2024-05-22 REL PDBe PDBe PDBe 2019-08-13 2020-01-01 2020-01-01 2024-05-22 Cryo-EM structure of the RecBCD Chi recognised complex Cheng K Wilkinson M DNA repair, Homologous recombination, ATP hydrolysis, Helicase, Nuclease, translocation, HYDROLASE Cheng K Wilkinson M Chaban Y Wigley DB A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair. Nat.Struct.Mol.Biol. US 27 71 77 2020 31907455 doi:10.1038/s41594-019-0355-2 1545-9985 6sjb FULLOVERLAP RecBCD bound to a forked Chi-containing DNA substrate RecBCD bound to a forked Chi-containing DNA substrate 0 1 2 3 4 0.355 RecBCD 1 1 2 3 Escherichia coli DNA substrate 1 4 synthetic construct RecBCD enzyme subunit RecB Escherichia coli 0.134123688 1 Escherichia coli BL21(DE3) LEVO GMSDVAETLDPLRLPLQGERLIEASAGTGKTFTIAALYLRLLLGLGGSAAFPRPLTVEELLVVTFTEAATAELRGRIRSN IHELRIACLRETTDNPLYERLLEEIDDKAQAAQWLLLAERQMDEAAVFTIHGFCQRMLNLNAFESGMLFEQQLIEDESLL RYQACADFWRRHCYPLPREIAQVVFETWKGPQALLRDINRYLQGEAPVIKAPPPDDETLASRHAQIVARIDTVKQQWRDA VGELDALIESSGIDRRKFNRSNQAKWIDKISAWAEEETNSYQLPESLEKFSQRFLEDRTKAGGETPRHPLFEAIDQLLAE PLSIRDLVITRALAEIRETVAREKRRRGELGFDDMLSRLDSALRSESGEVLAAAIRTRFPVAMIDEFQDTDPQQYRIFRR IWHHQPETALLLIGDPKQAIYAFRGADIFTYMKARSEVHAHYTLDTNWRSAPGMVNSVNKLFSQTDDAFMFREIPFIPVK SAGKNQALRFVFKGETQPAMKMWLMEGESCGVGDYQSTMAQVCAAQIRDWLQAGQRGEALLMNGDDARPVRASDISVLVR SRQEAAQVRDALTLLEIPSVYLSNRDSVFETLEAQEMLWLLQAVMTPERENTLRSALATSMMGLNALDIETLNNDEHAWD VVVEEFDGYRQIWRKRGVMPMLRALMSARNIAENLLATAGGERRLTDILHISELLQEAGTQLESEHALVRWLSQHILEPD SNASSQQMRLESDKHLVQIVTIHKSKGLEYPLVWLPFITNFRVQEQAFYHDRHSFEAVLDLNAAPESVDLAEAERLAEDL RLLYVALTRSVWHCSLGVAPLVRRRGDKKGDTDVHQSALGRLLQKGEPQDAAGLRTCIEALCDDDIAWQTAQTGDNQPWQ VNDVSTAELNAKTLQRLPGDNWRVTSYSGLQQRGHGIAQDLMPRLDVDAAGVASVVEEPTLTPHQFPRGASPGTFLHSLF EDLDFTQPVDPNWVREKLELGGFESQWEPVLTEWITAVLQAPLNETGVSLSQLSARNKQVEMEFYLPISEPLIASQLDTL IRQFDPLSAGCPPLEFMQVRGMLKGFIDLVFRHEGRYYLLAYKSNWLGEDSSAYTQQAMAAAMQAHRYDLQYQLYTLALH RYLRHRIADYDYEHHFGGVIYLFLRGVDKEHPQQGIYTTRPNAGLIALMDEMFAGMTLEEA A0A024LB08 3.1.11.5 RecBCD enzyme subunit RecC Escherichia coli 0.12897410199999998 1 Escherichia coli BL21(DE3) LEVO MLRVYHSNRLDVLEALMEFIVERERLDDPFEPEMILVQSTGMAQWLQMTLSQKFGIAANIDFPLPASFIWDMFVRVLPEI PKESAFNKQSMSWKLMTLLPQLLEREDFTLLRHYLTDDSDKRKLFQLSSKAADLFDQYLVYRPDWLAQWETGHLVEGLGE AQAWQAPLWKALVEYTHQLGQPRWHRANLYQRFIETLESATTCPPGLPSRVFICGISALPPVYLQALQALGKHIEIHLLF TNPCRYYWGDIKDPAYLAKLLTRQRRHSFEDRELPLFRDSENAGQLFNSDGEQDVGNPLLASWGKLGRDYIYLLSDLESS QELDAFVDVTPDNLLHNIQSDILELENRAVAGVNIEEFSRSDNKRPLDPLDSSITFHVCHSPQREVEVLHDRLLAMLEED PTLTPRDIIVMVADIDSYSPFIQAVFGSAPADRYLPYAISDRRARQSHPVLEAFISLLSLPDSRFVSEDVLALLDVPVLA ARFDITEEGLRYLRQWVNESGIRWGIDDDNVRELELPATGQHTWRFGLTRMLLGYAMESAQGEWQSVLPYDESSGLIAEL VGHLASLLMQLNIWRRGLAQERPLEEWLPVCRDMLNAFFLPDAETEAAMTLIEQQWQAIIAEGLGAQYGDAVPLSLLRDE LAQRLDQERISQRFLAGPVNICTLMPMRSIPFKVVCLLGMNDGVYPRQLAPLGFDLMSQKPKRGDRSRRDDDRYLFLEAL ISAQQKLYISYIGRSIQDNSERFPSVLVQELIDYIGQSHYLPGDEALNCDESEARVKAHLTCLHTRMPFDPQNYQPGERQ SYAREWLPAASQAGKAHSEFVQPLPFTLPETVPLETLQRFWAHPVRAFFQMRLQVNFRTEDSEIPDTEPFILEGLSRYQI NQQLLNALVEQDDAERLFRRFRAAGDLPYGAFGEIFWETQCQEMQQLADRVIACRQPGQSMEIDLACNGVQITGWLPQVQ PDGLLRWRPSLLSVAQGMQLWLEHLVYCASGGNGESRLFLRKDGEWRFPPLAAEQALHYLSQLIEGYREGMSAPLLVLPE SGGAWLKTCYDAQNDAMLDDDSTLQKARTKFLQAYEGNMMVRGEGDDIWYQRLWRQLTPETMEAIVEQSQRFLLPLFRFN QS P07648 3.1.11.5 RecBCD enzyme subunit RecD Escherichia coli 0.066990367 1 Escherichia coli BL21(DE3) LEVO MKLQKQLLEAVEHKQLRPLDVQFALTVAGDEHPAVTLAAALLSHDAGEGHVCLPLSRLENNEASHPLLATCVSEIGELQN WEECLLASQAVSRGDEPTPMILCGDRLYLNRMWCNERTVARFFNEVNHAIEVDEALLAQTLDKLFPVSDEINWQKVAAAV ALTRRISVISGGPGTGKTTTVAKLLAALIQMADGERCRIRLAAPTGKAAARLTESLGKALRQLPLTDEQKKRIPEDASTL HRLLGAQPGSQRLRHHAGNPLHLDVLVVDEASMIDLPMMSRLIDALPDHARVIFLGDRDQLASVEAGAVLGDICAYANAG FTAERARQLSRLTGTHVPAGTGTEAASLRDSLCLLQKSYRFGSDSGIGQLAAAINRGDKTAVKTVFQQDFTDIEKRLLQS GEDYIAMLEEALAGYGRYLDLLQARAEPDLIIQAFNEYQLLCALREGPFGVAGLNERIEQFMQQKRKIHRHPHSRWYEGR PVMIARNDSALGLFNGDIGIALDRGQGTRVWFAMPDGNIKSVQPSRLPEHETTWAMTVHKSQGSEFDHAALILPSQRTPV VTRELVYTAVTRARRRLSLYADERILSAAIATRTERRSGLAALFSSRE A0A061K747 3.1.11.5 DNA fork substrate synthetic construct 0.026159659999999998
We created a splayed duplex substrate with unpaired single strand overhangs by annealing two chemically synthesised oligonucleotides.
1 (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DG)(DA)(DG)(DC)(DG) (DA)(DC)(DT)(DG)(DC)(DA)(DC)(DT)(DA)(DC)(DA)(DA)(DC)(DA)(DG)(DA)(DA)(DC)(DC)(DA) (DT)(DG)(DG)(DT)(DT)(DC)(DT)(DG)(DT)(DT)(DG)(DT)(DA)(DG)(DT)(DG)(DC)(DA)(DG)(DT) (DC)(DG)(DC)(DT)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DG)(DC)(DT)(DG)(DG)(DT)(DG) (DG)(DT)(DT)(DT)(DT) DNA singleParticle particle 0.2 8.0 20.0 TRIS-HCl 50.0 NaCl 0.5 TCEP Quantifoil R2/2 COPPER 300 GRAPHENE OXIDE CONTINUOUS PLASMA CLEANING ETHANE 90 277 FEI VITROBOT MARK IV
1s blot before plunging.
FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 2.7 1.3 2.5 130000.0 47755.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GATAN K2 SUMMIT (4k x 4k) COUNTING 3838 3710 1-25 1 3721 10.0 45.0 1 1053451 5LD2
Initially low-pass filtered to 30 Angstrom
 C1 3.7 FSC 0.143 CUT-OFF RELION 3 74496 MAXIMUM LIKELIHOOD RELION 3 MAXIMUM LIKELIHOOD RELION 3 emd_10214.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 251.28001 251.28001 251.28001 90.0 90.0 90.0 X Y Z -0.023604069 0.05393631 0.000028449525 0.002595831 1.047 1.047 1.047 0.0116 AUTHOR EM map of the Chi-recognised structure of RecBCD 5LD2 PDB experimental model
Rounds of manual structure editing in coot and real-space refinement with Phenix
REAL