Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.
We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.
Oubridge
Chris
C
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, England, UK.
Krummel
Daniel A Pomeranz
DA
Leung
Adelaine K-W
AK
Li
Jade
J
Nagai
Kiyoshi
K
D001483:Base Sequence; D001665:Binding Sites; D001965:Bromides / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008715:Methionine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000032:analysis; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000032:analysis*; D012542:Scattering, Radiation; D012645:Selenomethionine / Q000032:analysis; D013635:Tantalum / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry; D014961:X-Ray Diffraction; D055517:snRNP Core Proteins / Q000737:chemistry*
D001965:Bromides; D010455:Peptides; D017412:Ribonucleoprotein, U1 Small Nuclear; D055517:snRNP Core Proteins; D013879:Thioredoxins; D012313:RNA; D013635:Tantalum; D012645:Selenomethionine; D008715:Methionine