X-ray Crystallography Facility, University Park

Crystallization

• You provide the sample (native gel electrophoresis should be performed to demonstrate purity)
• Screens are performed (eleven 48-conditions, seven 24-conditions, three 96-conditions from Hampton; six 96-condition suites from Nextal)
• Case-specific optimization grids are used to obtain diffraction-quality crystals from initial screens

Crystal screening for X-ray diffraction

• Crystals are screened on X-ray machines to determine diffraction limit, mosaicity and optimization of freezing conditions
• Crystals are characterized; unit cell dimensions and space group are determined

X-ray data collection

• For crystals that have been screened successfully, X-ray data acquisition is attempted.
• In cases where the diffraction is of low resolution, data collection using the “mail-in” facility at a synchrotron can be explored.
• In special cases a data collection trip is planned to collect data at one of the national synchrotron facilities in person.

3D structure determination and analysis

• X-ray structure determination, display and analysis of macromolecular structure are done using a variety of software.

Access to Protein Data Bank

• The Protein Data Bank is the single worldwide repository for the processing and distribution of 3-D structure data of large molecules of proteins and nucleic acids. We can visualize and analyze structures deposited in this database using a range of software.

Dynamic light scattering

• Analysis of variables such as aggregation, folding and conformation as a function or time and/or treatment.

Manuscript and grant preparation

• We will assist investigators with manuscript and preparation (including figures) for projects that involve 3D structure determination by X-ray crystallography at the facility.

Announcements

New equipment has arrived

We have just acquired new X-ray instrumentation and a crystallization robot. The robot is up and running. The X-ray instrumentation is being installed.

Collaborations

Examples of the types of collaboration we are involved with:

• Structural studies of protein complexes involved in yeast and T4 DNA replication, in collaboration with Prof. Stephen J. Benkovic.
• Elucidation of the crystal structure of a complex maize cell wall protein, expansin (EXP B1), with its polysaccharide substrate, in collaboration with Prof. Daniel Cosgrove.

Sample publications

• Gurjar AA, Yennawar NH, Yennawar HP, Rajashankar KR, Hegde NV, Jayarao BM (2007) Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens beta2-toxin. Acta Crystallograph Sect F Struct Biol Cryst Commun. 63(6):484-7.
• Yennawar NH, Islam MM, Conway M, Wallin R, Hutson SM (2006) Human mitochondrial branched chain aminotransferase isozyme: Structural role of the CXXC center in catalysis. J Biol Chem. 281(51):39660-71.
• Yennawar NH, Li LC, Dudzinski DM, Tabuchi A, Cosgrove DJ (2006) Crystal structure and activities of EXPB1 (Zea m 1), a beta-expansin and group-1 pollen allergen from maize. PNAS 103(40):14664-71.