt-hours
per year
• SPring 8 synchrotron - 137,000
megawatt-hours in FY 2010.
• NSLS-II will draw up to 20
megawatt (48,000 megawatthours per year) of power, or as
much as needed to power
about 300,000 light bulbs or
4,000 homes
• Canadian Light Source Facility
Electrical Budget: $1.6 million
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
29
Cutting Edge
•
Coherent diffractive imaging (CDI) is a “lensless” technique for 2D or 3D reconstruction
of nanoscale structures including proteins
 A highly coherent beam of x-rays illuminates an object.
 The beam scattered by the object produces a diffraction pattern.
 The recorded diffraction pattern is then used to reconstruct an image via an
iterative feedback algorithm.
o Here, the objective lens in a typical microscope is replaced with software to reconstruct
the real space image from the reciprocal space diffraction pattern.
o The advantage in using no lenses is that the final image is aberration–free and so
resolution is only diffraction and dose limited (dependent on wavelength, aperture size
and exposure).
 A simple Fourier transform retrieves only the intensity information and so is
insufficient for creating an image from the diffraction pattern due to the phase
problem.
•
Current limitation for high intensity coherent X-ray sources is being removed using
bright 3rd and 4th generation synchrotrons or XFEL sources
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
30
Ptychography : Coherent Diffractive Imaging
Ptychography is an experimental method developed
in the 1970s for electron microscopy which consists
of measuring multiple diffraction patterns by
scanning an extended sample through the coherent
X-ray beam while adjacent probe spots overlap
significantly in the object plane. Overlap between
adjacent illumination positions provides
overdetermination in the data (redundancy).
The Ptychographical Iterative (Engine) algorithm (PIE) uses the redundant information
provided by the overlap of many diffraction patterns (novel oversampling condition)
• Advantages are noise tolerance,
high speed of convergence and
high resolution of reconstructed
images.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
31
FIRST HIGH RESOLUTION 3D X-RAY DIFFRACTION MICROSCOPY of a VIRUS
Coherent x-ray diffraction
patterns were obtained from a
single, unstained herpesvirus
virion, and then directly inverted
to yield quantitative and high
contrast electron density maps
with a resolution of 22 nm. The
quantitative structure of the viral
capsid inside the virion was
visualized.
Schematic layout of the x-ray diffraction microscope. A 20-µm pinhole
was used to define the incident xray beam. The virion specimen was
positioned at a distance of 1 m from the pinhole. A silicon guard slit
with beveled edges was used to eliminate the parasitic scattering from
the pinhole. The oversampled diffraction pattern, recorded on a liquidnitrogen-cooled CCD camera, was directly inverted to a high-contrast
image using an iterative phase retrieval by the guided hybrid-inputoutput algorithm (GHIO)
Song et al. Quantitative imaging of single, unstained viruses with
coherent x rays. Phys Rev Lett. 2008 Oct 10;101(15):158101.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
Present resolution (circa 2010) was limited
by the coherent x-ray flux, higher
resolutions should be achievable by using
more brilliant synchrotron radiation sources
32
Ptychography : Protein nanocrystals and 2D-crystals
• In an extension of ptychography to 2D-crystal diffraction, a large (~ several μm) 2dcrystal is illuminated at randomly chosen positions with the tightly-focused (100 nm)
coherent XFEL beam.
• The resulting scattering is a convolution of crystalline Bragg peaks and finite-aperture
coherent scattering features.
• Each exposure represents a different alignment between the periodic lattice and the
illumination function describing the spot, and although it locally destroys the sample,
successive exposures from undamaged regions of the sample provide partiallyredundant information.
• Giewekemeyer et al. have demonstrated that this multiple exposure method aids in
solving the phase problem and hence facilitates the recovery of the projected highresolution structure of a membrane protein.
• By repeated scans at different tilt angles of the 2D-crystal, it may be possible to obtain
the 3D-protein structure.
Giewekemeyer K, Thibault P, Kalbfleisch S, Beerlink A, Kewish CM, Dierolf M, Pfeiffer F, Salditt
T. Quantitative biological imaging by ptychographic x-ray diffraction microscopy. Proc Natl
Acad Sci U S A. 2010 Jan 12;107(2):529-34.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
33
Speckle and lensless imaging
(a)
Whereas the scattering of incoherent
radiation, e.g. from a synchrotron, yields only
the average spacing d of a collection of
scatterers (a), the scattering of coherent
radiation (with beam diameter a) produces a
rich speckle pattern (b), which can be inverted
to obtain the exact distribution of scatterers.
(b)
Van der Veen JF & Pfeiffer F. Coherent x-ray
scattering,” J. Phys. Condens. Matter 16, 5003-5030
(2004)
 The phase-retrieval process is performed in an iterative manner, by making an initial
assumption for the phases and repeatedly Fourier and inverse-Fourier transforming
between the real and scattering (reciprocal) space representations.
 Constraints are applied in each iteration cycle.
o In reciprocal space, it is required that the calculated scattering intensity agree with
the measurements.
o In addition, one or more real-space constraints are applied: such as that scattering
from the sample is zero outside a delimiting boundary, that the sample’s electron
density is non-negative, or that the scattering is concentrated in isolated “atoms”.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
34
Fluctuation X-ray
Scattering
http://www.camera.lbl.gov/#!fluctuation-scattering/w29pl
• Although X-ray crystallography has been successful at determining several thousands of
macromolecular structures, it is limited to studying objects which can be formed into crystals.
• Furthermore the structure of biological nanomachines may have a different conformation in their
natural environment than what is determined through crystallography.
• Therefore, as an alternative and complementary technique, structural biologists often gather
diffraction patterns from particles in solution.
• However, in these so called small- and wide-angle X-ray scattering experiments, the particles rotate
during the imaging process, which yields angularly isotropic data and results in a loss of information
that often ultimately leads to poorly determined structural hypotheses.
• One solution to overcome the limitations of SAXS and WAXS is to perform the imaging process below
rotational diffusion times, so that the particles are frozen in position and orientation during imaging.
• From these so-called fluctuation X-ray scattering (FXS) experiments, speckle patterns emerge which
have angularly varying intensities.
• By computing angular correlations of these speckle patterns, one can extract information several
orders of magnitude larger than traditional SAXS and WAXS, which allows one to obtain medium-tohigh resolution three-dimensional reconstructions of macromolecular structure.
Donatelli JJ, Zwart PH, Sethian JA. Iterative phasing for fluctuation X-ray scattering. Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10286-91.
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
35
SAXS and WAXS:
Fluctuation X-ray
Scattering
Model of Model of the X-ray projection of the
pentameric ligand-gated ion channel (pLGIC).
Reconstruction from 3D FXS data using M-TIP.
Resolution 4.8Å
Malmerberg E, Kerfeld CA, Zwart PH. Operational
properties of fluctuation X-ray scattering data.
IUCrJ. 2015 Mar 20;2(Pt 3):309-16.
Université de Montréal
Faculté de Médecine
Département de biochimie
• X-ray scattering images collected on timescales
shorter than rotation diffusion times using a
(partially) coherent beam result in a significant
increase in information content in the scattered data.
• These fluctuation X-ray scattering (FXS)
measurements which are typically performed on an
X-ray free-electron laser (XFEL) can provide detailed
structure of biological molecules beyond what can be
obtained with standard X-ray scattering techniques.
• In order to understand, use and validate experimental
FXS data, the availability of basic data characteristics
and operational properties is essential.
• Recently, a computational approach has been
developed to interpret FXS data and properties that
shows:
o How FXS data can be used to derived structural models
o Its generalization to the Guinier and Porod laws
o How to plan experiments and assess the quality of
experimental data
BCM 6200
36
Free-electron laser instability
I. Electrons, propagating through the undulator, interact with the electromagnetic
field generated by other electrons. The interaction changes the electron energy,
and the change is modulated with the same period of the radiation wavelength, .
II. In the undulator magnetic field the trajectory of electrons with larger (smaller)
energy is bent less (more). The length of the electron trajectory changes and the
electrons within one radiation wavelength tend to get nearer to each other.
III. Since electrons are now near to each other within a wavelength, their
electromagnetic fields superimpose in phase, and the total field has a larger
amplitude. This in turn means that the interaction between the electrons and the
electromagnetic field becomes stronger the mechanism for getting them together
becomes more effective. The result is that the amplitude of the electromagnetic
field grows exponentially. The rate at which it grows is called the gain length, and is
the most important parameter characterizing the instability. The exponential
growth saturates when all the electrons are well ordered, and sing perfectly in
tune!
http://www-ssrl.slac.stanford.edu/stohr/xfels.pdf
Université de Montréal
Faculté de Médecine
Département de biochimie
BCM 6200
37