2020-07-29 REL PDBe PDBe PDBe 2018-10-30 2019-02-13 2019-02-13 2020-07-29 European Research Council 261151 United Kingdom Medical Research Council (United Kingdom) MC_U105192715 United Kingdom European Research Council 725685 United Kingdom Cryo-EM map of the Ysh1-Mpe1 nuclease complex Hill CH Boreikaite V Kumar A Casanal A Kubik P Degliesposti G Maslen S Mariani A von Loeffelholz O Girbig M Skehel M Passmore LA Hill CH Boreikaite V Kumar A Casanal A Kubik P Degliesposti G Maslen S Mariani A von Loeffelholz O Girbig M Skehel M Passmore LA Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex. Mol.Cell US 73 1217 1231.e11 2019 30737185 doi:10.1016/j.molcel.2018.12.023 1097-2765 2168 MOCEFL EMD-0325 associated EM volume Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation. Ysh1-Mpe1 complex Highly anisotropic due to severe preferred orientation. 0 1 2
The trimeric Ysh1-Mpe1-Yjr141w complex was analysed, however several regions are flexible/disordered. This map corresponds to the Ysh1 N-terminal nuclease domain in complex with the Mpe1 N-terminal ubiquitin-like domain. These are the only ordered, globular densities present in the above sample.
Saccharomyces cerevisiae unidentified baculovirus Sf9 0.057 Ysh1 Saccharomyces cerevisiae unidentified baculovirus LEVO MERTNTTTFKFFSLGGSNEVGRSCHILQYKGKTVMLDAGIHPAYQGLASLPFYDEFDLSK VDILLISHFHLDHAASLPYVMQRTNFQGRVFMTHPTKAIYRWLLRDFVRVTSIGSSSSSM GTKDEGLFSDEDLVDSFDKIETVDYHSTVDVNGIKFTAFHAGHVLGAAMFQIEIAGLRVL FTGDYSREVDRHLNSAEVPPLSSNVLIVESTFGTATHEPRLNRERKLTQLIHSTVMRGGR VLLPVFALGRAQEIMLILDEYWSQHADELGGGQVPIFYASNLAKKCMSVFQTYVNMMNDD IRKKFRDSQTNPFIFKNISYLRNLEDFQDFGPSVMLASPGMLQSGLSRDLLERWCPEDKN LVLITGYSIEGTMAKFIMLEPDTIPSINNPEITIPRRCQVEEISFAAHVDFQENLEFIEK ISAPNIILVHGEANPMGRLKSALLSNFASLKGTDNEVHVFNPRNCVEVDLEFQGVKVAKA VGNIVNEIYKEENVEIKEEIAAKIEPIKEENEDNLDSQAEKGLVDEEEHKDIVVSGILVS DDKNFELDFLSLSDLREHHPDLSTTILRERQSVRVNCKKELIYWHILQMFGEAEVLQDDD RVTNQEPKVKEESKDNLTNTGKLILQIMGDIKLTIVNTLAVVEWTQDLMNDTVADSIIAI LMNVDSAPASVKLSSHSCDDHDHNNVQSNAQGKIDEVERVKQISRLFKEQFGDCFTLFLN KDEYASNKEETITGVVTIGKSTAKIDFNNMKILECNSNPLKGRVESLLNIGGNLVTPLC Mpe1 Saccharomyces cerevisiae unidentified baculovirus LEVO MSSTIFYRFKSQRNTSRILFDGTGLTVFDLKREIIQENKLGDGTDFQLKIYNPDTEEEYD DDAFVIPRSTSVIVKRSPAIKSFSVHSRLKGNVGAAALGNATRYVTGRPRVLQKRQHTAT TTANVSGTTEEERIASMFATQENQWEQTQEEMSAATPVFFKSQTNKNSAQENEGPPPPGY MCYRCGGRDHWIKNCPTNSDPNFEGKRIRRTTGIPKKFLKSIEIDPETMTPEEMAQRKIM ITDEGKFVVQVEDKQSWEDYQRKRENRQIDGDETIWRKGHFKDLPDDLKCPLTGGLLRQP VKTSKCCNIDFSKEALENALVESDFVCPNCETRDILLDSLVPDQDKEKEVETFLKKQEEL HGSSKDGNQPETKKMKLMDPTGTAGLNNNTSLPTSVNNGGTPVPPVPLPFGIPPFPMFPM PFMPPTATITNPHQADASPKK singleParticle particle 7.9 150.0 sodium chloride 10.0 HEPES Quantifoil, UltrAuFoil GOLD 300 PLASMA CLEANING OTHER ETHANE 100 277 FEI VITROBOT MARK IV
blotted for 10 s.
350 nM complex was used.
 FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN VOLTA PHASE PLATE hexapole Cs-corrector GIF Bioquantum 20 GATAN K2 SUMMIT (4k x 4k) SUPER-RESOLUTION 1-42 1 994 9.0 45.0 1 509298
Initially using Gaussian-blob based picking, then after clean-up and enrichment of rare views, conducted template-based autopick to obtain 429703 better centred particles.
 Gctf
Gctf was also used to fit and refine the phase shift per image created by the Volta phase plate
 6I1D
The pdb file was converted to an EM volume and low-pass filtered to 20 angstroms resolution prior to any refinement
 1 C1 FOURIER SPACE 6.0 FSC 0.143 CUT-OFF RELION 2.0
Even though the gold-standard FSC cutoff indicated a resolution of 4.8A, the map was filtered to 6A in post-processing. This dataset had a severe preferred orientation leading to highly anisotropic reconstructions. The main purpose of the 3D refinement was to compare with the crystal structure (truncated proteins) to see if any more ordered domains were visible when imaging full-length proteins.
43308 MAXIMUM LIKELIHOOD RELION 2.0 MAXIMUM LIKELIHOOD RELION 2.0 emd_0325.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 139.52 139.52 139.52 90.0 90.0 90.0 X Y Z -0.047686033 0.079657555 0.000808997 0.005417154 1.09 1.09 1.09 0.025 AUTHOR Cryo-EM map of the Ysh1-Mpe1 complex 6I1D RIGID BODY FIT REAL emd_0325_msk_1.map emd_0325_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 139.52 139.52 139.52 90.0 90.0 90.0 X Y Z -0.032876905 0.051323276 0.00033737477 0.0036264732 1.09 1.09 1.09 AUTHOR half-map 2 emd_0325_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 139.52 139.52 139.52 90.0 90.0 90.0 X Y Z -0.036708575 0.049105708 0.0003384246 0.0036925056 1.09 1.09 1.09 AUTHOR half-map 1