2019-07-24RELPDBePDBePDBe2019-03-072019-05-082019-05-082019-07-24Cas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas systemWilkinson MDrabavicius GSilanskas AGasiunas GSiksnys VWigley DBWilkinson MDrabavicius GSilanskas AGasiunas GSiksnys VWigley DBStructure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.Mol.CellUS7590101.e5201931080012doi:10.1016/j.molcel.2019.04.0201097-27652168MOCEFLEMD-4668associated EM volumeCas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas system6qxfFULLOVERLAPCas1-Cas2-Csn2-DNA monomer complexCas1-Cas2-Csn2-DNA monomer complex0123450.53Cas1-Cas2-Csn21123Streptococcus thermophilusEscherichia coli BL21(DE3)DNA145Escherichia coli BL21(DE3)CRISPR-associated protein Csn2Streptococcus thermophilus0.0255334738Escherichia coli BL21(DE3)LEVOMKINFSLLDEPMEVNLGTVLVIEDVSVFAQLVKEFYQYDEQSNLTIFDSKIRSIRSSELLLITDILGYDINTSQVLKLLH
TDIVSQLNDKPEVRSEIDSLVSLITDIIMAECIENELDIEYDEITLLELIKALGVRIETKSCTVFEKIFEILQIFKYLVK
KRILVFVNSLSYFSKDEIYQILEYTKLSQADVLFLEPRQIEGIQQFILDKDYILMPYNNCRISPR-associated endonuclease Cas1Streptococcus thermophilus0.0353634618Escherichia coli BL21(DE3)LEVOMAGWRTVVVNIHSKLSYKNNHLIFRNSYKTEMIHLSEIDILLLETTDIVLTTMLVKRLVDENILVIFCDDKRLPTAFLTP
YYARHDSSLQIARQIAWKENVKCEVWTAIIAQKILNQSYYLGECSFFEKSQSIMELYHGLERFDPSNREGHSARIYFNTL
FGNDFTRESDNDINAALDYGYTLLLSMFAREVVVCGCMTQIGLKHANQFNQFNLASDIMEPFRPIIDRIVYQNRHNNFVK
IKKELFSIFSETYLYNGKEMYLSNIVSDYTKKVIKALNQLGEEIPEFRILESGWSHPQFEKA3.1.-.-CRISPR-associated endoribonuclease Cas2Streptococcus thermophilus0.013431564Escherichia coli BL21(DE3)LEVOMSYRYMRMILMFDMPTDTAEERKAYRKFRKFLLSEGFIMHQFSVYSKLLLNHTANTAMVGRLKANNPKKGNITILTVTEK
QFARMIYLYGDKNTSIANSEERLVFLGDNYCDED3.1.-.-DNA (25-MER)Escherichia coli BL21(DE3)0.007559863Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.1(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)
(DT)(DT)(DT)(DT)(DT)DNADNA (25-MER)Escherichia coli BL21(DE3)0.007785214Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.1(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)
(DA)(DA)(DA)(DA)(DA)DNACALCIUM ION4.0078e-058CAsingleParticleparticle0.057.525.0Tris150.0NaClSodium chloride1.0TCEPBuffer solution was filteredC-flat-2/1COPPER400GRAPHENE OXIDECONTINUOUSGraphene oxide sheets were applied to the grid prior to sample applicationETHANE90277FEI VITROBOT MARK IV15 s wait time, 1 s blot time.. Sample was DNaseI treated followed by gel filtration. Peak fractions were concentrated prior to making grids.FEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN300100.02.71.32.875000.129032.FEI TITAN KRIOS AUTOGRID HOLDERNITROGENFEI FALCON III (4k x 4k)INTEGRATING14.0149081.092.8Images were collected in movie mode with 39 frames1853391Reproductions of an initial ab initio generated 3D reconstruction were used as templates for auto pickingGctfGenerated ab initio from the data with cryoSPARCReconstruction was low-pass filtered to at least 30 Angstroms prior to starting classification/refinement runs5C13.6FSC 0.143 CUT-OFFRELION3.0306470OTHERRELION3.0OTHERRELION3.0657000.0RELION3.03D classification without alignment after running 3D refinement. A low-pass filtered soft mask around the complex was used with D2 symmetry enforced.3V7F5XVNA5XVNB5XVNEOTHERRounds of manual fitting in Coot, jelly-body refinement in REFMAC and real-space refinement in PHENIXCorrelation coefficient and visual inspectionREALemd_4668_additional.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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00296296296321.16321.16321.1690.090.090.0XYZ-0.048677350.161885380.00001125927250.00640932331.0851.0851.085Supplementary map refined with D2 symmetry to aid model building