Fast data collection for high throughput screening
As an example of a fast data collection, applications scientists at Bruker AXS recorded data on a crystal of a cyclin-dependent kinase (CDK) using a MetalJet X-ray source mounted on a Bruker D8 VENTURE system.
The complete experiment lasted 200 seconds and consisted of 100° of data with the resulting 1.95 Å data allowing for a structure solution by molecular replacement.
• Exposure: 1 second • Multiplicity: 3.68
• Crystal size: 0.1 x 0.08 x 0.05 mm3 • Rmerge : 6.58%
• Completeness: 97.5% • Rpim : 3.58%
Membrane proteins in-house
The successful data collection and structure solution of membrane proteins is notoriously difficult and is rarely achieved. Even rarer is the successful determination of a membrane protein structure using an in-house X-ray diffractometer system, rather than a high brilliance synchrotron radiation source.
A small crystal of GPCR (Human Orexin receptor Ox1R-StaR®) has been successfully measured at Bruker AXS using an in-house MetalJet X-ray source mounted on a D8 VENTURE diffractometer.
Data were collected in a total experiment time of ~2.5 hours to 2.77 Å resolution and the structure was successfully solved by molecular replacement.
• Scan width: 0.1° • I/sigma: 8.0
• Exposure time: 6 seconds • Rpim : 7.62%
• Crystal size: 0.08 x 0.08 x 0.05 mm3 • Rwork/ RFree : 0.244 / 0.272
• Multiplicity: 3.2
Ligand-enzyme co-crystallisation studies
Researchers at the University of Wisconsin (Department of Biochemistry) and the National Research Council Canada, Human Health Therapeutics, recently solved the structure of WlaRA (TDP-fucose-3,4-ketoisomerase) from Campylobacter jejuni using data collected with a gallium MetalJet D2+ X-ray source.
• Resolution: 2.15 Å • Rmerge : 7.1%
• Completeness: 99.3% • Multiplicity: 7.1
SAD phasing in-house
Application scientists at Bruker AXS have determined the crystal structure of Thaumatin obtained from Thaumatococcus danielii by sulphur-SAD phasing methods using data collected in-house on a D8 VENTURE diffraction system with a MetalJet X-ray source.
Using one 70 μm crystal, a complete data set was collected to 1.65 Å in <3 hours. The experimental phases were derived from the anomalous signal of the sulphur atoms and these allowed 95% of the protein backbone to be traced.
In-situ X-ray Crystallography
In-situ crystallography is a technique in which protein crystals undergo X-ray diffraction screening and or data collection whilst in a multi-well crystallisation plate and their original growth media/conditions. Typically, X-rays are directed from one side of the multi-well plate, pass through the plate and crystal and out the opposite side of the plate, where the diffraction data are collected on an X-ray sensitive detector.
In-situ X-ray diffraction offers the following benefits:
- Automated, rapid screening and identification of large numbers of potential protein crystals without risk of damage to the crystals
- The identification of protein crystals from non-crystalline objects, salt crystals and other impurity crystals
- The identification of the best protein crystals for further X-ray study
Multi-well crystallisation plates are typically of plastic construction and exhibit low X-ray transparency, high X-ray absorption and significant X-ray background scatter, all of which serve to reduce and/or obscure the X-ray diffraction signal to be studied.
The MetalJet is the ideal choice of X-ray source for in-situ X-ray diffraction in the home laboratory due to the following unique combination of technical features:
- The potential to tune the X-ray beam size through the software, means the X-ray beam may be matched to the size of the crystal and precisely focused on to the crystal of interest, rather than a group of crystals. This also means that a much smaller area of plastic plate is illuminated by the X-ray beam leading to reduced background scatter.
- The higher X-ray brilliance of the MetalJet combined with the lower X-ray absorption of gallium radiation; when compared with copper X-ray sources, means that the X-ray signal obtained from in-situ diffraction is greater for the MetalJet.