The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae.
BACKGROUND
Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search.


RESULTS
Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS.


CONCLUSIONS
The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution.
Sohi
Maninder K
MK
Randall Division of Cell and Molecular Biophysics, School of Biomedical and Health Sciences, King's College London, London, United Kingdom.
Veselkov
Dennis A
DA
Laponogov
Ivan
I
Pan
Xiao-Su
XS
Fisher
L Mark
LM
Sanderson
Mark R
MR
D001671:Biochemistry / Q000379:methods; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D027101:DNA Topoisomerase IV / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D003902:Detergents; D027101:DNA Topoisomerase IV; D003545:Cysteine
Acta Crystallogr D Biol Crystallogr. 2001 Oct;57(Pt 10):1367-72
11567147
Nat Struct Biol. 1996 Feb;3(2):109-12
8564532
J Mol Graph. 1996 Feb;14(1):33-8, 27-8
8744570
FEBS Lett. 2000 Jan 14;465(2-3):161-4
10631326
Eur J Biochem. 1990 Apr 20;189(1):1-23
2185018
Biochim Biophys Acta. 1998 Oct 1;1400(1-3):29-43
9748489
Annu Rev Biochem. 2001;70:369-413
11395412
Nature. 1990 Dec 6;348(6301):515-20
2147232
Biochem Soc Trans. 2005 Dec;33(Pt 6):1460-4
16246146
Antimicrob Agents Chemother. 1997 Feb;41(2):471-4
9021211
Microbiol Mol Biol Rev. 1997 Sep;61(3):377-92
9293187
Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
15299374
Nature. 1989 Feb 23;337(6209):745-9
2645526
Nat Rev Mol Cell Biol. 2002 Jun;3(6):430-40
12042765
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Methods Enzymol. 1997;276:307-26
27754618
Biochem Soc Trans. 2005 Dec;33(Pt 6):1465-70
16246147
J Mol Biol. 1989 Aug 20;208(4):661-7
2553982
PLoS One. 2007 Mar 21;2(3):e301
17375187
Biochim Biophys Acta. 1998 Oct 1;1400(1-3):3-18
9748476
Annu Rev Biophys Biomol Struct. 2004;33:95-118
15139806
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Nov 1;62(Pt 11):1164-7
17077506
Antimicrob Agents Chemother. 1999 May;43(5):1129-36
10223925
J Biol Chem. 2005 Apr 8;280(14):14252-63
15659402