Bio-crystallography Laboratory

The laboratory takes advantage of a comprehensive equipment setup allowing the formation of bio-molecular monocrystals (e.g. protein), synthesis of protein complexes with small bioactive particles, and performance of structural tests on said systems at the molecular level by means of X-ray diffraction. The bio-crystallography laboratory is the first facility of its kind to be operated by a university or research institute in Lublin.

Comprehensive analyses of compound bioactivity mechanisms require  molecular structure data at the atomic level to allow SAR (structure-activity relationship) correlation. Reliable information on the structure of molecules - both "small" and macromolecules - can be obtained by means of monocrystal diffraction. The most common experimental diffraction technique relies on x-ray structure analysis.

Planned research:

Identification of receptor protein binding sites, in particular in substances with atypical activity mechanisms - allosteric modulators, dualsteric modulators, compounds with multidirectional activity, and chimeras. The results may facilitate the discovery of new, innovative drugs for the treatment of pain, depression, addiction and other CNS diseases, as well as cancer. Moreover, co-crystallisation of ligands and receptor proteins will allow an insight into the mechanisms of protein sensitisation and desensitisation, as well as homo- and hetero-oligomerization. It is pioneering research in the Polish context, on a par with the most advanced research conducted anywhere in the world.
It has been made possible by the laboratory's unique equipment configuration.

Laboratory equipment:

A. Equipment for diffraction analyses of monocrystals comprising a diffractometer by Rgaku XtaLAB with a high-intensity MicroMax-007 HF X-ray radiation source and Pilatus surface detector for protein crystallography applications, as well as a Cryostream 800 crystal cooling system by Oxford Cryosystems.

To allow the analysis of the smallest an poorly dispersing protein crystals, a 'microfocus' x-ray generator with a rotating anode is used, with 75 µm electron beam diameter on the anode. Coupled with the monochromatizing and focusing optics, this facilitates obtaining a high-intensity Cu Kα beam on the crystal (source brightness at the sample of at least  3.4 ´ 109 photons/sec/mm2/mrad2).
The second key element of the diffractometer is the x-ray HPAD (hybrid pixel array detector) combining the functions of counting individual photons with the hybrid-pixel technology for the direct detection of X photons. The detector's active field is approximately 80 x 70 mm² to ensure high resolution. The detector readout time is under 8 msec at full resolution, which minimises the total measurement time.

The diffractometer runs under HKL-3000R software responsible for device control as well as data collection, integration and analysis. The CrysAlisPRO software package is also utilised at the laboratory.

XtaLAB series x-ray diffractometers are used for analysing a wide range of crystalline samples, from small molecules, through metalorganic networks, to biological macromolecules. The key component of this series is the HPAD (hybrid pixel array detector). The HPAD technology facilitates near-perfect detection and greatly extends the capacity of monocrystal diffractometers, both in terms of data collection speed and greater measurement precision for weak diffraction data. The XtaLAB system features a MicroMax-007 HF generator - a second generation generator with a rotating anode and true electron beam microfocus capacity. Combined with VeriMax optics, the uniquely narrow electron beam on the anode generates an intensive, monochromatic x-ray beam on the sample, which allows for signal level to noise ratio optimisation for small protein crystals currently used in research laboratories.

Rotating anode generator with a microfocus MicroMax-007HF drive
- load - 1200 W,
- copper anode with direct drive,
- focus diameter of approximately 70 microns
- two high-speed rotating shutters

Optimum diffraction efficiency for samples of 300 microns or smaller.
The VariMaxTM-HF x-ray optics focusing system
 ensures maximum beam brightness and features a divergence adjustment functionality to allow regulation of the beam diameter depending on the crystal network constants.

The system includes:
- Osmic VariMax High Flux optics with housing,
- optic translator (2 rotational axes, 3 manually adjusted travel axes, divergence adjustment slits),
- system connecting the optics to the shutter window,
- vacuum housing for optics protection and minimisation of beam scatter in air.

PILATUS 200K 2D detector is built using the HPAD technology, which combines pixelated silicon sensors with electronic CMOS readout circuitry. It is used for the most demanding crystallography examinations using x-ray beams. The key features of the detector include:

- active area of 83.8 ´ 70.0 mm2,
- direct detection of x-ray beams by counting individual photons,
- high dynamics range (> 1,000,000:1 per pixel),
- short readout time (7 msec), high image frequency, high-speed count,
- elimination of background and readout noise and excellent pixel resolution (point-spread function). 

Pilatus 200 is a small-size, user friendly device. It is cooled with air, which allows for low operating requirements.
4-circle AFC11 Partial-chi goniometer facilitates movement along the ϕ, ω,
c, 2θ  axes and adjustable distance between the sample and the detector.

The advantages of this 4-circle goniometer include:
- automatic adjustment of the distance between the sample and the detector: 27 to 287 mm
- maximum accessibility of Ewald's sphere ensures high scan completeness, also for low-symmetry crystals.
- the capacity for circular motion in various angles facilitates more ergonomic strategies for fast data collection.

Cryostream 800 is a temperature extension (the device is fuelled with liquid nitrogen acting as the coolant) which allows for experiments conducted in the temperature range of 80 300 K.

B. Nikon Eclipse LV100ND metallographic polarised light microscope is an additional device necessary for the assessment of the processes of crystallisation and monitoring diffraction analysis specimens. A polarised light metallographic microscope allows visual observation in both transmitted and reflected light; it is fitted with a trinocular eyepiece. It allows the samples to be photographed directly on the crystallisation slide, which facilitates quicker selection of specimens and optimisation of the crystallisation conditions.

C. Temperature controlled room for storage of the protein crystallisation sets and performance of crystallisation processes.

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