Preparation of crystals of T7 RNA polymerase suitable for high-resolution X-ray structure analysis

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The crystallization of bacteriophage T7 RNA polymerase is described. A number of features of the crystallization methodology are worthy of note: (1) Preparation of crystals suitable for X-ray analysis depended on removal of oligomeric forms of the enzyme which formed during purification and were not detectable by denaturing gel electrophoreris. (2) By increasing the protein supersaturation, changes in the relative interfacial growth rates were induced, resulting in increases in crystal thickness and diffraction to higher resolution. (3) The stability of the crystalline versus amorphous phase of the solid protein was shifted by the presence of glycerol in the mother liquor: crystallization was dependent on the presence of at least 15% glycerol. The high density and viscosity of glycerol mother liquors reduced convective and diffusive flow and eliminated crystal sedimentation. The implications and possible mechanisms of the glycerol effect on crystallization are discussed and the generality and extension of this effect is suggested.

Original languageEnglish (US)
Pages (from-to)237-246
Number of pages10
JournalJournal of Crystal Growth
Volume110
Issue number1-2
DOIs
StatePublished - Mar 1 1991
Externally publishedYes

Fingerprint

Crystallization
glycerols
RNA
Glycerol
crystallization
X rays
preparation
Crystals
high resolution
crystals
x rays
proteins
Proteins
bacteriophages
Bacteriophages
convective flow
Supersaturation
X ray analysis
supersaturation
Sedimentation

ASJC Scopus subject areas

  • Condensed Matter Physics

Cite this

Preparation of crystals of T7 RNA polymerase suitable for high-resolution X-ray structure analysis. / Sousa, Rui J; Lafer, Eileen M; Wang, B. C.

In: Journal of Crystal Growth, Vol. 110, No. 1-2, 01.03.1991, p. 237-246.

Research output: Contribution to journalArticle

@article{f92bb74360194a10b664b700ac96d95b,
title = "Preparation of crystals of T7 RNA polymerase suitable for high-resolution X-ray structure analysis",
abstract = "The crystallization of bacteriophage T7 RNA polymerase is described. A number of features of the crystallization methodology are worthy of note: (1) Preparation of crystals suitable for X-ray analysis depended on removal of oligomeric forms of the enzyme which formed during purification and were not detectable by denaturing gel electrophoreris. (2) By increasing the protein supersaturation, changes in the relative interfacial growth rates were induced, resulting in increases in crystal thickness and diffraction to higher resolution. (3) The stability of the crystalline versus amorphous phase of the solid protein was shifted by the presence of glycerol in the mother liquor: crystallization was dependent on the presence of at least 15{\%} glycerol. The high density and viscosity of glycerol mother liquors reduced convective and diffusive flow and eliminated crystal sedimentation. The implications and possible mechanisms of the glycerol effect on crystallization are discussed and the generality and extension of this effect is suggested.",
author = "Sousa, {Rui J} and Lafer, {Eileen M} and Wang, {B. C.}",
year = "1991",
month = "3",
day = "1",
doi = "10.1016/0022-0248(91)90891-8",
language = "English (US)",
volume = "110",
pages = "237--246",
journal = "Journal of Crystal Growth",
issn = "0022-0248",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Preparation of crystals of T7 RNA polymerase suitable for high-resolution X-ray structure analysis

AU - Sousa, Rui J

AU - Lafer, Eileen M

AU - Wang, B. C.

PY - 1991/3/1

Y1 - 1991/3/1

N2 - The crystallization of bacteriophage T7 RNA polymerase is described. A number of features of the crystallization methodology are worthy of note: (1) Preparation of crystals suitable for X-ray analysis depended on removal of oligomeric forms of the enzyme which formed during purification and were not detectable by denaturing gel electrophoreris. (2) By increasing the protein supersaturation, changes in the relative interfacial growth rates were induced, resulting in increases in crystal thickness and diffraction to higher resolution. (3) The stability of the crystalline versus amorphous phase of the solid protein was shifted by the presence of glycerol in the mother liquor: crystallization was dependent on the presence of at least 15% glycerol. The high density and viscosity of glycerol mother liquors reduced convective and diffusive flow and eliminated crystal sedimentation. The implications and possible mechanisms of the glycerol effect on crystallization are discussed and the generality and extension of this effect is suggested.

AB - The crystallization of bacteriophage T7 RNA polymerase is described. A number of features of the crystallization methodology are worthy of note: (1) Preparation of crystals suitable for X-ray analysis depended on removal of oligomeric forms of the enzyme which formed during purification and were not detectable by denaturing gel electrophoreris. (2) By increasing the protein supersaturation, changes in the relative interfacial growth rates were induced, resulting in increases in crystal thickness and diffraction to higher resolution. (3) The stability of the crystalline versus amorphous phase of the solid protein was shifted by the presence of glycerol in the mother liquor: crystallization was dependent on the presence of at least 15% glycerol. The high density and viscosity of glycerol mother liquors reduced convective and diffusive flow and eliminated crystal sedimentation. The implications and possible mechanisms of the glycerol effect on crystallization are discussed and the generality and extension of this effect is suggested.

UR - http://www.scopus.com/inward/record.url?scp=0026119322&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026119322&partnerID=8YFLogxK

U2 - 10.1016/0022-0248(91)90891-8

DO - 10.1016/0022-0248(91)90891-8

M3 - Article

AN - SCOPUS:0026119322

VL - 110

SP - 237

EP - 246

JO - Journal of Crystal Growth

JF - Journal of Crystal Growth

SN - 0022-0248

IS - 1-2

ER -