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Title: A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements

Abstract

A technique is described for generating large well diffracting crystals from conditions that yield microcrystals. Crystallization using this technique is both rapid (crystals appear in <1 h) and robust (48 out of 48 co-crystallized with a fragment library, compared with 26 out of 48 using conventional hanging drop). Agarose gel is used to exclude nucleation inducing elements from the remaining crystallization cocktail. The chemicals in the crystallization cocktail are partitioned into high concentration components (presumed to induce aggregation by reducing water activity) and low concentration nucleation agents (presumed to induce nucleation through direct interaction). The nucleation agents are then combined with 2% agarose gel and deposited on the crystallization shelf of a conventional vapor diffusion plate. The remaining components are mixed with the protein and placed in contact with the agarose drop. This technique yielded well diffracting crystals of lysozyme, cubic insulin, proteinase k, and ferritin (ferritin crystals diffracted to 1.43 Å). The crystals grew rapidly, reaching large size in less than one hour (maximum size was achieved in 1–12 h). This technique is not suitable for poorly expressing proteins because small protein volumes diffuse out of the agarose gel too quickly. Nonetheless, it is a useful technique for situationsmore » where crystals must grow rapidly (such as educational applications and preparation of beamline test specimens) and in situations where crystals must grow robustly (such as co-crystallization with a fragment library).« less

Authors:
 [1]; ORCiD logo [2];  [3]; ORCiD logo [4]; ORCiD logo [5];  [6];  [7]; ORCiD logo [7];  [7]; ORCiD logo [7]
  1. Brookhaven National Laboratory (BNL), Upton, NY (United States); Stony Brook Univ., Stony Brook, NY (United States)
  2. Brookhaven National Laboratory (BNL), Upton, NY (United States); State Univ. of New York, Old Westbury, NY (United States)
  3. Brookhaven National Laboratory (BNL), Upton, NY (United States); College of William and Mary, Williamsburg, VA (United States)
  4. Brookhaven National Laboratory (BNL), Upton, NY (United States); General Douglas MacArthur High School, Levittown, NY (United States)
  5. Brookhaven National Laboratory (BNL), Upton, NY (United States); Ross School, East Hampton, NY (United States)
  6. Brookhaven National Laboratory (BNL), Upton, NY (United States); Shoreham-Wading River High School, Shoreham, NY (United States)
  7. Brookhaven National Laboratory (BNL), Upton, NY (United States)
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1502804
Report Number(s):
BNL-211432-2019-JAAM
Journal ID: ISSN 2073-4352
Grant/Contract Number:  
SC0012704
Resource Type:
Accepted Manuscript
Journal Name:
Crystals
Additional Journal Information:
Journal Volume: 8; Journal Issue: 12; Journal ID: ISSN 2073-4352
Publisher:
MDPI
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; drug discovery; education; crystallization; crystallography; nucleation; micro-crystals; agarose; ferritin; lysozyme; proteinase k; insulin

Citation Formats

Blackburn, Adafih, Partowmah, Shahla, Brennan, Haley, Mestizo, Kimberly, Stivala, Cristina, Petreczky, Julia, Perez, Aleida, Horn, Amanda, McSweeney, Sean, and Soares, Alexei. A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements. United States: N. p., 2018. Web. doi:10.3390/cryst8120464.
Blackburn, Adafih, Partowmah, Shahla, Brennan, Haley, Mestizo, Kimberly, Stivala, Cristina, Petreczky, Julia, Perez, Aleida, Horn, Amanda, McSweeney, Sean, & Soares, Alexei. A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements. United States. doi:10.3390/cryst8120464.
Blackburn, Adafih, Partowmah, Shahla, Brennan, Haley, Mestizo, Kimberly, Stivala, Cristina, Petreczky, Julia, Perez, Aleida, Horn, Amanda, McSweeney, Sean, and Soares, Alexei. Wed . "A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements". United States. doi:10.3390/cryst8120464. https://www.osti.gov/servlets/purl/1502804.
@article{osti_1502804,
title = {A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements},
author = {Blackburn, Adafih and Partowmah, Shahla and Brennan, Haley and Mestizo, Kimberly and Stivala, Cristina and Petreczky, Julia and Perez, Aleida and Horn, Amanda and McSweeney, Sean and Soares, Alexei},
abstractNote = {A technique is described for generating large well diffracting crystals from conditions that yield microcrystals. Crystallization using this technique is both rapid (crystals appear in <1 h) and robust (48 out of 48 co-crystallized with a fragment library, compared with 26 out of 48 using conventional hanging drop). Agarose gel is used to exclude nucleation inducing elements from the remaining crystallization cocktail. The chemicals in the crystallization cocktail are partitioned into high concentration components (presumed to induce aggregation by reducing water activity) and low concentration nucleation agents (presumed to induce nucleation through direct interaction). The nucleation agents are then combined with 2% agarose gel and deposited on the crystallization shelf of a conventional vapor diffusion plate. The remaining components are mixed with the protein and placed in contact with the agarose drop. This technique yielded well diffracting crystals of lysozyme, cubic insulin, proteinase k, and ferritin (ferritin crystals diffracted to 1.43 Å). The crystals grew rapidly, reaching large size in less than one hour (maximum size was achieved in 1–12 h). This technique is not suitable for poorly expressing proteins because small protein volumes diffuse out of the agarose gel too quickly. Nonetheless, it is a useful technique for situations where crystals must grow rapidly (such as educational applications and preparation of beamline test specimens) and in situations where crystals must grow robustly (such as co-crystallization with a fragment library).},
doi = {10.3390/cryst8120464},
journal = {Crystals},
number = 12,
volume = 8,
place = {United States},
year = {2018},
month = {12}
}

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