SCSB Crystallography Facilities

The mission of the Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory is to provide access to state of the art X-ray diffraction instrumentation and the associated support facilities for crystallographic research at UTMB. 

The Center has x-ray diffraction and solution scattering instrumentation in the facility. This equipment starts with the Rigaku Ultimate Home Lab® which comprises the unsurpassed high-brilliance FR-E++DW Superbright x-ray generator, with the industry standard RAXIS-IV++ crystallography system with both Cu and Cr optics. In addition, we have acquired the innovative Rigaku BioSAXS-1000 with a 96-well automatic sample changer. This is the first SAXS instrument dedicated to biological research in the southern mid-western states, once again marking UTMB as a leader in structural biology resources.

The SCSB X-ray Crystallography resources currently consists of two x-ray area detector systems. The first area detector is a Rigaku R-AXIS-IV++ dual 30cm Imaging Plate detector mounted on the Rigaku FR-E++DW Superbright x-ray generator. The second detector is a Brüker D8 Venture with the high intensity TURBO TXS source and a 4-axis goniometer which can reach upto 0.81 Å resolution. For enhanced data we have available a choice of sample cooling systems. Both systems are equipped with Crysosystems, the R-AXIS a Cryo Industries of America CRYOCOOLER, the Brüker an Oxford Cryostream 800. A 4 °C Cold Air refrigerated cooling system is available for samples which cannot be frozen, or do not require freezing.

The Rigaku BioSAXS-1000 2D-Kratky camera is mounted on the left port of our Rigaku FR-E++DW Superbright x-ray generator. The camera is equiped with a 96-well automatic sample changer (ASC-96) with an in-vacu quartz flow-cell. In addition, we have several quartz sample cells for manual loading of smaller sample volumes or detergent samples, which are not compatible with the ASC-96 liquid handling system. The quality of SAXS data obtained with the BioSAXS camera has been exceptional. Using our in-house SAXNS web-services for data processing and analysis the optimal quality data can be collected. It is not unusual to collect useful data to the limit of the detector (q~0.7 Å-1). This camera offers unsurpassed solution scattering data for macromolecular samples. For more information visit our SAXNS site.

Instrument scheduling, for qualified users, is available online.
To discuss a collaboration or potential project please contact the manager or one of our faculty members.



PROTOCOLS

These are a collection of documents from the SCSB X-ray Crystallography Center and our research groups.

X-RAY Policy (UTMB) (State)
R-AXIS-IV Guide BioSAXS Guide General Rad Safety
R-AXIS-IV Logbook BioSAXS Logbook nbs111 Rad Safety Rules
D8 UserManual BioSAXS Manual Radiation Hazards
D8 Logbook Proteum Manual TX DSHS Radiation Safety
Cryo how to Crystal Cryo Log Radiation Safety *Test*

ROBOTICS Policy
PHOENIX Quick Start Zetasizer QS Robotics *Test*
PHOENIX Manual ZS Logbook ZS Software Download
PHOENIX Logbook Olympus-DP20+UV DLS Manual (8MB)
PHOENIX Sample Form HPLC Column Care DIP User Manual
Sealing DWBs Alchemist Guide DIP Logbook
Nano Drop Log Minstrel Use Crystal Trak Trays

Administrative Docs
Faculty Use Form (MOU) Emergency Plan Data archive info
MOU Instructions
EHS: DPS Regulations EHS: Spill Procedures EHS: Eyewash Log

Tuorials and Manuals (Local Access)
Brüker Power Points Brüker Manuals Rigaku Manuals


Crystallographic Education

Teachers: Please provide feed-back when using these materials in your courses. These courses are only published on the web. Please support this free publishing effort by registering your use. Educational users may download and edit these tutorials to suit their needs provided that the original author is given credit. Any comments or suggestions you may have to improve these tutorials will be greatly appreciated.

Thank you,
Mark A. White


Tutorials developed at UTMB:

Links to Tutorials at other sites:


Radiation Safety

Table Of Contents

Radiation Safety For X-ray Diffraction


Safe Practices:

Telltale Lights: Users should check the Power and Shutter lights every time they walk into the room. Each light operates in an interlock circuit such that if the light is off, you are guaranteed that either the Power is off, or the Shutter is closed.
Beamstops: Beamstops serve two functions: to shorten the path of air scatter, and to protect the image plate. Users should check that the beamstop is in place whenever x-ray power is on, regardless of the shutter status. If you wish to remove the beamstop to check the direct beam position, you must remain in the room while you are doing the experiment. Protecting Your Fingers from the Direct Beam: Always CLOSE the shutters before working in the enclosures. When you manipulate anything in the vicinity of the goniometer, you should assume that the shutter is open and that there is no beamstop: therefore do not put your fingers in the path of the direct beam.
Generator Room Access: Do not work behind the X-ray Generators. Generator rooms are not shielded from air scatter.
Beam-confinement Labyrinths: Labyrinths are junctions between two pieces of equipment through which the beam passes. Any small disturbance of the x-ray optics can ruin the alignment, and reduce the beam intensity. In this event, call Mark White for assistance. Furthermore, if the equipment is severely distorted, dangerous amounts of x-rays can leak out. Visually check that the beam path is properly intact before opening the shutter.
Survey Meters: Users should know how to conduct radiation survey with portable meter.
X-ray Shutter: When data are not being collected the shutter switch should be closed. Before opening the shutter, double check that the beamstop and shields are in place, and that the labyrinths are properly aligned.
Visitors: Visitors must be escorted by an authorized user. It is considered to be a risk factor to bring visitors into the generator rooms.

RADIATION SAFETY FOR X-RAY UNITS

NATURE OF ANALYTICAL X-RAYS

Analytical x-ray machines produce intense beams of ionizing radiation that are used for diffraction and fluorescence studies. The most intense part of a beam is that corresponding to the K emission of the target material and is called characteristic radiation. In addition to the characteristic radiation, a continuous radiation spectrum of low intensity is produced ranging from a very low energy to the maximum kV-peak setting. This is referred to as 'bremsstrahlung' or white radiation. Undesirable wavelengths may be filtered out using a monochromator.

X-ray diffraction wavelengths (w) are selected so as to roughly correspond to the inter-atomic distances within the sample, and to minimize fluorescence. Wavelengths commonly used are 1.54 Å (Cu targets), 0.71 Å (Mo targets), 0.56 Å (Ag targets), and 2.3 Å (Cr targets). The relationship between wavelength and x-ray photon energy is determined by the equation

E = hc/w

where
E = energy in ergs (1eV = 1.6E-12 erg)
h = Planck's constant = 6.614E-27 erg-sec
c = velocity of light = 3E10 cm/sec
w = wavelength in cm (1Å = 1E-8 cm)

X-rays emitted from an open, uncollimated port form a cone of about 30 degrees. The x-ray flux can produce a radiation field at one meter on the order of 10,000 R/hr. A collimator reduces the beam size to about 1 millimeter diameter.

X-RAY HAZARDS AND BIOLOGICAL EFFECTS

X-rays produced by diffraction machines are readily absorbed in the first few millimeters of tissue, and therefore do not contribute any dose to the internal organs of the body. However, the lens of the eye can receive a dose from x-rays of this energy. Overexposure of lens tissue can lead to the development of lens opacities and cataracts.

Absorbed doses of a few hundred rad may produce a reddening of the skin (erythema) which is transitory in nature. Higher doses -- 10,000 rad and greater -- may produce significant cellular damage resulting in pigment changes and chronic radiation dermatitis. Exposure to erythema doses may not result in immediate skin reddening. The latent period may be from several hours to several days.

(Note: X-rays used for medical diagnosis are about one order of magnitude shorter in wavelength. Diagnostic rays are designed for tissue penetration and are carefully filtered to avoid x-ray damage to the skin caused by the longer, more readily absorbed wavelengths).

SOURCES OF IONIZING RADIATION

The primary beam is not the only source of ionizing radiation. Any high voltage discharge is a potential source of x-rays. Faulty high-voltage vacuum-tube rectifiers may emit x-rays of twice the voltage applied to the x-ray tube. Other sources of ionizing radiation are:

    • Secondary emissions and scattering from the sample, shielding material, and fluorescent screens.
    • Leakage of primary or scattered x-rays through gaps and cracks in shielding.
    • Penetration of the primary beam through or scattering from faulty shutters, beam traps, or collimator couplings.

SAFETY PRECAUTIONS AND NOTES

The shielding, safety equipment and safety procedures prescribed for x-ray diffraction equipment are applicable only for up to 75 kV-peak x-rays. Additional or greater precautions are necessary for machines operating at higher voltages.

The PI has the basic responsibility for providing a safe working environment by ensuring that equipment is operationally safe and that users understand safety and operating procedures.

The equipment operator is responsible for his own safety and the safety of others when using an analytical x-ray machine.

Prior to removing shielding or working in the sample area, the operator must check both the warning lights and the current (mA) meter on the console. Never trust a warning light unless it is on! Always use a survey meter to check that the shutters are actually closed if current is still being supplied to the tube. It is possible for a shutter to be stuck partially open even when the indicator shows that it is shut. The best way to avoid an accidental exposure is to turn the machine off before working in the sample area.

Never put any part of the body in the primary beam. Exposure of any part of the body to the collimated beam for even a fraction of a second may result in damage to the exposed tissue.

A person not knowledgeable about x-ray equipment should not attempt to make repairs or remedy malfunctions. If you suspect a machine is malfunctioning, turn it off or unplug it. Place a note on the control panel and inform the PI or his designated representative.

Repairs to the high voltage section must not be made unless the primary leads are disconnected from the high voltage transformer and a signed and dated notice is posted near the x-ray ON switch. Turning off a circuit breaker is not sufficient.

Bare feet are not permitted in the laboratory or around electrical equipment. Even slightly moist skin is an excellent electrical conductor and contact with faulty, ungrounded equipment may result in severe injury or death.

Do not attempt to align x-ray cameras without first consulting an experienced person. Alignment procedures require special training and knowledge.

Special care is required when one power supply is connected to more than one x-ray tube.

EYE PROTECTION

The use of safety glasses or prescription lenses is encouraged when working with analytical x-rays. While glasses cannot be depended upon to provide complete protection to the eyes, they can reduce x-ray exposure. Glass provides about 10 times the protection of plastic. Neither, however, will adequately protect the eye from direct exposure to the primary beam.

FLUORESCENT SCREENS

It is unsafe to inspect an x-ray beam with a fluorescent screen without special precautionary measures. Notify the Safety Office before performing a procedure using a fluorescent screen.

TUBE STATUS INDICATORS

There must be a visual indication located on or near the tube head to indicate when x-rays are being produced This is usually an assembly consisting of two red bulbs, wired in parallel and labeled X-RAYS ON. If one of the lights is burned out, the operator should either replace it before leaving the room, or leave a note on the light assembly indicating that the bulb is burned out. An unlit warning bulb does not necessarily mean that x-rays are not being produced. Always check the control panel.

SAFETY DEVICES

Interlock switches are used to prevent inadvertent access to the beam. They should not be bypassed. Interlocks should be checked periodically to insure that they are functioning properly.

Interlocks and other safety devices and warning systems are not foolproof or fail-safe. A safety device should be used as a back-up to minimize the risk of radiation exposure -- never as a substitute for proper procedures and good judgment.




Crystallographic Facilities

    Detector Type

      Detector Size image

      30 cm square

      10 cm W x 14 cm H

      Cycle time

      2 minutes

      shutterless

      Crystal-Det distance

      102 - 450 mm

      45 - 300 mm

      X-Ray Source

      FR-E++DW 2.475 kW Cu/Cr 70 micron micro-focus

      TURBO TXS 2.5kW Cu ultra-fine focus

      X-Ray Optics

      MSC Varimax Confocal Cu/Cr

      Helios Cu (CML)
      Collimators: 17, 10, 6 mRad
      beam intensity: 1, ½, ⅕

      Cooling system -170 C

    85°K CryoCooler-II

    100°K Cryostream 800

      On-line training

      Other Shared Instruments supported by the SCSB

    AUC (SBL BSB 6.656)

    BIACORE T100 (SBL BSB 6.656)

    Malvern Zetasizer micro V Dynamic Light Scattering (Robotics Lab BSB 6.656)

    THERMOFLUOR (SBL BSB 6.656)




Rigaku R-AXIS-IV++

Our R-AXIS IV++ is the primary X-ray area detector for macromolecular crystallography. Its two large active area imaging plates with fast readout speed and a wide dynamic range is combined with the FR-E++DW microfocus x-ray source, making it the most popular system for all aspects of macromolecular crystallography, from screening, to data collection, and phasing.

Data Collection

See the R-AXIS-IV User Manual

Instrument scheduling, for qualified users, is available online.
To discuss a collaboration or potential project please contact the manager or one of our faculty members.




Brüker D8 Venture


Our Brüker D8 Venture system offers a modern high speed data collection system for Macromolecular and chemical crystallography. The modern Venture system, with the new Photon II-14 detector, offers superior resolution for macromolecular and small molecule (drug) crystallography. Its fast, realtime, readout provides quick feed-back for initial screening, and low noise for acurate data collection from weakly diffracting crystals. The 'shutterless' data collection eliminates deadtime when fine-slicing data collections. The TURBO TXS high intensity, 2.5 kW, x-ray source combined with the small 96 micron pixel size and 3D profiling, in SAINT, permits data collection on very long unit cells ~400 Å.

Data Collection

See the D8 User Guide and the Brüker Manuals

Instrument scheduling, for qualified users, is available online.
To discuss a collaboration or potential project please contact the manager or one of our faculty members.




Rigaku BioSAXS-1000


Solution Studies of Macromolecules and their Complexes The Rigaku BioSAXS-1000 offers the ability to gain fundamental information about the behavior of a sample in solution with minimal time & effort. A simple dilution series can be collected from 140µl of sample. Basic analysis can determine the size and shape, Mw, and oligomeric state of your sample. Sophisticated analyses can determine the orientations of domains or complexe constituents, and if they are dynamic, the extent of their disorder.

Data Collection

See the BioSAXS User Manual and the SAXNS group site.

Instrument scheduling, for qualified users, is available online.
To discuss a collaboration or potential project please contact the manager or one of our faculty members.




CIA CRYO-COOLER

Instruction Manual

See the X-ray Data Collection How-to.


Cryo-Loops

See the Cryo-Loops How-to.


Our Cryo Industries of America (CIA) Cryo-Coolers offer one-button operation, fast cool-down times, low LN2 consumption, good temperature stability, and low sample temperatues.



Crystallization Robotics

Crystallization Screens:
(Maintained by The MXL Sharing-Group)

The faculty of the MXL has joined together to share in the cost of maintaining this set of crystalization screens. If you wish to participate in the sharing group please contact the manager (mawhite@utmb.edu).

Robotics Protocols

DLS Zetasizer Micro V & Nano-drop

Crystallization Rooms & Incubator

  • The MXL maintains two large walk-in envionmental rooms for crystallization trials:
    • 20 °C Room 6.656A - Houses the Olympus DP-20 microsope/camera with UV light, and a Nikon SMZ1000 microscope, in addition to 10x5 full metro-shelves
    • 4 °C Room 6.650A - Houses an Olympus SZ-60 microscope in addition to 4x5 full 24" deep metro-shelves (Inside X-ray Lab)
    • 4 °C Room 6.624A - Departmental Cold room houses several metro-shelves
  • The MXL currently has one large incubator for crystallization trials:
    • 4 ° Incubator 6.656 - A 30Cuft Thermo Scientific Precision Incubator is available for trials at low temperature. (4 °C microscope is in room 6.650)

Microscopes for Crystals

The MXL maintains Microscopes for Crystal handling, Tray screening, and Imaging (All with Polarizers):
  • Crystal handling:
    • Olympus SZ-60 microscope in X-ray Lab, Room 6.650 by R-AXIS-IV
    • Olympus SZ-60 microscope in X-ray Lab, Room 6.650 by D8-Venture
  • Crystal Tray screening and Imaging:
    • Olympus DP-20 ∨ SZH-10 microsope/DP-20 camera with LED/Filter base and UV-light in 20 °C Room 6.656A
    • Lieca M205C microscope with DFC225/PC high resolution color camera in 20 °C Room 6.656A
    • Lieca M205C microscope with IC80HD/SSD high resolution color camera in 4 °C Room 6.650A
    • Leica MZ125 microscope/Spot Insight Color camera in Solutions Biophysics Lab, Room 6.656 by nanodrop-1000 and Phoenix













Crystallization Room Temperature Monitor (19°C)

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