MacCHESS is involved in phasing of macromolecular data using MAD phasing techniques, direct methods and more recently multiple-beam diffraction. In order to implement MAD phasing at CHESS we have an ongoing effort for hardware and software development. Station F-2 was equipped with double crystal, focusing optics, a rotation camera, CCD detector, liquid nitrogen cooling apparatus and scintillation detectors for fluorescent measurements during energy scans of sample and reference materials. New software was implemented to rapidly change energies while maintaining the alignment of the x-ray beam relative to the sample. At F-2, the energy resolution is about 3-4 eV and exposure times are typically 20-30 seconds per degree of crystal rotation. These parameters are well suited to MAD phasing experiments. During the past year we have focused on analyzing MAD structure of greater and greater complexity. The include 5'-methylthioadenosine phosphorylase (9 Se atoms), thiamin phosphate synthase (16 Se atoms), glycinamide ribonucleotide reductase (14 Se atoms), aminoimidazole ribonucleotide reductase (28 Se atoms) and S-adenosylmethionione decarboxylase (24 Se atoms). All five of these structures have been determined using MAD data. More recently, we have located used MAD data to locate the 66 Se atoms of a previously unknown epimerase structure. The phasing process is just now underway. We have also used direct methods for direct structure determination. We have developed procedures for measuring high resolution data using CHESS stations A-1 or F-1, cryocrystallography, CCD detectors. Using 1.0 ? resolution data for triclinic lysosyme (1200 atoms including ordered solvent), we have determined the structure with the Shake and Bake procedure developed by Hauptman, et al. We have also measured data for xylan esterase (1500 atoms) and the structure analysis is currently underway. Experimental measurement of phases using multiple beam diffraction is just now underway at CHESS. Preliminary experiments have lead to a new procedure for data collection in which the crystal is rotated about a reference beam and analysis of the results are encouraging.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001646-20
Application #
6667788
Study Section
Project Start
2002-09-30
Project End
2003-08-14
Budget Start
Budget End
Support Year
20
Fiscal Year
2002
Total Cost
$142,703
Indirect Cost
Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Kozlov, Guennadi; Wong, Kathy; Gehring, Kalle (2018) Crystal structure of the Legionella effector Lem22. Proteins 86:263-267
Ménade, Marie; Kozlov, Guennadi; Trempe, Jean-François et al. (2018) Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations. J Biol Chem 293:12832-12842
Xu, Jie; Kozlov, Guennadi; McPherson, Peter S et al. (2018) A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy. J Biol Chem 293:4566-4574
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Chen, Yu Seby; Kozlov, Guennadi; Fakih, Rayan et al. (2018) The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity. J Biol Chem 293:19998-20007
Xu, Caishuang; Kozlov, Guennadi; Wong, Kathy et al. (2016) Crystal Structure of the Salmonella Typhimurium Effector GtgE. PLoS One 11:e0166643
Cogliati, Massimo; Zani, Alberto; Rickerts, Volker et al. (2016) Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population. Fungal Genet Biol 87:22-9
Oot, Rebecca A; Kane, Patricia M; Berry, Edward A et al. (2016) Crystal structure of yeast V1-ATPase in the autoinhibited state. EMBO J 35:1694-706
Lucido, Michael J; Orlando, Benjamin J; Vecchio, Alex J et al. (2016) Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 55:1226-38
Bauman, Joseph D; Harrison, Jerry Joe E K; Arnold, Eddy (2016) Rapid experimental SAD phasing and hot-spot identification with halogenated fragments. IUCrJ 3:51-60

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