Microbatch macromolecular crystallization in micropipettes

Luft, Joseph R.; Rak, Dawn; DeTitta, George T.

doi:10.1016/s0022-0248(98)00864-1

Cited by 22 publications

(10 citation statements)

References 14 publications

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“…The protein solution was incubated for 18 h with NADPH and PT653 at 277 K, after which it was concentrated to 6 mg ml À1 . Crystallization experiments were conducted using a capillary microbatch method (Luft et al, 1999a) set up on a polythermal gradient (Luft et al, 1999b). Crystals were grown in a 0.7 mm diameter special glass X-ray capillary that contained 3 mg ml À1 protein solution, 20%(w/v) PEG 2000 MME, 100 mM KCl, 50 mM MES buffer pH 6.0.…”

Section: Experimental Section 21 Crystallization and X-ray Data Colmentioning

confidence: 99%

Structure-based enzyme inhibitor design: modeling studies and crystal structure analysis ofPneumocystis cariniidihydrofolate reductase ternary complex with PT653 and NADPH

Cody¹,

Galitsky²,

Luft³

et al. 2002

Acta Crystallogr D Biol Cryst

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Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz [b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could ®t better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, re®ned to 2.4 A Ê resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously , J. Med. Chem. 42, 4853±4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most signi®cant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A Ê away from the inhibitor of residues 61±66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes , Biochemistry, 38, 4303±4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced ®t of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could in¯uence the enhanced selectivity of PT653 for tgDHFR.

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Section: Experimental Section 21 Crystallization and X-ray Data Colmentioning

confidence: 99%

Structure-based enzyme inhibitor design: modeling studies and crystal structure analysis ofPneumocystis cariniidihydrofolate reductase ternary complex with PT653 and NADPH

Cody¹,

Galitsky²,

Luft³

et al. 2002

Acta Crystallogr D Biol Cryst

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“…This allows the crystallographer to assess the quality of the crystals grown before alteration by the cryoprotection process (Luft et al, 1999;McPherson, 2000;Ng et al, 2003;Yadav et al, 2005;Lunde et al, 2008) and to collect data for structure determination from robust crystals. The CrystalCard is sufficiently transparent to X-rays to be mounted directly onto a goniometer for X-ray diffraction experiments at room temperature.…”

Section: In Situ X-ray Diffractionmentioning

confidence: 99%

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

Gerdts

Elliott

Lovell

et al. 2008

Acta Crystallogr D Biol Cryst

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The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, $10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive 'hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.

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“…Screening for protein crystallization includes many repetitive and reproducible pipetting operations. To ease this manual and time-consuming task, several automatic methods have been introduced [4,5]. Despite such efforts at reducing protein volumes, these processes still consume a significant amount of protein samples (in the microliter range) and they are labor-intensive.…”

Section: Introductionmentioning

confidence: 99%