Data Collection
Diffraction experiment:
X-ray source
Synchrotron
Lab sources (Sealed
tube, rotating anode)
Optics
Focusing mirrors
Monochromators
 select Kα or Kβ
Monochromatic X-ray beam
Crystal
30μm
100μm
2D Detector
Orthogonal to the
rotation axis and
typically but not
necessary to the
incident beam.
2 between diffracted and unaffected beam
Standard method of data collection for macromolecules:
- Copper rotating anode
- rotation method
- area detector
X-ray sources:
Seald tube
vacuum
A = anode of copper or molybdenum cooled by water (Win – Wout)
X = X-rays obtained through Beryllium windows
K = Katode emitting a beam of high speed electrons (blue points; energy = 10,000 eV) due to high
negative potential with respect to the anode
U = 30 to 150kV between K and A– accelerates the electrons
Disadvantage:
limited intensity because of melting of the anode at higher tension
Rotating anode
Rotation allows better cooling of the anode and therefore application of higher powers (12-18kV)
generating higher intensities.
Disadvantage:
continuous pumping to maintain the vacuum
Synchrotron
85% macromolecular structures
rings with UHV (ultra high vacuum) and radius of 10-100m in which electrons circulate at velocities
99,9% of light speed
Magnetic fields bend the electron beam (acceleration toward the center of the ring) which then
emittes X-rays: wide wavelength region depending on particle energy and field strength
3rd generation
elmag. radiation
collision of particles
Advantages:
-
-
High flux = many photons (macromolecular crystals show weak diffraction due to big
dimensions of proteins (fewer in the crystal than of small molecules) and due to small
number of electrons in light elements like N, O and C are)
High collimated and small beam:
instead of
(brilliance = ?radiance = Strahlendichte) => small macromolecular crystals
Tunablility = ability to choose wavelength (MAD)
polarized electromagnetic radiation
Time structure (radiation is produced in flashes: allows time resolved experiments i.e.
snapshots of reaction intermediates)
Devices:
-
-
Bending magnets (guide electrons in their orbit: dependent of their energy and the
magnetic field)
Wavelength shifter (-> shorter wavelength = higher energy due to increased field
strength)
Multipole wiggler = a series of wavelength shifter (increasing of intensity due to
constructive interference of the radiation of single periods -> fluxtotal = 2N*fluxsingle period
with N periods)
-
Undulator (for collimation on the orbit plane of the electrons, nearly monochromatic
radiation = narrow engery bands compared to wiggler due to strong interference
because of the moderate magnetic field and poles close together, tunability by
adjustment of the pole distance, intensity total= N2*intensitysingle periode)
Crystal Mounting:
Avoid precession (Kreiselbewegung) upon spindle axis rotation. You have to center the crystal on the
goniometer.
-
-
Capillary Mounting:
o If crystal decay is no problem
o At Room temperature
1. Link a thin-walled capillary tube with a syringe or pipette with the help of a flexible
adaptor.
2. Soak up the crystal.
3. Close the end of the capillary needle with resin(Harz)/clay(Lehm).
4. Remove the excess water by a thin glass capillary.
5. Break off the needle and close the second end with resin/clay.
6. Mark the position of the crystal with a felt tip pen.
Loop Mountin:
o Safe option for synchrotron data collection
o Cryocooling: Flash freeze crystal soaked with cryoprotectant
 Nitrogen steam at 100K and dry air (or nitrogen) at 300K (0°C = 273K)
perpendicular to the X-ray beam
o Kinds of loops:
 Litholoops
 Micromounts
o A thin liquid spans the loop whose size approximate the size of the crystal. By this
way the crystal is fixed. The surface tension has to be big enough.
Tools:
-
-
Manual mount:
o Crystalwands (Stäbe)
o Cryotons
o Reverse pincers (Kneifzange)
crystal mounting and transfer:
o loops homemade and bought
o arcs (Bögen)
o tongs
o
vials and special vials and holders
Transfer the crystal to the diffractometer:
1.
2.
3.
4.
5.
6.
Precool tongs and plunge (abschrecken) the crystal.
Clasp (ergreifen) the mounting pin.
Remove the pin holder.
Carry it to the diffractometer.
Transfer it to the diffractometer.
Open the tongs
Dewar for storage:
-
A dewar is a double walled vessel for isolation and vacuum with mirrored glass for storage of
very hot or very cold liquid.
You fill it with liquid nitrogen.
Storage of crystals in vials stored in baskets in a canister.
Automated crystal transfer from the dewar on the diffractometer:
 Reproducible
 Fast
 expensive
Cryocooling:
Resolution and overall data quality can be improved by flash-cooling the crystals to 100K. For this we
use liquid nitrogen. We mount the crystal in rayon (Kunstseide) and put it in the nitrogen steam





reduced thermal motion
reduced conformational disorder
enhanced signal-to-noise-ratio
higher limiting resolution
SUPPRESSION OF RADIATION DAMAGE
Ad radiation damage:
When X-rays interact with matter free radicals and electrons are formed. This radicals and electrons
can attack the protein. They diffuse through the water channels of crystals.
This diffusion can be prevented by decreasing the temperature. But it does not inhibit the formation
of the radicals and electrons.
Cryoprotectants are added before crystallization:
-
Glycerol
Ethylene glycol
PEG of low molecular weight
Sucrose
They prevent ice formation and support vitrification (vitrifies = amorphous ice).
 increase in viscosity
 depression of freezing temperature
Sudden freezing (flash freezing or shock cooling) also prevents ice formation.
Surrounding the cold gas steam by a warm and dry steam of air or nitrogen avoids the accumulation
of ice on the crystal and the diffraction instrument. Adjusting the flow speeds of the steams prevents
turbulences between them. Further protection against ice formation is enclosing the apparatus in a
box.
Rotation method:
The crystal is fixed on a goniometer for rotation of crystal (Eulerian or Kappa geometry) around an
axis orthogonal to the incident beam.
2.0° > Δ > 0.1°
 Planes of the reciprocal lattice are brought into diffraction conditions.
Rotations is repeated for contiguous angles until at least the independent part (not already
measured because of symmetry) of the reciprocal lattice is completely scanned.
independent
 A set of reciprocal lattice planes will never meet diffraction conditions. This set is collected as
symmetry related. Only if the symmetry axis lies along the or near the rotation axis there will
be a blind region. Kappa goniometer can help.
Advantage:
fast
Disadvantage:
distorted image of reciprocal lattice planes.
Diffraction pattern: lunes
To avoid spots superposition the rotation angle has to be small enough so that the lunes are well
resolved.
Quality Indicators of rotation methode:
-
-
Completeness:
indicates the percentage of reflections experimentally determined with respect to the
theoretical ones for that resolution (should be as near as possible to 100%).
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑢𝑛𝑖𝑞𝑢𝑒 𝑟𝑒𝑓𝑙𝑒𝑐𝑡𝑖𝑜𝑛𝑠 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑢𝑛𝑖𝑞𝑢𝑒 𝑟𝑒𝑓𝑙𝑒𝑐𝑡𝑖𝑜𝑛𝑠
Check the low resolution completeness.
Ideally, every independent (unique) reflection for a chosen resolution should be detected,
but in reality there are always a few reflections omitted.
<I>/σ<I>:
indicates the signal to noise ratio (how strong the diffraction from the crystal is compared to
the backround).
-
-
<I> is the mean intensity.
σ<I> is the mean standard deviation of the measurements.
Hight resolution limit = |I/σ| = for example 2. That means <I> has fallen to twice the mean
standard deviation.
Redundancy:
indicates the number of times that the reflections are measured as such or as symmetry
related.
The higher redundancy the better the reflection intensity estimation.
Rmerge = Σhkl Σi |Ii(hkl) - <I(hkl)>|/Σhkl |<I(hkl)>|
Ii(hkl) are measurements of a symmetry related reflection I(hkl). The number of
measurement is indicated by i.
<I(hkl)> is the mean value of I(hkl). It is ΣIi(hkl)/n
R(merge) can be regarded as the mean error of an intensity measurement compared to the
mean intensity.
Detectors:
 Measure intensities of diffracted beams (-> amplitude of structure factor)
 Number of X-photons
 Energy in a given time
Quality Indicators:
-
-
-
-
Detection Quantum Efficiency:
indicates signal to noise ratio (fraction of photons absorbed from the detector and the
detector intrinsic noise)
= fraction/percentage of the total radiation impinging (auswirkend) on an image receptor
that is actually detected by the receptor.
the higher the better
Dynamic range:
The ability to acquire (annehmen) very strong and very weak spots at the same time.
the wider the better
Detector response:
relationship between incoming photons and detected photons
linear over the entire dynamic range
uniform all over the surface
Point spread function.
ability to discriminate very close spots
Types of detectors:
a) Photon counting:
- Scintillation counters
measurement of individual diffracted beams: one beam at a time
- Multi-wire proportional gas chambers = 2D detectors
slow
fast saturation
outdated technology
b) Energy in a given time -> 2D detectors:
-
-
Films
low sensibility
non-linear response
narrow dynamic range
a lot of work
IP = Image plates
CCD = Charge-coupled devices
Image Plates:
1.) X-ray-photons excite electrons in a thin layer of small crystals of photostimulable phosphor.
2.) Electrons are excited to a conduction band (Leitungsband).
only in semiconductor
(Halbleiter) and
isolators
3.) In the conduction band the electrons are trapped in Br- and F- vacancies into a metastable
state.
4.) A laser scans the plate and the visible light stimulates the electrons
5.) The electrons drop back to the valance band and thereby emit photons.
6.) Photons are detected by a photomultiplier.
Advantages:
- good Detection Quantum Efficiency
- high Dynamic Range
- good Point Spread Function
Disadvantage:
Readout time of minutes
-> poor duty cycle especially for short exposures
Wikipedia:
Duty cycle is the proportion of time during which a component, device, or system is operated.[1]
Suppose a disk drive operates for 1 second, and is shut off for 99 seconds, then is run for 1 second
again, and so on. The drive runs for one out of 100 seconds, or 1/100 of the time, and its duty cycle is
therefore 1/100, or 1 percent.
Charge coupled Device:
1.) X-rays excite a phosphor layer on a fiberoptic taper.
2.) Phosphor emits visible light.
3.) Light is transferred through the fiberoptic taper to the CCD.
4.) In CCD photons of energy greater than 1.1 eV allows a valance electron of the semiconductor
to move into the conduction band.
5.) The free electrons are collected and counted by an electronic system.
Advantages:
- good Detection Quantum Efficiency
- high Dynamic Range
- good Point Spread Funktion
- fast readout
- small, smaller than IP
Properties
Size
Speed of reading
Price
X-ray-source
Exposure time
Image Plates
Small
10 sec
120,000 Euros
Rotating anodes
Few to over 10 min
Charge coupled devices
bigger
< few sec.
250,000 – 1,000,000 Euros
Synchrotron
Few sec