DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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 Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
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. https://doi.org/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. https://doi.org/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 = {Wed Dec 12 00:00:00 EST 2018},
month = {Wed Dec 12 00:00:00 EST 2018}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

A novel dialysis procedure for the crystallization of proteins
journal, January 1989

  • Thomas, D. H.; Rob, A.; Rice, D. W.
  • "Protein Engineering, Design and Selection", Vol. 2, Issue 6
  • DOI: 10.1093/protein/2.6.489

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density
journal, July 2015

  • Teplitsky, Ella; Joshi, Karan; Ericson, Daniel L.
  • Journal of Structural Biology, Vol. 191, Issue 1
  • DOI: 10.1016/j.jsb.2015.05.006

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology
journal, January 2018

  • Gorrec, Fabrice; Löwe, Jan
  • Journal of Visualized Experiments, Issue 131
  • DOI: 10.3791/55790

Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

Novel Protein Crystal Growth Electrochemical Cell For Applications In X-ray Diffraction and Atomic Force Microscopy
journal, September 2011

  • Gil-Alvaradejo, Gabriela; Ruiz-Arellano, Rayana R.; Owen, Christopher
  • Crystal Growth & Design, Vol. 11, Issue 9
  • DOI: 10.1021/cg200485v

Introduction to protein crystallization
journal, December 2013

  • McPherson, Alexander; Gavira, Jose A.
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 70, Issue 1
  • DOI: 10.1107/S2053230X13033141

Ferritin: A Versatile Building Block for Bionanotechnology
journal, January 2015

  • Jutz, Günther; van Rijn, Patrick; Santos Miranda, Barbara
  • Chemical Reviews, Vol. 115, Issue 4
  • DOI: 10.1021/cr400011b

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule
journal, September 2015

  • folzer, Emilien; diepold, Katharina; bomans, Katrin
  • Journal of Pharmaceutical Sciences, Vol. 104, Issue 9
  • DOI: 10.1002/jps.24509

What’s happened over the last five years with high-throughput protein crystallization screening?
journal, April 2018


X-ray diffraction measurement of cosolvent accessible volume in rhombohedral insulin crystals
journal, December 2017


Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes
journal, April 2014

  • Yin, Xingyu; Scalia, Alexander; Leroy, Ludmila
  • Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 5
  • DOI: 10.1107/S1399004713034603

Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase
journal, February 2016

  • Ericson, Daniel L.; Yin, Xingyu; Scalia, Alexander
  • Journal of Laboratory Automation, Vol. 21, Issue 1
  • DOI: 10.1177/2211068215616365

Crystal growth in gels: Principle and applications
journal, July 1988


Macromolecular crystallization in microgravity
journal, March 2005


Protein crystal growth in gels and stationary magnetic fields
journal, March 2007

  • Moreno, A.; Quiroz-García, B.; Yokaichiya, F.
  • Crystal Research and Technology, Vol. 42, Issue 3
  • DOI: 10.1002/crat.200610805

A simple technique to reduce evaporation of crystallization droplets by using plate lids with apertures for adding liquids
journal, November 2014

  • Zipper, Lauren E.; Aristide, Xavier; Bishop, Dylan P.
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 70, Issue 12
  • DOI: 10.1107/S2053230X14025126

Phagocytes, Immunology and Inflammation
journal, March 2002


Unity in the Biochemistry of the Iron-Storage Proteins Ferritin and Bacterioferritin
journal, November 2014

  • Honarmand Ebrahimi, Kourosh; Hagedoorn, Peter-Leon; Hagen, Wilfred R.
  • Chemical Reviews, Vol. 115, Issue 1
  • DOI: 10.1021/cr5004908

Crystallization of protein–ligand complexes
journal, December 2006

  • Hassell, Anne M.; An, Gang; Bledsoe, Randy K.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 1
  • DOI: 10.1107/S0907444906047020

Ferritin, cellular iron storage and regulation: FERRITIN AND IRON STORAGE
journal, March 2017

  • Arosio, Paolo; Elia, Leonardo; Poli, Maura
  • IUBMB Life, Vol. 69, Issue 6
  • DOI: 10.1002/iub.1621

Application of gel growth to hanging drop technique
journal, March 1991


Screening of Nucleation Conditions Using Levitated Drops for Protein Crystallization
journal, April 2003

  • Santesson, Sabina; Cedergren-Zeppezauer, Eila S.; Johansson, Thomas
  • Analytical Chemistry, Vol. 75, Issue 7
  • DOI: 10.1021/ac020496y

A free interface diffusion technique for the crystallization of proteins for X-ray crystallography
journal, August 1972


Automated refinement of protein models
journal, January 1993

  • Lamzin, V. S.; Wilson, K. S.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 49, Issue 1
  • DOI: 10.1107/S0907444992008886

Comparison of the Structures of the Cubic and Tetragonal Forms of Horse-Spleen Apoferritin
journal, September 1997

  • Granier, T.; Gallois, B.; Dautant, A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 5
  • DOI: 10.1107/S0907444997003314

On the limited ability of superoxide to release iron from ferritin
journal, November 1990


Ferritin, cellular iron storage and regulation: FERRITIN AND IRON STORAGE
journal, March 2017

  • Arosio, Paolo; Elia, Leonardo; Poli, Maura
  • IUBMB Life, Vol. 69, Issue 6
  • DOI: 10.1002/iub.1621

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density
journal, July 2015

  • Teplitsky, Ella; Joshi, Karan; Ericson, Daniel L.
  • Journal of Structural Biology, Vol. 191, Issue 1
  • DOI: 10.1016/j.jsb.2015.05.006

X-ray diffraction measurement of cosolvent accessible volume in rhombohedral insulin crystals
journal, December 2017


Unity in the Biochemistry of the Iron-Storage Proteins Ferritin and Bacterioferritin
journal, November 2014

  • Honarmand Ebrahimi, Kourosh; Hagedoorn, Peter-Leon; Hagen, Wilfred R.
  • Chemical Reviews, Vol. 115, Issue 1
  • DOI: 10.1021/cr5004908

Chaperone salts, polyethylene glycol and rates of equilibration in vapor-diffusion crystallization
journal, September 1995

  • Luft, J. R.; DeTitta, G. T.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 51, Issue 5
  • DOI: 10.1107/s0907444995002277

From screen to structure with a harvestable microfluidic device
journal, July 2011

  • Stojanoff, Vivian; Jakoncic, Jean; Oren, Deena A.
  • Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 67, Issue 8
  • DOI: 10.1107/s1744309111024456

On the limited ability of superoxide to release iron from ferritin
journal, November 1990


Works referencing / citing this record:

Crystal Structure of Chaperonin GroEL from Xanthomonas oryzae pv. oryzae
journal, August 2019


Biological Crystallization
journal, August 2019

  • Gómez-Morales, Jaime; Falini, Giuseppe; García-Ruiz, Juan Manuel
  • Crystals, Vol. 9, Issue 8
  • DOI: 10.3390/cryst9080409