2020-12-02RELPDBePDBePDBe2019-05-092019-09-252019-09-252020-12-02The Francis Crick InstituteFC0010061United KingdomThe Francis Crick InstituteFC0010065United KingdomPFV intasome - nucleosome strand transfer complexRenault LMaskell DPWilson MDCherepanov PCosta AWilson MDRenault LMaskell DPGhoneim MPye VENans ARueda DSCherepanov PCosta ARetroviral integration into nucleosomes through DNA looping and sliding along the histone octamer.Nat CommunUK1041894189201931519882doi:10.1038/s41467-019-12007-w2041-1723Wilson MDRenault LMaskell DPGhoneim MPye VENans ARueda DSCherepanov PCosta ARetroviral integration into nucleosomes through DNA looping and sliding along the histone octamer.Nat CommunUK1041894189201931519882doi:10.1038/s41467-019-12007-w2041-1723EMD-4692other EM volumeD02 nucleosome in absence of PFV intasomeEMD-4960associated EM volumePFV intasome - nucleosome strand transfer complex6rnyFULLOVERLAPNucleosome core particle bound by PFV intasome post in the post catalytic stateNucleosome core particle bound by PFV intasome post in the post catalytic state012345678910human histones refolded as an octamer with native human D02 sequence.
PFV intasome formed form short annealed oligos and PFV integrase.
complex formed by mixing and separated by SEC. Catalysis initiated by addition of Magnesium ions0.399Nucleosome11234NucleosomeHomo sapiensEscherichia coliIntegrase15IntegraseHuman spumaretrovirusEscherichia coliHuman DNA16789Human DNAHomo sapienssynthetic constructDNA110DNAPyrobaculum filamentous virus 1synthetic constructHistone H3.3Homo sapiens0.0153609832Escherichia coliLEVOMARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFK
TDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERAHistone H4Homo sapiens0.0113944262Escherichia coliLEVOMSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRK
TVTAMDVVYALKRQGRTLYGFGGHistone H2A type 1Homo sapiens0.0141215372Escherichia coliLEVOMSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRII
PRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGKHistone H2B type 1-C/E/F/G/IHomo sapiens0.0139372132Escherichia coliLEVOMPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASR
LAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSKIntegraseHuman spumaretrovirus0.0444566954Escherichia coliLEVOGPGCNTKKPNLDAELDQLLQGHYIKGYPKQYTYFLEDGKVKVSRPEGVKIIPPQSDRQKIVLQAHNLAHTGREATLLKIA
NLYWWPNMRKDVVKQLGRCQQCLITNASNKASGPILRPDRPQKPFDKFFIDYIGPLPPSQGYLYVLVVVDGMTGFTWLYP
TKAPSTSATVKSLNVLTSIAIPKVIHSDQGAAFTSSTFAEWAKERGIHLEFSTPYHPQSSGKVERKNSDIKRLLTKLLVG
RPTKWYDLLPVVQLALNNTYSPVLKYTPHQLLFGIDSNTPFANQDTLDLTREEELSLLQEIRTSLYHPSTPPASSRSWSP
VVGQLVQERVARPASLRPRWHKPSTVLKVLNPRTVVILDHLGNNRTVSIDNLKPTSHQNGTTNDTATMDHLEKNE2.7.7.49DNA (128-MER)Homo sapiens0.0391890941(DG)(DC)(DG)(DA)(DA)(DA)(DT)(DT)(DC)(DC)(DA)(DT)(DG)(DA)(DC)(DA)(DC)(DT)(DA)(DC)
(DC)(DT)(DT)(DC)(DC)(DC)(DA)(DG)(DG)(DA)(DA)(DA)(DC)(DA)(DG)(DG)(DT)(DT)(DT)(DC)
(DA)(DC)(DC)(DA)(DG)(DC)(DC)(DA)(DG)(DG)(DC)(DC)(DT)(DT)(DG)(DA)(DA)(DT)(DG)(DC)
(DA)(DA)(DT)(DT)(DG)(DT)(DC)(DT)(DT)(DA)(DC)(DT)(DA)(DG)(DG)(DA)(DA)(DT)(DA)(DT)
(DT)(DT)(DG)(DG)(DA)(DC)(DT)(DT)(DC)(DC)(DC)(DC)(DA)(DC)(DC)(DT)(DA)(DC)(DC)(DA)
(DT)(DT)(DC)(DA)(DG)(DG)(DT)(DA)(DA)(DC)(DT)(DT)(DG)(DA)(DT)(DA)(DC)(DA)(DA)(DA)
(DC)(DA)(DC)(DA)(DG)(DC)(DC)(DC)DNADNA (108-MER)Homo sapiens0.0335814531(DG)(DG)(DG)(DC)(DT)(DG)(DT)(DG)(DT)(DT)(DT)(DG)(DT)(DA)(DT)(DC)(DA)(DA)(DG)(DT)
(DT)(DA)(DC)(DC)(DT)(DG)(DA)(DA)(DT)(DG)(DG)(DT)(DA)(DG)(DG)(DT)(DG)(DG)(DG)(DG)
(DA)(DA)(DG)(DT)(DC)(DC)(DA)(DA)(DA)(DT)(DA)(DT)(DT)(DC)(DC)(DT)(DA)(DG)(DT)(DA)
(DA)(DG)(DA)(DC)(DA)(DA)(DT)(DT)(DG)(DC)(DA)(DT)(DT)(DC)(DA)(DA)(DG)(DG)(DC)(DC)
(DT)(DG)(DG)(DC)(DT)(DG)(DG)(DT)(DG)(DA)(DA)(DA)(DC)(DC)(DT)(DG)(DT)(DT)(DT)(DC)
(DC)(DT)(DG)(DG)(DG)(DA)(DA)(DG)DNADNA (33-MER)Homo sapiens0.0101145231(DG)(DT)(DC)(DC)(DA)(DG)(DG)(DT)(DT)(DC)(DT)(DC)(DC)(DC)(DT)(DG)(DT)(DG)(DG)(DT)
(DG)(DA)(DA)(DA)(DA)(DC)(DC)(DA)(DA)(DC)(DT)(DA)(DA)DNADNA (53-MER)Homo sapiens0.0163975311(DG)(DC)(DG)(DA)(DA)(DA)(DT)(DT)(DC)(DC)(DA)(DT)(DG)(DA)(DC)(DA)(DG)(DT)(DA)(DG)
(DT)(DT)(DA)(DG)(DT)(DT)(DG)(DG)(DT)(DT)(DT)(DT)(DC)(DA)(DC)(DC)(DA)(DC)(DA)(DG)
(DG)(DG)(DA)(DG)(DA)(DA)(DC)(DC)(DT)(DG)(DG)(DA)(DC)DNADNA (5'-D(*AP*TP*TP*GP*TP*CP*AP*TP*GP*GP*AP*AP*TP*TP*TP*CP*GP*C)-3')Pyrobaculum filamentous virus 10.0058347942(DA)(DT)(DT)(DG)(DT)(DC)(DA)(DT)(DG)(DG)(DA)(DA)(DT)(DT)(DT)(DC)(DG)(DC)(DA)DNAMAGNESIUM ION2.4305e-052MGsingleParticleparticle0.171.0DTTC-flat-1/1COPPER400CARBONHOLEYPLASMA CLEANINGETHANE80277LEICA EM CPC1 min incubation 3.5s blot. FEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN30070.02.70.00150.003559000.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENMicroscope was modified with a Cs corrector (NeCEN netherlands)FEI FALCON II (4k x 4k)INTEGRATING249161.656.013125CTFFIND1.4initial model based EMDB-2992 low pass filtered to 50A3C1FOURIER SPACE3.9FSC 0.143 CUT-OFFRELION1.3177155MAXIMUM LIKELIHOODRELION1.3MAXIMUM LIKELIHOODRELION1.3459000RELION1.3Two rounds of 3D classification. First with 8 classes coarse sampling, second with 4 classes.3UTB3OS0The initial model was placed in the density using Chimera. Manual building was performed in Coot and final refinement was carried out using phenix.real_space_refine and namdinator. Additional restraints describing protein secondary structure, DNA base pairing and stacking were used in Phenix.REALemd_4960_half_map_2.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
240
240240
0
00240240240268.8268.8268.890.090.090.0XYZ-0.024331560.080862634-0.000060545530.00464244231.121.121.12Half map 1 of Nucleosome core particle bound by PFV intasome post catalytic stateemd_4960_half_map_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
240
240240
0
00240240240268.8268.8268.890.090.090.0XYZ-0.0248365160.08269778-0.000060940110.0046388651.121.121.12Half map 1 of Nucleosome core particle bound by PFV intasome post catalytic state