Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.
We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.
Tan
S
S
Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, ETH-Zürich, CH-8093, Switzerland.
Hunziker
Y
Y
Pellegrini
L
L
Richmond
T J
TJ
D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D026341:Minichromosome Maintenance 1 Protein; D008958:Models, Molecular; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004271:DNA, Fungal; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; C062928:MATA2 protein, S cerevisiae; D026341:Minichromosome Maintenance 1 Protein; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C037405:heparin-sepharose; D012685:Sepharose