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- EMDB-3584: Cryo-EM structure of the human Separase-Securin complex at medium... -

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Basic information

Entry
Database: EMDB / ID: EMD-3584
TitleCryo-EM structure of the human Separase-Securin complex at medium resolution
Map dataCryo-EM structure of the human Separase-Securin complex
Sample
  • Complex: Homo sapiens Separase-Securin complex
    • Protein or peptide: Separase-Securin complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsBoland A / Martin TG / Zhang Z / Yang J / Bai XC / Chang L / Scheres SHW / Barford D
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution.
Authors: Andreas Boland / Thomas G Martin / Ziguo Zhang / Jing Yang / Xiao-Chen Bai / Leifu Chang / Sjors H W Scheres / David Barford /
Abstract: Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle ...Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase-securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase-securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.
History
DepositionJan 31, 2017-
Header (metadata) releaseFeb 15, 2017-
Map releaseMar 8, 2017-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3584.png

Downloads & links

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Map

FileDownload / File: emd_3584.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the human Separase-Securin complex
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.0043986565 - 0.022666683
Average (Standard dev.)0.00015417936 (±0.0012133757)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0040.0230.000

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Supplemental data

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Sample components

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Entire : Homo sapiens Separase-Securin complex

EntireName: Homo sapiens Separase-Securin complex
Components
  • Complex: Homo sapiens Separase-Securin complex
    • Protein or peptide: Separase-Securin complex

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Supramolecule #1: Homo sapiens Separase-Securin complex

SupramoleculeName: Homo sapiens Separase-Securin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: H. sapiens Separase-Securin complex at medium resolution refined with Relion. Sample exhibits strong preferred orientation of particles.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightTheoretical: 255 KDa

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Macromolecule #1: Separase-Securin complex

MacromoleculeName: Separase-Securin complex / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRSFKRVNFG TLLSSQKEAE ELLPALKEFL SNPPAGFPSS RSDAERRQAC DAILRACNQQ LTAKLACPR HLGSLLELAE LACDGYLVST PQRPPLYLER ILFVLLRNAA AQGSPEATLR L AQPLHACL VQCSREAAPQ DYEAVARGSF SLLWKGAEAL LERRAAFAAR ...String:
MRSFKRVNFG TLLSSQKEAE ELLPALKEFL SNPPAGFPSS RSDAERRQAC DAILRACNQQ LTAKLACPR HLGSLLELAE LACDGYLVST PQRPPLYLER ILFVLLRNAA AQGSPEATLR L AQPLHACL VQCSREAAPQ DYEAVARGSF SLLWKGAEAL LERRAAFAAR LKALSFLVLL ED ESTPCEV PHFASPTACR AVAAHQLFDA SGHGLNEADA DFLDDLLSRH VIRALVGERG SSS GLLSPQ RALCLLELTL EHCRRFCWSR HHDKAISAVE KAHSYLRNTN LAPSLQLCQL GVKL LQVGE EGPQAVAKLL IKASAVLSKS MEAPSPPLRA LYESCQFFLS GLERGTKRRY RLDAI LSLF AFLGGYCSLL QQLRDDGVYG GSSKQQQSFL QMYFQGLHLY TVVVYDFAQG CQIVDL ADL TQLVDSCKST VVWMLEALEG LSGQELTDHM GMTASYTSNL AYSFYSHKLY AEACAIS EP LCQHLGLVKP GTYPEVPPEK LHRCFRLQVE SLKKLGKQAQ GCKMVILWLA ALQPCSPE H MAEPVTFWVR VKMDAARAGD KELQLKTLRD SLSGWDPETL ALLLREELQA YKAVRADTG QERFNIICDL LELSPEETPA GAWARATHLV ELAQVLCYHD FTQQTNCSAL DAIREALQLL DSVRPEAQA RDQLLDDKAQ ALLWLYICTL EAKMQEGIER DRRAQAPGNL EEFEVNDLNY E DKLQEDRF LYSNIAFNLA ADAAQSKCLD QALALWKELL TKGQAPAVRC LQQTAASLQI LA ALYQLVA KPMQALEVLL LLRIVSERLK DHSKAAGSSC HITQLLLTLG CPSYAQLHLE EAA SSLKHL DQTTDTYLLL SLTCDLLRSQ LYWTHQKVTK GVSLLLSVLR DPALQKSSKA WYLL RVQVL QLVAAYLSLP SNNLSHSLWE QLCAQGWQTP EIALIDSHKL LRSIILLLMG SDILS TQKA AVETSFLDYG ENLVQKWQVL SEVLSCSEKL VCHLGRLGSV SEAKAFCLEA LKLTTK LQI PRQCALFLVL KGELELARND IDLCQSDLQQ VLFLLESCTE FGGVTQHLDS VKKVHLQ KG KQQAQVPCPP QLPEEELFLR GPALELVATV AKEPGPIAPS TNSSPVLKTK PQPIPNFL S HSPTCDCSLC ASPVLTAVCL RWVLVTAGVR LAMGHQAQGL DLLQVVLKGC PEAAERLTQ ALQASLNHKT PPSLVPSLLD EILAQAYTLL ALEGLNQPSN ESLQKVLQSG LKFVAARIPH LEPWRASLL LIWALTKLGG LSCCTTQLFA SSWGWQPPLI KSVPGSEPSK TQGQKRSGRG R QKLASAPL RLNNTSQKGL EGRGLPCTPK PPDRIRQAGP HVPFTVFEEV CPTESKPEVP QA PRVQQRV QTRLKVNFSD DSDLEDPVSA EAWLAEEPKR RGTASRGRGR ARKGLSLKTD AVV APGSAP GNPGLNGRSR RAKKVASRHC EERRPQRASD QARPGPEIMR TIPEEELTDN WRKM SFEIL RGSDGEDSAS GGKTPAPGPE AASGEWELLR LDSSKKKLPS PCPDKESDKD LGPRL RLPS APVATGLSTL DSICDSLSVA FRGISHCPPS GLYAHLCRFL ALCLGHRDPY ATAFLV TES VSITCRHQLL THLHRQLSKA QKHRGSLEIA DQLQGLSLQE MPGDVPLARI QRLFSFR AL ESGHFPQPEK ESFQERLALI PSGVTVCVLA LATLQPGTVG NTLLLTRLEK DSPPVSVQ I PTGQNKLHLR SVLNEFDAIQ KAQKENSSCT DKREWWTGRL ALDHRMEVLI ASLEKSVLG CWKGLLLPSS EEPGPAQEAS RLQELLQDCG WKYPDRTLLK IMLSGAGALT PQDIQALAYG LCPTQPERA QELLNEAVGR LQGLTVPSNS HLVLVLDKDL QKLPWESMPS LQALPVTRLP S FRFLLSYS IIKEYGASPV LSQGVDPRST FYVLNPHNNL SSTEEQFRAN FSSEAGWRGV VG EVPRPEQ VQEALTKHDL YIYAGHGAGA RFLDGQAVLR LSCRAVALLF GCSSAALAVR GNL EGAGIV LKYIMAGCPL FLGNLWDVTD RDIDRYTEAL LQGWLGAGPG APLLYYVNQA RQAP RLKYL IGAAPIAYGL PVSLR

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Glow discharging was performed before applying graphene oxide solution onto the grid. Grid was washed three times, dried and sample was applied. For detailed information see: https: ...Details: Glow discharging was performed before applying graphene oxide solution onto the grid. Grid was washed three times, dried and sample was applied. For detailed information see: https://figshare.com/articles/Graphene_Oxide_Grid_Preparation/3178669
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Details: Custom Manual Plunger.
DetailsMonodisperse sample

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: -20 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 1.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: NONE / Details: Initial model was created using SIMPLE PRIME
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 103696

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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