PDBsum entry 4tpy

Hydrolase

PDB id: 4tpy

Contents

Protein chain

223 a.a.

Ligands

BEN

TRS

EDO ×7

Metals

_CA

_NA ×6

_BR ×26

Waters ×385

PDB id:

4tpy

Name:

Hydrolase

Title:

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density

Structure:

Cationic trypsin. Chain: a. Synonym: beta-trypsin. Other_details: bromine derivative identified using acoustic droplet ejection to screen 1728 distinct conditions on each micro plate.

Source:

Bos taurus. Bovine. Organism_taxid: 9913

Resolution:

1.30Å R-factor: 0.144 R-free: 0.172

Authors:

E.Teplitsky,K.Joshi,J.D.Mullen,A.S.Soares

Key ref:

E.Teplitsky et al. (2015). High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density. J Struct Biol, 191, 49-58. PubMed id: 26027487 DOI: 10.1016/j.jsb.2015.05.006

Date:

10-Jun-14 Release date: 22-Oct-14

Protein chain

P00760  (TRY1_BOVIN) -  Serine protease 1 from Bos taurus

Seq: 246 a.a.

Struc: 223 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

DOI no: 10.1016/j.jsb.2015.05.006

J Struct Biol 191:49-58 (2015)

PubMed id: 26027487

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.

E.Teplitsky, K.Joshi, D.L.Ericson, A.Scalia, J.D.Mullen, R.M.Sweet, A.S.Soares.

ABSTRACT

We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5nL of each component.