Hopefully this will evolve over the coming months into something that is useful for everyone in the lab. The guide is roughly divided into three areas: Garman group pages, other LMB pages and external groups. At the moment the latter two consist mainly of links that maybe useful, if people want to suggest changes/additions let me know. Please bear in mind that at the moment this is very much work in progress.

The following have been written by members of the Garman group. Feel free to use them, but if you do and find them useful, please reference them or cite the relevant paper. There is some repetition/duplication between the below, but hopefully this means that they all stand alone as useful guides.

Garman group guides

  1. Freezing and storing a crystal using an Arc
  2. Retrieving a frozen sample from a Dewar and mounting it on a goniometer using an Arc
  3. Freezing and storing a crystal using CryoTongs
  4. Retrieving a frozen sample from a Dewar and mounting it on a goniometer using CryoTongs
  5. Flash-cooling a sample in liquid nitrogen and storage
  6. General notes to add to the above and dry shipping Dewar care
  7. Xenon guide
  8. RADDOSE users guide
  9. T5 generator calibration

LMB Guides

The S9 users page is particularly useful, with users guides to the crystallisation robot, microscopes, particle size detector and SPECTRAFluor.

Computing on synapse information is here.

  • Back
  • One: Cryo-cooling and crystal storage

  • Next
  1. Establish a 'standard' pin length that will keep the crystal in the centre of the Arc as it is rotated. Ensure that all your pins and Top Hats conform to the standard length.
  2. Mount the crystal on the Arc base and cool it using a Cryostream.
  3. Once the sample is frozen and ready to store, label a Cryovial and Cryocane.
  4. Fill a 2 litre Intermediate Dewar with liquid nitrogen.
  5. Take a Glass Sample Handling Dewar and also fill it with liquid nitrogen. Place the labelled vial in the Vial Holder (or the Vial Tweezers) and submerge them both in the liquid nitrogen, contained in the Glass Sample Handling Dewar.
  6. Rotate the goniometer so that the Arc attachment screw hole is upwards. Attach the removable Arc. If the Cryostream nozzle is in the way, rack it back to create more room for maneuvering.
  7. Rotate the Top Hat on the Arc so that it is pointing downwards by pushing the horizontal metal bar around gently.
  8. Put on a pair of Cryogloves.
  9. Bring the Glass Sample Handling Dewar, containing the Vial Holder and vial, as close to the goniometer as possible. Inside the generator housing is ideal.
  10. Remove the Vial Holder from the Glass Sample Handling Dewar and place the vial full of liquid nitrogen in the Vial Holder.
  11. Bring the Vial Holder and vial full of liquid nitrogen up under the pin and carefully enclose the Top Hat in the vial. Move the Vial Holder SIDEWAYS to break the magnetic hold of the Magnetic Base on the Top Hat. If necessary use the forefinger of your free hand to nudge the Top Hat sideways from the other side of the holding magnet. Try not to panic at this stage. You have approximately 25 seconds to complete this stage before the liquid nitrogen will boil off to below the position of your crystal in the vial.
  12. As soon as the Top Hat and sample are inside the frozen vial, return the Vial Holder and vial to the Glass Sample Handling Dewar so that the vial and sample are fully submerged in liquid nitrogen.
  13. It is important to note that if a lid is to be placed on the vial, a blowhole should be drilled in the lid before it is used. It is recommended that the lid is placed on the vial if canes with lateral extrusions (to rest the vial base on) are to be used.
  14. Next, bring the Vial Holder up so that the top of the vial just breaks the surface of the liquid nitrogen. Put the lid on the end of the Lid Opener and screw it on the vial. Do NOT screw the lid on too tightly as vial lids sometimes freeze on and can be very hard to get off. Immerse the whole vial and lid in the liquid nitrogen until the surface of the liquid nitrogen goes flat and equilibrium is reached.
  15. Lift the Vial Holder and vial from the Glass Sample Handling Dewar and remove the vial. Put the Vial Holder down. With the free hand lift the pre-labelled cane out of the 2 litre Dewar and place the vial on it at the next free position, working from the bottom of the cane upwards.
  16. Make a note of the crystal shape, the marking on the vial and cane and the date. When the canes are full, transfer them to the main storage Dewar all at the same time. This avoids opening and closing the storage Dewar unnecessarily, preventing ingress of moisture from the air.

Copyright Garman group 2005

  1. Fill a 2 litre Intermediate Dewar and a Glass Sample Handling Dewar with liquid nitrogen.
  2. Put the removable Arc on the goniometer and position the magnet on it so that the Top Hat and Pin will point downwards when on the magnet.
  3. Check that you can gain access to the magnet with the Vial Holders by doing a dry run with an empty vial in the holder.
  4. Transfer the Cryocane, which is holding the desired crystal, from the storage Dewar or dry Dewar to the 2 litre Dewar.
  5. Put the Vial Holders into the small Dewar to cool down.
  6. Put on a pair of Cryogloves.
  7. Lift the Cryocane out of the 2 litre Dewar and take out the required vial with the other hand or with the Vial Tweezers.
  8. Place the vial in the Vial Holder (or the Vial Tweezers) and place both into the Glass Sample Handling Dewar.
  9. If there is a lid on the vial, remove it using the Lid Opener.
  10. Move the Glass Sample Handling Dewar as close to the goniometer as possible.
  11. Bring the Vial Holders up underneath the magnet till the Top Hat is in contact with the magnet.
  12. Carefully withdraw the vial to leave the crystal in the nitrogen stream.
  13. Move the crystal around the Arc by pushing on the metal bar on the Slider.
  14. Unscrew the removable part of the Arc and proceed with the final crystal alignment. Note: if the sliding part of the Arc is too loose, tightening the small screws in the underside of the blue curved piece can tighten it.

Copyright Garman group 2005

  1. Establish a standard pin length that will keep the crystal in the right place in the CryoTongs.
  2. Ensure that all your Pins and Top Hats conform to the standard length.
  3. Stream freeze a crystal onto the Magnetic Base on the goniometer.
  4. When you are ready to store the crystal, label a Cryovial.
  5. Fill a 2 litre Intwermediate Dewar with liquid nitrogen and label a Cryocane.
  6. Fill a Glass Sample Handling Dewar with liquid nitrogen.
  7. Place the labelled vial in the Vial Holder and submerge them both in the liquid nitrogen. Also place the CryoTongs in the Glass Sample Handling Dewar.
  8. Put on a pair of Cryogloves.
  9. Bring the small Dewar and Vial Holder and the vial as close to the goniometer as possible. Inside the generator housing is ideal.
  10. Bring the cold CryoTongs up to the crystal. Separate the CryoTongs and clasp the Top Hat and crystal in them. Move the CryoTongs sideways to break the magnetic hold of the Magnetic Base on the Top Hat.
  11. As soon as the Top Hat and sample are held in the CryoTongs, immerse the tongs and sample in the Glass Sample Handling Dewar.
  12. Transfer the crystal to the Magnetic Wand by placing the wand in the Dewar and letting it cool down. Bring the wand up to the base of the Top Hat and release the tongs carefully. Remove the tongs from the Dewar.
  13. Bring the Vial Holder and vial up underneath the pin and Top Hat.Locate the Top Hat into the top of the vial.
  14. Move the wand SIDEWAYS to break the magnetic hold between the Top Hat and wand.
  15. As soon as the Top Hat and sample are inside the frozen vial, return the Vial Holder and vial to the Glass Sample Handling Dewar so that the vial and sample are fully submerged in liquid nitrogen.
  16. It is important to note that if a lid is to be placed on the vial, a blowhole should be drilled in the lid before it is used. It is recommended that the lid is placed on the vial if canes with lateral extrusions (to rest the vial base on) are to be used.
  17. Next, bring the Vial Holder up so that the top of the vial just breaks the surface of the liquid nitrogen. Put the lid on the end of the Lid Opener and screw the lid on the vial. Do NOT screw the lid on too tightly as vial lids sometimes freeze on and can be very hard to get off.
  18. Immerse the whole vial and lid in the liquid nitrogen until the surface of the liquid nitrogen goes flat and equilibrium is reached.
  19. Lift the Vial Holder and vial from the Glass Sample Handling Dewar and remove the vial. Put the Vial Holder down.
  20. With the free hand lift the pre-labelled cane out of the 2 litre Dewar and place the vial on it at the next free position, working from the bottom of the cane upwards.
  21. Make a note of the crystal shape, the marking on the vial and cane and the date.
  22. When the canes are full, transfer them to the main storage Dewar all at the same time. This avoids opening and closing the storage Dewar unnecessarily, preventing ingress of moisture from the air. Note that the tongs should be warmed up and dried after each use to stop them freezing together.

Copyright Garman group 2005

  • Back
  • Four: Retrieving a frozen sample

  • Next
  1. Fill a 2 litre Intermediate Dewar and a Glass Sample Handling Dewar with liquid nitrogen.
  2. Check that you can gain access to the magnet with the CryoTongs by doing a dry run with an empty Top Hat in the tongs.
  3. Put the tongs in the small Dewar.
  4. Transfer the Cryocane, which is holding the desired crystal from the storage Dewar or dry Dewar to the 2 litre Dewar.
  5. Place the Vial Holder into the small Dewar to cool down.
  6. Put on a pair of Cryogloves.
  7. Lift the Cryocane out of the 2 litre Dewar and take out the required vial with the other hand or with the Vial Tweezers.
  8. Place the vial in the Vial Holder (or the Vial Tweezers) and put them both into the small Dewar.
  9. If there is a lid on the vial, remove it using the Lid Opener.
  10. Place the Magnetic Wand in the small Dewar and wait for it to cool down.
  11. Put the wand on the Top Hat and withdraw it from the vial keeping the hat under the liquid nitrogen.
  12. Take the Vial Holder and empty the vial out of the small Dewar and put the tongs into the Dewar.
  13. When the tongs are cold (surface of the liquid nitrogen becomes calm) open them and grasp the Top Hat.
  14. Withdraw the wand sideways and put it down.
  15. Move the small Dewar as close to the goniometer as possible.
  16. Lift the tongs to the goniometer magnet and place the Top Hat base on the Magnetic Base.
  17. Release the tongs and carefully withdraw them.
  18. Proceed with crystal alignment.

Copyright Garman group 2005

  • Back
  • Five: Flash cooling a sample in liquid nitrogen

  • Next
  1. Establish a standard pin length that will keep the crystal in the centre when it is rotated on the Arc during retrieval, or alternatively, fit into your tongs if using this method.
  2. Ensure that all your pins and Top Hats conform to the standard length.
  3. Label a Cryovial. Fill a 2 litre Intermediate Dewar with liquid nitrogen and label a Cryocane. Fill a Glass Sample Handling Dewar with liquid nitrogen.
  4. Place the labelled vial in the Vial Holder (or the Vial Tweezers) and place them both into the Glass Sample Handling Dewar.
  5. Place the small Dewar as close to the microscope as possible.
  6. Hold the Top Hat on the Magnetic Wand.
  7. Fish the crystal and plunge it immediately into the liquid nitrogen. The shorter the time the crystal is travelling through the air and dehydrating, the better.
  8. Wait until the liquid nitrogen surface is still. Bring the Vial Holder and vial up underneath the pin and the Top Hat. Locate the Top Hat into the top of the vial.
  9. Move the wand SIDEWAYS to break the magnetic hold between the Top Hat and wand.
  10. As soon as the Top Hat and sample are inside the frozen vial, return the Vial Holder and vial to the Glass Sample Handling Dewar so that the vial and sample are fully submerged in liquid nitrogen.
  11. It is important to note that if a lid is to be placed on the vial, a blowhole should be drilled in the lid before it is used. It is recommended that the lid is placed on the vial if canes with lateral extrusions (to rest the vial base on) are to be used.
  12. Next, bring the Vial Holder up so that the top of the vial just breaks the surface of the liquid nitrogen.
  13. Put the lid on the end of the Lid Opener and screw the lid on the vial. Do NOT screw the lid on too tightly as vial lids sometimes freeze on and can be very hard to get off.
  14. Immerse the whole vial and lid in the liquid nitrogen until the surface of the liquid nitrogen goes flat and equilibrium is reached. Lift the Vial Holder and vial from the Glass Sample Handling Dewar and remove the vial.
  15. Put the Vial Holder down. With the free hand lift the pre-labelled Cryocane out of the 2 litre Intermediate Dewar and place the vial on it at the next free position, working from the bottom of the Cryocane, upwards.
  16. Make a note of the crystal shape, the marking on the vial and cane and the date. When the canes are full, transfer them to the main storage Dewar all at the same time. This avoids opening and closing the storage Dewar unnecessarily, preventing ingress of moisture from the air.

Copyright Garman group 2005

  1. When fishing for crystals in the cryoprotectant, have the microscope on fairly low magnification so that the whole drop is visible and the depth of field is larger than on high magnification.
  2. Also when fishing, rest the fourth finger of the "fishing" hand on the edge of the crystallisation tray to steady your hand and act as an anchor point.
  3. When manipulating crystals and vials under liquid nitrogen, wait until the liquid has stopped bubbling and is calm before trying to see where the objects are under the liquid. Also, it is easier to move only one hand at a time and rest the side of the stationary hand on the edge of the Dewar top.
  4. The dry shipper should be properly dried out after each use, especially if it was opened and closed many times at the synchrotron. Moisture can get into the sorption material and seriously compromise its cooling capacity.
  5. The next time the Dewar is cooled with liquid nitrogen, the water freezes to ice in the material preventing it from being able to sorb the nitrogen.
  6. Be sure to refer to any manufacturer's instructions supplied with the Shipper.

Copyright Garman group 2005

Valves

  1. Stop cock on top of Xe cylinder
  2. Cock between high and low pressure
  3. Stop cock that releases low pressure gas to cell
  4. Butterfly tap inlet to cell
  5. Bleeding tap outlet from cell

Before use

  1. Put filter between the metal diffusers. Soak filter rounds (originally punched out rounds in workshop from Whatman) in cryo-buffer (4 to 6 of them). [Home device: When replace it put cone UPPERMOST rather than flat rivet uppermost. (cotton wool pad was tried originally but did not work.)]
  2. Screw the filter holder on.
  3. Check crystal O-ring. Grease if necessary.
  4. Push it home and lock it in (checking).

Cylinder

  1. Connect device to xenon cylinder using snap fitting via the "stapler" hand pump if the cylinder is getting empty.
  2. Have the bleeding valve, E, open when you connect the cylinder.

Method for pressurisation

At this point Xe is in low pressure chamber of regulator.

Copyright Garman group 2005

Given the crystal composition and beam parameters, the program RADDOSE calculates the dose (in Gray, Gy =Joules per kilogram) absorbed by a crystal. The calculation can be applied to any type of crystal in either a synchrotron or in-house X-ray beam.

This brief guide merely summarises the keyword input required for a RADDOSE calculation, highlighting possible problems and ambiguities. The new version of the program, which includes X-ray fluorescent escape routines is documented in the paper Absorbed dose calculations for macromolecular crystals: improvements to RADDOSE.

Citations: Paithankar et. al. (2009) J. Synch. Rad. 16, 152-162. Previous publications of RADDOSE include Murray et. al. (2004) and Murray et. al. (2005). Full details of these are on the publications page.

RADDOSE can be obtained by contacting Elspeth. We have tested it on Linux systems. The current version does NOT require a CCP4 environment.

Installation: (updated 22 September 2009)

1. The program needs a FORTRAN compiler. Supported compilers include g77, f77, and fort77. You may test your compiler with

[username@linux] g77 --version
gcc (Ubuntu 4.3.3-5ubuntu4) 4.3.3

Note that we do not support the compiler gfortran.

2. Extract the archive with

tar zxvf raddose*tar.gz
3. Since a lot of email servers do not allow executables in the email, we have disabled "exec flag" in the attachment. Hence, you have to
[username@linux] chmod +x build.com example.com
[username@linux] ./build.com
4. The executable of the program, raddose (output by build.com) is then available in the same directory. Modify example.com to suit your experiment.
Example script
#!/bin/bash

./raddose << EOF
!RANGE 6 20 0.001
ENERGY 12.7
REMARK  1UOX Acta D V59 118-126 2003 Girard et al.
CELL 78.9 95.162 104.087 90.0 90.0 90.0
NRES 295
NMON 8
!PATM S 4 Se 4
BEAM  0.1 0.1
CRYST 0.1 0.1 0.1
PHOSEC 1E12
EXPO 1.0
IMAGE 1
SPLINOR Se Se.splinor
!debug
!GAUSS 0.1 0.1
!oldprogram
END
EOF
Keywords
ENERGY
The energy of the beam in keV.
RANGE
If this is uncommented, RADDOSE will perform the calculation from 10keV to 15keV in steps of 0.005keV (all of these values can be changed as required). Good if you want to investigate absorption at or around an absorption edge.
CELL
The dimensions of the unit cell in Angstroms and angles in degrees (a, b, c, alpha, beta, gamma)
NRES, NMON
NRES is the number of amino acid residues in a single chain. The number and types of atoms are calculated assuming an average amino acid content defined as amino acid = 5 C + 1.35 N + 1.5 O + 8H
NMON refers to the number of monomers in the unit cell. (N.B. NOT the asymmetric unit).
PATM
Define atoms to added to the protein part of the scattering as no. of atoms per monomer. The number of sulphur atoms (i.e. number of cysteine residues + number of methionine residues) must be added explicitly here.
SATM
Define the concentration of elements (not including water) in the solvent in mM. Do not define these for oxygen and lighter elements (i.e. do not for PEG, but do define for ammonium sulphate).
BEAM, GAUSS
These define the extent and shape of the beam. BEAM defines the x and y beamsize in mm. The optional keyword GAUSS gives the full-width-half-maxima in mm of a Gaussian beam profile. If this keyword is not given, a uniform beam is assumed (i.e. top hat shape). nb, BEAM is not an optional keyword and should be given even if using GAUSS.
CRYST
Crystal dimensions x, y, z in mm.
PHOSEC
The flux of the beam in photons per second into the area defined by the BEAM keyword.
EXPO, IMAGE
Exposure time per image in seconds, and the number of images taken.


In the NEW version of the program there are additional keywords:
OLDPROGRAM
Run the program without X-ray fluorescence escape correction.
GRAPH
Works with the option RANGE. Prints the experimental dose limit for the entire energy range in step_keV
DEBUG
Detailed information on K and L1, L2, and L3 shell escape.


Important Change
The keyword HENDLI is replaced by USERLI and defaults to a value of 20 MGy unless changed by user. USERLI stands for USERLIMIT to specify an user-defined limit for dose.
Notes

For a full description of the KEYWORDS please see the file raddose.doc that is inside tar.gz (available from Elspeth).

Output

Below is the output of RADDOSE using the parameters in the example script given above. It should be noted that if a different dose limit is required, this can be changed using the keyword USERLI. Temperature changes should be treated with a certain amount of caution.



REMARK  1UOX Acta D V59 118-126 2003 Girard et al.                              

Element % contribution to TOTAL absorbed energy
C      9.5
N      5.3
O     57.6
S      1.9
SE    25.7

Energy (keV)    12.70
Beam non-uniformity intensity correction    1.000
Protein Monomer Concentration (mM)        17.0
Unit Cell Volume (A^3)   781514.6
Crystal Density (g/cm^3)          1.15
Solvent Content (%)               59.2
No. Water Molecules /cell        15311

Total absorbed dose (Gy)           0.421E+05
Absorbed dose per image (Gy)       0.421E+05
Diffracted intensity per absorbed dose (arbitrary units)     2.71
Attenuation Coefficient (mm^-1)    0.320

Cross-section due to Photoelectric effect (mm^-1)                   0.282
                     Inelastic X-ray (Compton) scattering (mm^-1)   0.019
                     Elastic X-ray (Rayleigh) scattering (mm^-1)     0.019
Absorption Coefficient (mm^-1)    0.28

Fraction of beam absorbed by the crystal  2.8%
Fraction of beam seen by the crystal     100%
Fraction of crystal seen by the beam      99%
Absorbed photons per unit cell per dataset      0.02
Absorbed photons per unit cell for experimental dose limit    15.47
System temperature time constant (sec)     0.03
* Temperature rise in the crystal (K)     2.52

Number of atoms by type in the unit cell:
  Z  NAME  PROTEIN   SOLVENT  TOTAL
  1   H    18880     30623     49503
  6   C    11800         0     11800
  7   N     3186         0      3186
  8   O     3540     15311     18851
 16   S       32         0        32
 34   SE      32         0        32

DOSE LIMITS:
   Time in sec to reach Henderson limit (20 MGy) calculated from electron diffraction      474
** Time in sec to reach experimental dose limit (30 MGy)      712

  • Back
  • Nine: T5 Calibration or Myrtle Calibration

  • Top

T5 calibration curve

Update: This generator is not in use anymore. See here.

The flux provided by the in-house RU200H generator. The plot allows conversion from ion chamber 2 reading to photons per second. This calibration was produced using a silicon photodiode (Hamamatsu, model S3204-, diode thickness of 500 microns).
It should be noted that this calibration is valid only for the generator used in T5, LMB, Oxford. It is extremely unlikely that the relationship between ionchamber reading and photon flux will be the same on different sources with different optical elements.

Last updated 24 Feb 2009