2020-07-15 REL PDBe PDBe PDBe 2020-04-06 2020-05-20 2020-05-20 2020-07-15 Cancer Research UK C47547/A21536 United Kingdom Wellcome Trust 200818/Z/16/Z United Kingdom Negative stain reconstruction of grafix crosslink human condensin II in the presence of ATPyS Cutts EE Beuron F Morris E Vannini A Kong M Cutts EE Pan D Beuron F Kaliyappan T Xue C Morris EP Musacchio A Vannini A Greene EC Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA. Mol.Cell US 79 99 114.e9 2020 32445620 doi:10.1016/j.molcel.2020.04.026 1097-2765 2168 MOCEFL EMD-10833 associated EM volume Human condensin II pentamer composed of SMC2, SMC4, CapH2, CapD3 and CapG2 Human condensin II pentamer composed of SMC2, SMC4, CapH2, CapD3 and CapG2 0 1 2 3 4 5 Homo sapiens Trichoplusia ni 0.657 SMC2 Homo sapiens Trichoplusia ni LEVO MHIKSIILEGFKSYAQRTEV NGFDPLFNAITGLNGSGKSN ILDSICFLLGISNLSQVRAS NLQDLVYKNGQAGITKASVS ITFDNSDKKQSPLGFEVHDE ITVTRQVVIGGRNKYLINGV NANNTRVQDLFCSVGLNVNN PHFLIMQGRITKVLNMKPPE ILSMIEEAAGTRMYEYKKIA AQKTIEKKEAKLKEIKTILE EEITPTIQKLKEERSSYLEY QKVMREIEHLSRLYIAYQFL LAEDTKVRSAEELKEMQDKV IKLQEELSENDKKIKALNHE IEELEKRKDKETGGILRSLE DALAEAQRVNTKSQSAFDLK KKNLACEESKRKELEKNMVE DSKTLAAKEKEVKKITDGLH ALQEASNKDAEALAAAQQHF NAVSAGLSSNEDGAEATLAG QMMACKNDISKAQTEAKQAQ MKLKHAQQELKNKQAEVKKM DSGYRKDQEALEAVKRLKEK LEAEMKKLNYEENKEESLLE KRRQLSRDIGRLKETYEALL ARFPNLRFAYKDPEKNWNRN CVKGLVASLISVKDTSATTA LELVAGERLYNVVVDTEVTG KKLLERGELKRRYTIIPLNK ISARCIAPETLRVAQNLVGP DNVHVALSLVEYKPELQKAM EFVFGTTFVCDNMDNAKKVA FDKRIMTRTVTLGGDVFDPH GTLSGGARSQAASILTKFQE LKDVQDELRIKENELRALEE ELAGLKNTAEKYRQLKQQWE MKTEEADLLQTKLQQSSYHK QQEELDALKKTIEESEETLK NTKEIQRKAEEKYEVLENKM KNAEAERERELKDAQKKLDC AKTKADASSKKMKEKQQEVE AITLELEELKREHTSYKQQL EAVNEAIKSYESQIEVMAAE VAKNKESVNKAQEEVTKQKE VITAQDTVIKAKYAEVAKHK EQNNDSQLKIKELDHNISKH KREAEDGAAKVSKMLKDYDW INAERHLFGQPNSAYDFKTN NPKEAGQRLQKLQEMKEKLG RNVNMRAMNVLTEAEERYND LMKKKRIVENDKSKILTTIE DLDQKKNQALNIAWQKVNKD FGSIFSTLLPGANAMLAPPE GQTVLDGLEFKVALGNTWKE NLTELSGGQRSLVALSLILS MLLFKPAPIYILDEVDAALD LSHTQNIGQMLRTHFTHSQF IVVSLKEGMFNNANVLFKTK FVDGVSTVARFTQCQNGKIS KEAKSKAKPPKGAHVEV SMC4 Homo sapiens Trichoplusia ni LEVO MAHHHHHHHHHHLEVLFQGP RKGTQPSTARRREEGPPPPS PDGASSDAEPEPPSGRTESP ATAAETASEELDNRSLEEIL NSIPPPPPPAMTNEAGAPRL MITHIVNQNFKSYAGEKILG PFHKRFSCIIGPNGSGKSNV IDSMLFVFGYRAQKIRSKKL SVLIHNSDEHKDIQSCTVEV HFQKIIDKEGDDYEVIPNSN FYVSRTACRDNTSVYHISGK KKTFKDVGNLLRSHGIDLDH NRFLILQGEVEQIAMMKPKG QTEHDEGMLEYLEDIIGCGR LNEPIKVLCRRVEILNEHRG EKLNRVKMVEKEKDALEGEK NIAIEFLTLENEIFRKKNHV CQYYIYELQKRIAEMETQKE KIHEDTKEINEKSNILSNEM KAKNKDVKDTEKKLNKITKF IEENKEKFTQLDLEDVQVRE KLKHATSKAKKLEKQLQKDK EKVEEFKSIPAKSNNIINET TTRNNALEKEKEKEEKKLKE VMDSLKQETQGLQKEKESRE KELMGFSKSVNEARSKMDVA QSELDIYLSRHNTAVSQLTK AKEALIAASETLKERKAAIR DIEGKLPQTEQELKEKEKEL QKLTQEETNFKSLVHDLFQK VEEAKSSLAMNRSRGKVLDA IIQEKKSGRIPGIYGRLGDL GAIDEKYDVAISSCCHALDY IVVDSIDIAQECVNFLKRQN IGVATFIGLDKMAVWAKKMT EIQTPENTPRLFDLVKVKDE KIRQAFYFALRDTLVADNLD QATRVAYQKDRRWRVVTLQG QIIEQSGTMTGGGSKVMKGR MGSSLVIEISEEEVNKMESQ LQNDSKKAMQIQEQKVQLEE RVVKLRHSEREMRNTLEKFT ASIQRLIEQEEYLNVQVKEL EANVLATAPDKKKQKLLEEN VSAFKTEYDAVAEKAGKVEA EVKRLHNTIVEINNHKLKAQ QDKLDKINKQLDECASAITK AQVAIKTADRNLQKAQDSVL RTEKEIKDTEKEVDDLTAEL KSLEDKAAEVVKNTNAAEES LPEIQKEHRNLLQELKVIQE NEHALQKDALSIKLKLEQID GHIAEHNSKIKYWHKEISKI SLHPIEDNPIEEISVLSPED LEAIKNPDSITNQIALLEAR CHEMKPNLGAIAEYKKKEEL YLQRVAELDKITYERDSFRQ AYEDLRKQRLNEFMAGFYII TNKLKENYQMLTLGGDAELE LVDSLDPFSEGIMFSVRPPK KSWKKIFNLSGGEKTLSSLA LVFALHHYKPTPLYFMDEID AALDFKNVSIVAFYIYEQTK NAQFIIISLRNNMFEISDRL IGIYKTYNITKSVAVNPKEI ASKGLC CapH2 Homo sapiens Trichoplusia ni LEVO EDVEARFAHLLQPIRDLTK NWEVDVAAQLGEYLEELDQI CISFDEGKTTMNFIEAALLI QGSACVYSKKVEYLYSLVYQ ALDFISGKRRAKQLSSVQED RANGVASSGVPQEAENEFLS LDDFPDSRTNVDLKNDQTPS EVLIIPLLPMALVAPDEMEK NNNPLYSRQGEVLASRKDFR MNTCVPHPRGAFMLEPEGMS PMEPAGVSPMPGTQKDTGRT EEQPMEVSVCRSPVPALGFS QEPGPSPEGPMPLGGGEDED AEEAVELPEASAPKAALEPK ESRSPQQSAALPRRYMLRER EGAPEPASCVKETPDPWQSL DPFDSLESKPFKKGRPYSVP PCVEEALGQKRKRKGAAKLQ DFHQWYLAAYADHADSRRLR RKGPSFADMEVLYWTHVKEQ LETLRKLQRREVAEQWLRPA EEDHLEDSLEDLGAADDFLE PEEYMEPEGADPREAADLDA VPMSLSYEELVRRNVELFIA TSQKFVQETELSQRIRDWED TVQPLLQEQEQHVPFDIHTY GDQLVSRFPQLNEWCPFAEL VAGQPAFEVCRSMLASLQLA NDYTVEITQQPGLEMAVDTM SLRLLTHQRAHKRFQTYAAP SMAQPENLYFQSWSHPQFEK GGGSGGGSGGGSWSHPQFEK CapD3 Homo sapiens Trichoplusia ni LEVO VALRGLGSGLQPWCPLDLR LEWVDTVWELDFTETEPLDP SIEAEIIETGLAAFTKLYES LLPFATGEHGSMESIWTFFI ENNVSHSTLVALFYHFVQIV HKKNVSVQYREYGLHAAGLY FLLLEVPGSVANQVFHPVMF DKCIQTLKKSWPQESNLNRK RKKEQPKSSQANPGRHRKRG KPPRREDIEMDEIIEEQEDE NICFSARDLSQIRNAIFHLL KNFLRLLPKFSLKEKPQCVQ NCIEVFVSLTNFEPVLHECH VTQARALNQAKYIPELAYYG LYLLCSPIHGEGDKVISCVF HQMLSVILMLEVGEGSHRAP LAVTSQVINCRNQAVQFISA LVDELKESIFPVVRILLQHI CAKVVDKSEYRTFAAQSLVQ LLSKLPCGEYAMFIAWLYKY SRSSKIPHRVFTLDVVLALL ELPEREVDNTLSLEHQKFLK HKFLVQEIMFDRCLDKAPTV RSKALSSFAHCLELTVTSAS ESILELLINSPTFSVIESHP GTLLRNSSAFSYQRQTSNRS EPSGEINIDSSGETVGSGER CVMAMLRRRIRDEKTNVRKS ALQVLVSILKHCDVSGMKED LWILQDQCRDPAVSVRKQAL QSLTELLMAQPRCVQIQKAW LRGVVPVVMDCESTVQEKAL EFLDQLLLQNIRHHSHFHSG DDSQVLAWALLTLLTTESQE LSRYLNKAFHIWSKKEKFSP TFINNVISHTGTEHSAPAWM LLSKIAGSSPRLDYSRIIQS WEKISSQQNPNSNTLGHILC VIGHIAKHLPKSTRDKVTDA VKCKLNGFQWSLEVISSAVD ALQRLCRASAETPAEEQELL TQVCGDVLSTCEHRLSNIVL KENGTGNMDEDLLVKYIFTL GDIAQLCPARVEKRIFLLIQ SVLASSADADHSPSSQGSSE APASQPPPQVRGSVMPSVIR AHAIITLGKLCLQHEDLAKK SIPALVRELEVCEDVAVRNN VIIVMCDLCIRYTIMVDKYI PNISMCLKDSDPFIRKQTLI LLTNLLQEEFVKWKGSLFFR FVSTLIDSHPDIASFGEFCL AHLLLKRNPVMFFQHFIECI FHFNNYEKHEKYNKFPQSER EKRLFSLKGKSNKERRMKIY KFLLEHFTDEQRFNITSKIC LSILACFADGILPLDLDASE LLSDTFEVLSSKEIKLLAMR SKPDKDLLMEEDDMALANVV MQEAQKKLISQVQKRNFIEN IIPIIISLKTVLEKNKIPAL RELMHYLREVMQDYRDELKD FFAVDKQLASELEYDMKKYQ EQLVQEQELAKHADVAGTAG GAEVAPVAQVALCLETVPVP AGQENPAMSPAVSQPCTPRA SAGHVAVSSPTPETGPLQRL LPKARPMSLSTIAILNSVKK AVESKSRHRSRSLGVLPFTL NSGSPEKTCSQVSSYSLEQE SNGEIEHVTKRAISTPEKSI SDVTFGAGVSYIGTPRTPSS AKEKIEGRSQGNDILCLSLP DKPPPQPQQWNVRSPARNKD TPACSRRSLRKTPLKTAN CapG2 Homo sapiens Trichoplusia ni LEVO MEKRETFVQAVSKELVGEFL QFVQLDKEASDPFSLNELLD ELSRKQKEELWQRLKNLLTD VLLESPVDGWQVVEAQGEDN METEHGSKMRKSIEIIYAIT SVILASVSVINESENYEALL ECVIILNGILYALPESERKL QSSIQDLCVTWWEKGLPAKE DTGKTAFVMLLRRSLETKTG ADVCRLWRIHQALYCFDYDL EESGEIKDMLLECFININYI KKEEGRRFLSCLFNWNINFI KMIHGTIKNQLQGLQKSLMV YIAEIYFRAWKKASGKILEA IENDCIQDFMFHGIHLPRRS PVHSKVREVLSYFHHQKKVR QGVEEMLYRLYKPILWRGLK ARNSEVRSNAALLFVEAFPI RDPNLHAIEMDSEIQKQFEE LYSLLEDPYPMVRSTGILGV CKITSKYWEMMPPTILIDLL KKVTGELAFDTSSADVRCSV FKCLPMILDNKLSHPLLEQL LPALRYSLHDNSEKVRVAFV DMLLKIKAVRAAKFWKICPM EHILVRLETDSRPVSRRLVS LIFNSFLPVNQPEEVWCERC VTLVQMNHAAARRFYQYAHE HTACTNIAKLIHVIRHCLNA CIQRAVREPPEDEEEEDGRE KENVTVLDKTLSVNDVACMA GLLEIIVILWKSIDRSMENN KEAKLYTINKFASVLPEYLK VFKDDRCKIPLFMLMSFMPA SAVPPFSCGVISTLRSREEG AVDKSYCTLLDCLCSWGQVG HILELVDNWLPTEHAQAKSN TASKGRVQIHDTRPVKPELA LVYIEYLLTHPKNRECLLSA PRKKLNHLLKALETSKADLE SLLQTPGGKPRGFSEAAAPR AFGLHCRLSIHLQHKFCSEG KVYLSMLEDTGFWLESKILS FIQDQEEDYLKLHRVIYQQI IQTYLTVCKDVVMVGLGDHQ FQMQLLQRSLGIMQTVKGFF YVSLLLDILKEITGSSLIQK TDSDEEVAMLLDTVQKVFQK MLECIARSFRKQPEEGLRLL YSVQRPLHEFITAVQSRHTD TPVHRGVLSTLIAGPVVEIS HQLRKVSDVEELTPPEHLSD LPPFSRCLIGIIIKSSNVVR SFLDELKACVASNDIEGIVC LTAAVHIILVINAGKHKSSK VREVAATVHRKLKTFMEITL EEDSIERFLYESSSRTLGEL LNS singleParticle particle 8 20.0 C8H18N2O4S Hepes 200.0 KCl Potassium Chloride
Grafix crosslinked sample was gel filtered on a superose 6 column into EM buffer (20 mM HEPES [pH 8], 200 mM KCl)
 NEGATIVE Uranyl Acetate
The grids were glow discharged for 1 minute at 15 mA, the sample applied for 10 seconds and then washed twice with water before staining with 2% uranyl acetate for 1 minute before blotting and drying.
 Quantifoil R1.2/1.3 COPPER CARBON CONTINUOUS GLOW DISCHARGE FEI TECNAI F20 FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 TVIPS TEMCAM-F416 (4k x 4k) 50.0 1 3251
Relion initial model
 C1 20.5 FSC 0.143 CUT-OFF RELION 3.0 1288 MAXIMUM LIKELIHOOD RELION 3.0 MAXIMUM LIKELIHOOD RELION 3.0 emd_10833.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 200 200 200 0 0 0 200 200 200 1038.0 1038.0 1038.0 90.0 90.0 90.0 X Y Z -0.03836988 0.13957503 0.00002846297 0.0012712684 5.19 5.19 5.19 0.0125 AUTHOR Human condensin II negative stain reconstruction, crosslinked with glutaraldehyde in the presence of ATPyS in a glycerol gradient