Cryo-crystallography and Data Collection 
Structural Molecular Biology Laboratory, ChemM230D
A one degree oscillation photograph
Suggested Reading Materials

1) Cryocrystallography: A Practical Approach a movie by Steve Ealick, Matt Slaybaugh and Rick Walter, edited for 10 minute run time.

2) Some Notes on Choices in Data Collection by Philip R. Evans, Acta Crystallographica D55 1771-1772 (1999). Or, for a more in-depth treatment, see Data Collection Strategies by Zbigniew Dauter, Acta CrystallographicaD55, 1703-1717 (1999).

3) Safe Handling of Liquid Nitrogen by Duilio Cascio.

4) Radiation Safety Guidance from the Department of Environment, Health & Safety, Radiation Safety Division, UCLA.

5) A table specifying the minimum concentration of glycerol to be added to solution 1 to 50 of the Hampton Screen is available in PDF format. Original reference is Garman & Mitchel,J. Appl. Cryst. (1996). 29 584-587, and included in a cryo kit from Hampton

6) Macromolecular cryocrystallography--methods for cooling and mounting protein crystals at cryogenic temperatures. Pflugrath JW. Methods. 2004 Nov;34(3):415-23. and webinar available here.

 

Laboratory Procedures
 
Part One:
Cryogenic Crystal Recovery Techniques
Illustrations
Objective: To store a crystal for shipping to a synchrotron and then restore it to the goniometer head undamaged. 

Caution: Wear gloves during the crystal storage for protection from the extreme cold of liquid nitrogen. 

Procedures:
 To remove the crystal from the goniometer head for storage, fill a short dewar with liquid nitrogen.  Place the head of the cryotongs in the nitrogen  until it stops boiling.  Clamp the tongs around the crystal and quickly return the crystal to the liquid nitrogen in the dewar.  Clamp the cryovial with a pair of forceps and dunk it in the liquid nitrogen.  The cryo-pin is pierced with small holes (to allow gaseous nitrogen to escape without popping the cryopin off the top of the vial.)  With another pair of forceps clamp the base of the cryopin, and release the crystal from the cryotongs.  Transfer the crystal to the cryovial.  The crystal must remain under liquid nitrogen surface during the entire procedure.  Slight warming of the crystal after it is frozen will often cause loss of diffraction. Screw the cryopin onto the vial using the magnetic wand. You may need to use your fingers to tighten the screw.

 
Part Two:
Cryocooling Procedures
Illustrations
Objective: To learn how to screen for cryo-protection conditions.  Cryoprotect crystals. Flash freeze crystals for data collection, and store them in vials for synchrotron trips.

Method: Cryoprotectant (in this case glycerol) is introduced into conditions resembling the mother liquor composition.  The effectiveness of cryoprotection is evaluated by looking for a clear (not opaque) freeze of the cryoprotected solution in the cryostream.
Once a suitable condition is found, the crystal is quickly dipped in the cryoprotected mother liquor and judged for diffraction quality.  Remove the crystal, place it in a cryo vial and store it in a nitrogen dewar.

Caution: Wear gloves during the crystal storage for protection from the extreme cold of liquid nitrogen.

Procedures:
1) Watch the 10 minute video on cryocrystallography from the Ealick lab at Cornell.

2) At the microscope, select a crystal that you would like to use for data collection.   Gently lift the cover-slip and remove 7 uL of reservoir.  Deposit the reservoir solution on a glass cover slip .  Take a cryoloop and dip it in the cryoprotected solution.  Place the loop on the goniometer head under the cryo stream.  Look through the microscope or video monitor to see if the film in the loop is clear or opaque.  If it is opaque, add 3-3.5 microliters of glycerol to the drop and check again for clarity upon freezing.  normally, one would like to use the minimum amount of glycerol required to provide cryoprotection because too much glycerol could destroy the crystal. 

3)  Select a crystal, and lift it out of the drop with a cryoloop.  Transfer the crystal to the cryoprotected reservoir solution and swish for 1 second.  Lift the crystal out of solution and bring it near the cryostream.  Block the stream with a ruler or buisness card.  Place the loop on the goniometer head.  Then quickly unblock the cryostream.  Cryocooling is successful only when done quickly, hence the term flash-freezing.  The speed of flash freezing prevents formation of crystalline ice, which disrupts the crystal lattice and degrade the data quality. 

4)Take a diffraction image to check for crystal quality.

5) To remove the crystal from the goniometer head for storage, fill a short dewar with liquid nitrogen.  Place the head of the cryotongs in the nitrogen  until it stops boiling.  Clamp the tongs around the crystal and quickly transfer the crystal to the liquid nitrogen in the dewar.  Clamp the cryovial with a pair of forceps and dunk it in the liquid nitrogen.  The cryovial should be clearly labeled. Use a magnetic wand to hold the base of the cryopin, and release the crystal from the cryotongs.  Transfer the crystal to the cryovial.  The crystal must remain under liquid nitrogen surface during the entire procedure.  Slight warming of the crystal after it is frozen will often cause loss of diffraction. 

Tools and Equipment

 


A crystal mounted in a cryoloop, of a cryopin placed in the cryostream.
 
 

 
Part Three:
X-ray Equipment and safety
Illustrations
Objective: To understand the operation of x-ray equipment and use it safely. 

Method: Read the description of the equipment used in the diffraction experiment.

Rigaku FR-E rotating anode X-ray generator : There are two types of x-ray generators commonly used by protein crystallographers today, rotating anode generators and synchrotron radiation sources. Crystallographers prefer to use synchrotron radiation because it is 10 to 1000 times more intense than radiation from rotating anode generators.  The result is stronger, higher resolution data.  The wavelength of synchrotron radiation is also tunable, which allows optimization of anomalous scattering data for MAD or MIRAS phasing techniques.  However, there are only six synchrotrons in the US suitable for protein crystallographers.  They are large (the size of a shopping mall), government funded facilities, operating with a full staff 24 hours a day.  Using a synchrotron is costly in time and travel expenses, and requires months of planning ahead.  Rotating anode generators have the advantage of being commercially available and fit in a typical lab space. 
     The production of X-radiation by rotating anode generators begins by passing a current through a tungsten filament (show filament), typically 50kV, 100mA. Electrons are ejected from the filament and accelerate toward a copper anode (show anode).  The energy from the electrons is absorbed by the copper atom thus ejecting an electron from the inner shell (K-shell).  An outer shell electron (L-shell) then falls into the inner orbit (K-shell), emitting radiation of 1.5418 Angstroms wavelength.  The particular wavlength emitted is a property of copper's L to K electronic transition and can be changed only by replacing the Cu anode with a different metal coating which will have a different electronic transition. 
     The intensity of the x-rays generated is limited by how quickly the heat can be dissipated.  The rotating x-ray generator you are using produces a more intense beam than most other generators because it has a larger than average copper anode.  The larger copper surface distributes the heat over a larger area, keeping the temperature down so that more xrays can be generated with a smaller focal size. The anode rotates so the heat can be dissipated over the entire cyllindrical surface and water is circulated through the inside of the (hollow) anode for further cooling.  The production of x-rays takes place in a vacuum because air molecules would interfere.  Maintenance can be difficult and time consuming because bitter enemies (high voltage, water, and vacuum) often refuse to coexist peacefully in a small space .
    A red light on the generator signifies that X-rays are being produced.  X-rays are continuously generated except during maintenance periods. X-rays are not permitted out of the sealed tower (see tower in the figure on the right) unless a shutter is open by flipping a switch.  A collimator and optics system help collimate and focus the beam on the crystal sample.  A beamstop absorbs all non-diffracted x-rays (see schematic on right).  Do not touch the x-ray collimator or beamstop, it could missalign the beam geometry.

Goniometer assembly: The crystal is held in a loop at the tip of the cryo-pin.  The metallic pin sits on a magnetic base which is attached to the goniometer head.  The goniometer head is a delicate instrument that can be translationally adjusted (in 3 dimensions) to bring the crystal into the x-ray beam and center it on an axis of rotation (omega) defined by the goniometer. The goniometer rotates omega slowly during data collection to bring different reflections into diffraction position.  It is absolutely crictical that the translation screws on the goniometer head be adjusted so that the crystal is centered in the x-ray beam throughout 360 degrees of rotation in omega. Mike or Duilio will demonstrate this centering procedure.

X-ray Detectors: You will be using an Raxis IV++ imaging plate detector (as pictured above).

Imaging Plate detector:  The measurement of diffracted x-rays with an imaging plate detector is a two step process.

Step 1. Exposure to x-rays and creation of the latent image.  An x-ray photon is absorbed by the phosphor matrix and the energy is transferred to a number of Eu2+ sites. Eu2+ is oxidized to Eu3+ and a photoelectron is ejected into the conduction band. The photoelectron becomes trapped in a lattice defect created by the absence of a halogen (F or X) counter ion. These vacancies are created during the manufacturing process andare called F-centers or color centers. 

Step 2. Recovery of the latent image.  HeNe laser light (l = 632nm) is used to irradiate the IP to generate the photostimulated luminescence.  The visible light photons excite the trapped photoelectron in the F-center into the conduction band where it recombines with the Eu3+ in less than 0.8 m seconds, releasing a visible light photon at l = 400 nm. The wavelength of the luminescence is well matched to the detection capabilities of bi-alkali photo-multiplier tubes (PMTs), which have a sensitivity range of about 300 nm to 600 nm. The readout process removes 80% to 90% of the stored image. In order to prepare the IP for reuse all the F-centers must be depopulated. This is accomplished by bleaching the IP with visible light whose spectrum has been adjusted to enhance this depopulation.
 

Comparison of imaging plate to CCD detector.
Image plate (IP) detectors are the general purpose workhorses in protein crystallography. They have a large active surface, sufficient spatial resolution, and are relatively affordable. Their main drawback is that readout (1 minute) is not as fast as the electronic detectors (15 seconds). This is not a real concern at home where exposure times (5-10 minutes) are much longer than the readout times. However, at modern synchrotrons IP detectors are just too slow. 

 CCD detectors are almost the opposite of IP detectors; they are very fast( 30-90second exposures), have a small active surface and are expensive. They also "suffer" from time-dependent noise; both dark current and zingers. CCD Detectors with a larger active surface are made by coupling the detector to a fiber optic, and by tiling multiple CCDs together. CCDs shine when very intense signals are collected very rapidly, i.e. at a synchrotron. In this situation time-dependent noise is small and you really need the readout speed. 

CCD detector: CCD stands for charge-coupled device. They operate on the principle of localized light-induced charge accumulation. The three main components of the Quantum CCD detector are phosphor screen (to convert X-rays to visible light), fiber-optic taper (to demagnify the light image down to the size of the CCD chip), and CCD chip to detect the light image as an electric charge image.  The electric charge image is read  out of the CCD chip and digitized (converted to binary numbers) then fed into a computer. After geometric and intensity corrections are applied, the resulting data are similar to data from other types of X-ray detectors and can be processed by most standard software packages.
 

-adapted from Bart Hazes diffraction tutorial.



Photo ofdiffraction equipment (as viewed from the side) with corresponding schematic (as viewed from above).
 
 
 


Goniometer head assembly.
 


How an imaging plate works 
at various stages of recording and image.
 
 


Contents of the R-axis IV++ imaging plate housing 
shown next to Duilio in Figure 1above.
 
 
 

 
 


 
Part Four:
Data Collection Strategy
Illustrations
Objective: To optimize data collection parameters to obtain measurements with high redundancy, high completeness, and high signal to noise.

Method: Once a satisfactory cryocooled crystal has been mounted, there are a number of practical considerations and decisions that have to be made about data collection strategy. These are listed below.

Detector distance 
       Where should you place the detector?  near or far from the crystal?  There are 3 factors to consider: the resolution of the diffraction, the pixel resolution of the detector, and the absorption of the x-rays by air.  If a crystal is diffracting well, you would like to move the detector close to the crystal to capture the high resolution spots --the closer the detector, the higher the Bragg angle intercepted by the detector.  However, at some point, the limited resolution of the pixels on the detector will be unable to separate one reflection from its neighbor.  This is epecially true if the unit cell of the crystal is large, the reciprocal lattice will be closely spaced.  Moving the detector far from the crystal may cause you to miss the high resolution reflections.   In this case, you would need to rotate the detector (2theta) angle to intercept the high resolution reflections. The solution is to move the detector to the point where you see a reasonable separation of spots.  If you need to move the detector further than 300mm from the crystal to resolve large unit cell, then you should consider using a helium box to lessen the absorption of x-rays by air.

Exposure time 
       How long should you expose per image?  Theoretically, the longer the exposure, the better the counting statistics (i.e improved signal to noise). The length of the exposure is especially important for weaker reflections at high resolution.  However, there are limits imposed by the physics of the detector, the patience of the crystallographer, and the impatience of the crystallographers waiting behind you for beam time.  Detectors can be saturated, this event is indicated by yellow pixels in the images taken on the CCD.  The dialog box also indicates the number of pixels with "overflows".  After 20 minutes, imaging plates begin to lose the latent image before the image is developed. Typical exposure times are in the range of  5-8 minutes on the imaging plate, 1-2 minutes on the CCD

Oscillation angle 
    Through how many degrees should the crystal rotate during a single exposure?  Data are usually collected in snapshots taken during a small rotation of the crystal (phi angle).  The larger the oscillation angle, the more reflections are collected on a single exposure. However, if the oscillation is too big, reflections will overlap on the film.  Those overlapped reflections would have to be omited from the final data set.  Overlap is especially a problem for large unit cells, where the reciprocal lattice points are closely spaced. Typical oscillation angles are 2 degrees for DNA crystals, 0.5-1 degrees for protein crystals.

Total number of degrees of data collected 
     Through how many degrees should the crystal rotate before ending data collection?  The minimum number of degrees of rotation is dictated by the space group symmetry and crystal orientation.  Knowing exactly how to collect the unique set of reflections in the minimal amount of time used to be of the utmost importance before the advent of cryocrystallography; x-rays quickly damage a crystal at room temperature . However, significant improvement in signal to noise can be gained by collecting more more data, up to 360 degrees.  With cryo cooling, decay is rarely a problem. 

Diffraction simulation: To develop your intuition about Ewald's sphere, and the reciprocal lattice, run excercise 1 and exercise 4 with XRayView, the interactive computer graphics software package.  Explore the inverse relationship between the unit cell and the reciprocal lattice.  Understand how overlap can occur during data collection.


XrayView exercise.

Well separated reflections, poorly separated reflections
Overloaded pixels in a reflection measurement


Smaller detector distances intercept higher Bragg angles.


Data collection software window

>
XRayView software.


A movie compiled from diffraction images. Each image (frame) contains the sum of diffracted intensities as the crystal is rotated over a 1 degree oscillation. In total, the movie covers 60 degrees of crystal rotation about a vertical axis. Taken from James Holton's web site http://ucxray.berkeley.edu/~jamesh/movies/.

Sphere of Reflection
Originated by Bernal but more popularly known as Ewald's sphere.


Powerpoint presentation
Instructor's preparations

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