skip to main content

SciTech ConnectSciTech Connect

Title: Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin. Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as themore » working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.« less
Authors:
 [1] ;  [2] ;  [3] ;  [1] ;  [2] ;  [1] ;  [4] ;  [4] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [1] ;  [2] ;  [1] ;  [2] ; more »; ;  [1] ;  [1] ;  [2] ; ;  [1] « less
  1. Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)
  2. (United States)
  3. (China)
  4. (Brazil)
Publication Date:
OSTI Identifier:
22347794
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 70; Journal Issue: Pt 5; Other Information: PMCID: PMC4014116; PMID: 24816088; PUBLISHER-ID: nj5173; OAI: oai:pubmedcentral.nih.gov:4014116; Copyright (c) Yin et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ACCURACY; CRYSTALLIZATION; CRYSTALLOGRAPHY; CRYSTALS; DROPLETS; ENVIRONMENT; EQUILIBRIUM; IRON; LIQUIDS; LYSOZYME; SCREENING; SYNCHROTRON RADIATION; TRAJECTORIES; VELOCITY