2019-12-25RELPDBePDBePDBe2019-08-052019-08-142019-10-092019-12-25Spanish Ministry of Science, Innovation, and UniversitiesSAF2017-82632-P o-funded by the European Regional Development FundSpainEuropean Regional Development FundP2018/NMT4443 co-funded by Autonomous Region of MadridSpainEuropean Regional Development FundY2018/BIO4747 co-funded by the Autonomous Region of MadridSpainBavarian State Ministry of Science and the Arts Funding OrganisationN-BM-2013-246GermanyEuropean UnionHorizon 2020, iNEXT (PID2907) , Grant number 653706SpainESX-3 core complex centred at the cytoplasmic region, conformation 2.Rivera-Calzada AFamelis NGeibel SLlorca OFamelis NRivera-Calzada ADegliesposti GWingender MMietrach NSkehel JMFernandez-Leiro RBottcher BSchlosser ALlorca OGeibel SArchitecture of the mycobacterial type VII secretion system.NatureUK576321325201931597161doi:10.1038/s41586-019-1633-11476-46870006NATUASEMD-10190associated EM volumeESX-3 core complex, conformation 2ESX-3 core complex, conformation 201234The sample consists of four protein components, EccB3:EccC3:EccD3:EccE3 in a 1:1:2:1 stoichiometry
Molecular weight of the complex without the amphipol micelle: 0.65 MDaMycolicibacterium smegmatis MC2 155Mycolicibacterium smegmatis MC2 1550.65EccB3Mycolicibacterium smegmatis MC2 155Mycolicibacterium smegmatis MC2 155LEVOMTGPVNPDDRRSFSSRTPVNENPDGVQYRRGFVTRHQVSGWRFVMRRIASGVALHDTRML
VDPLRTQSRAVLTGALILVTGLVGCFIFSLFRPGGVPGNNAILADRSTSALYVRVGEQLH
PVLNLTSARLISGSPDNPTMVKTSEIDKFPRGNLLGIPGAPERMVQNAATDAEWTVCDAV
GGANPGVTVIAGPLGADGERAAPLPPDHAVLVHSDAEPNPGDWLLWDGKRSPIDLADRAV
TDALGLGGQALAPRPIAAGLFNAVPAAPALTAPVIPDAGAAPQFELSLPVPVGAVVVAYD
ADNTARYYAVLSDGLQPISPVLAAILRNTDSHGFAQPPRLGPDEVARTPMSRGLDTSAYP
DNPVTLVEASAHPVTCAHWTKPSDAAESSLSVLSGAVLPLAEGLHTVDLVGAGAGGAANR
VALTPGTGYFVQTVGAEPGSPTAGSMFWVSDTGVRYGIDTAEDDKVVAALGLSTSPLPVP
WSVLSQFAAGPALSRGDALVAHDAVSTNPNSARMEASREccD3Mycolicibacterium smegmatis MC2 155Mycolicibacterium smegmatis MC2 155DEXTROMASWSHPQFEKGSMSENTVMPIVRVAVLAAGDDGGRLTEMALPSELPLREILPAVQRIVQPARENDGAADPAAAPNPVRLSLAPIGGAPFSLDATLDTVGVVDGDLLALQAVPSGPPAPRIVEDIADAAVIFSEARRRQWGPTHIARGAALALIGLILVGTGLSVAHRVITGDLLGQFIVSGIALATVIAALAVRNRSAVLATSLAVTALVPVAAAFALGVPGDFGAPNVLLAAAGVAAWSLISMAGSPDDRGIAVFTATAVTGVGVLLVAGAASLWVISSDVIGCALVLLGLIVTVQAAQLSAMWARFPLPVIPAPGDPTPAARPLSVLADLPRRVRVSQAHQTGVIAAGVLLGVAGSVALVSSANASPWAWYIVVAAAAGAALRARVWDSAACKAWLLGHSYLLAVALLVAFVIGDRYQAALWALAALAVLVLVWIVAALNPKIASPDTYSLPMRRMVGFLATGLDASLIPVMALLVGLFSLVLDREccC3Mycolicibacterium smegmatis MC2 155Mycolicibacterium smegmatis MC2 155DEXTROMSRLIFEHQRRLTPPTTRKGTITIEPPPQLPRVVPPSLLRRVLPFLIVILIVGMIVALFA
TGMRLISPTMLFFPFVLLLAATALYRGGDNKMRTEEVDAERADYLRYLSVVRDNVRAHAA
EQRAALEWSHPEPEVLATIPGTRRQWERDPRDRDFLVLRAGRHDVPLDAALKVKDTADEI
DLEPVAHSALRGLLDVQRTVRDAPTGLDVAKLARITVIGEADEARAAIRAWIAQAVTWHD
PTMLGVALAAPDLESGDWSWLKWLPHVDVPNEADGVGPARYLTTSTAELRERLAPALADR
PLFPAESGAALKHLLVVLDDPDADPDDIARKPGLTGVTVIHRTTELPNREQYPDPERPIL
RVADGRIERWQVGGWQPCVDVADAMSAAEAAHIARRLSRWDSNPGYIRSTSTGSATFTTL
LGIPDASALDVASLWAPRPRDEELRVPIGVTSTGEPLYFDLKDEAEGGMGPHGLMIGMTG
SGKSQTLMSILLSLLTTHPADRLIVIYADFKGEAGADIFRHFPQVVAVISNMAEKRSLAD
RFADTLRGEVARREQILKEAGRRVQGSAFNSVAEYESAIAAGHDLPPMPTLFVVADEFTL
MLAEHPEYADLFDYVARKGRSFRIHLLFASQTLDVGRIKDIDKNTSYRIGLKVASPSISR
QIIGVEDAYHIESGREHKGEGFLVPAPGAVPIKFRSTYVDGIYDPPRAEKSIVVHALPQP
QVFTAGRVEPEPDTVIATGDVEVHTAPPRKLIATIGDQLAAYGPKAPQLWLPPLDEPIAL
ADVLAGADVEPGQLRWPLGEIDKPFEMRRDVLVYDAHTAAANVLIHGGPRSGKSTALQAF
VLSAAALHSPRAITFYCLDYGGGKLADLADLAHVGSVATPLEPERIRRTFGELEQLLRAR
QRQGAVNRTGSYTDGYGEVFLVIDNLYAFSRDNTDTFNTRNPLLAKVTELANSGLAYGIH
VVITTPNWLEVPLAMRDGLGLRLELKLHDSHDSIVRVAGALRRPADSVPADQPGRGLTMA
AEHFLFAEPALSDIAVINARYPGVSAPPVRLLPTDLSPDALAPLYPAPETVVIGQREEDL
APVAVDFANHPLLMVFGDSKSGKTTLLRHIIRTVRENSTPDQVAFTVIDRRLHLVDEPLF
PDNEYTANIDRVLPAMLGLSALIEKRRPPAGLSAQELSRWTYTGHTHYLIVDDVDQIPDT
PAVSGPFVGQRPWTNIVGLLAEAADLGLRVIVTARATGSAHAVMTAPLLRRLNDLQATTL
MLSGNPTDSGKIRGHRFARFPAGRGLLLTDTDTPDHIQLVNPLGDAALSGNIGNNGNHNR
GGEYRGSMGGSHHHHHH
EccE3Mycolicibacterium smegmatis MC2 155Mycolicibacterium smegmatis MC2 155DEXTROMTARIALASLFVVAAVLAQPWQTTTQRWVLGVSIAAVIVLLAWWKGMFLTTRIGRALAMV
RRNRAEDTVETDAHRATVVLRVDPAAPAQLPVVVGYLDRYGITCDKVRITHRDAGGTRRS
WISLTVDAVDNLAALQARSARIPLQDTTEVVGRRLADHLREQGWTVTVVEGVDTPLPVSG
KETWRGVADDAGVVAAYRVKVDDRLDEVLAEIGHLPAEETWTALEFTGSPAEPLLTVCAA
VRTSDRPAAKAPLAGLTPARGRHRPALAALNPLSTERLDGTAVPLPAVVRTSVKGSVEHE
AAQEAGHPAsingleParticleparticle0.3830 mM Hepes pH 8.0, 150 mM NaClQuantifoil R1.2/1.3COPPER/RHODIUM400GLOW DISCHARGEETHANE100278FEI VITROBOT MARK IVBlotting time: 3s
Blotting force: -10. bound to Amphipol A8-35FEI TITAN KRIOSSPOT SCANBRIGHT FIELDFIELD EMISSION GUN3002.71.62.675000.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENFEI FALCON III (4k x 4k)COUNTING1190350.0Images acquired as 55 frames movies at a calibrated magnification of 1.0635 Angs/px1Images were collected in counting mode2066007Particles were extracted centred at the distal cytoplasmic tip instead than in the centre of mass in order to improve the resolution in that distal region.GctfC15.3FSC 0.143 CUT-OFFRELION69919MAXIMUM LIKELIHOODRELIONMAXIMUM LIKELIHOODRELION4NH0B401-772RIGID BODY FITA homology model based on pdb 4NH0 was created using Phyre2 and fitted into the density using Chimera.Map correlation coefficient