Journal: Cell host & microbe

Article Title: Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

doi: 10.1016/j.chom.2019.08.002

Figure Lengend Snippet: (D) Cryo-EM density map of AP2ΔμC bound to SIVsmm Nef and the SMM tetherin cytoplasmic tail (E) Ribbon model of AP2ΔμC bound to SIVsmm Nef and the SMM tetherin cytoplasmic tail build from the AP2ΔμC:SMM tetherin-SIVsmm Nef cryo-EM density map. See also Figure S1–3, Table S1

Article Snippet: Figure S2 shows the cryo-EM data processing workflow.

Techniques: Cryo-EM Sample Prep

Journal: Cell host & microbe

Article Title: Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

doi: 10.1016/j.chom.2019.08.002

Figure Lengend Snippet: (A) Tetherin 14GDIWK18 motif rests in a pocket created by the SIVsmm Nef dileucine loop. The GDIWK motif is further sandwiched by the AP2ΔμC β2 N-terminus and SIVsmm Nef H0 helix. The cryo-EM map density is depicted as a mesh around the SMM tetherin peptide while the Nef and AP-2 are depicted as space filling surface representations. (B) Ribbon representation of the SIVsmm Nef dileucine loop (190ExxxLV195) bound to the dileucine binding site of the AP2ΔμC α-σ2. SMM tetherin peptide is shown to highlight its position relative to the SIVsmm Nef dileucine loop. Fig 3B is rotated ~90° counterclockwise to Fig 3A. (C) Hydrophobic pocket or SIV Nef created by both the SIVsmm Nef dileucine loop and Nef core with the SMM tetherin GDIWK peptide bound and sandwiched by the AP2ΔμC β2 N-terminus and SIVsmm Nef H0 helix. Fig 3C is rotated ~20° clockwise to Fig 3B. Interacting residues are highlighted. (D) Dileucine loop alignment between SIVsmm Nef of the AP2ΔμC:SMM tetherin: SIVsmm Nef complex and HIV NL4–3 Nef bound to the AP-2 α-σ2 hemi complex (PDB: 4NEE). Alignment was performed between AP2ΔμC α-σ2 and AP-2 α-σ2 hemi. Fig 3D is rotated ~90° counterclockwise to Fig 3C. Disulfide bond is labeled S-S.

Article Snippet: Figure S2 shows the cryo-EM data processing workflow.

Techniques: Cryo-EM Sample Prep, Binding Assay, Labeling

Journal: Molecular cell

Article Title: RNA polymerase accommodates a pause RNA hairpin by global conformational rearrangements that prolong pausing

doi: 10.1016/j.molcel.2018.01.018

Figure Lengend Snippet: (A) The hisPEC structure is shown as an α-carbon backbone worm. The nucleic acids are shown in cartoon format (t-strand DNA, dark gray; nt-strand DNA, yellow; RNA, magenta). The protein is color-coded as a ramp (color-key shown below) according to the Cα(EC)-Cα(hisPEC) distance, where the two structures were superimposed via the structural core module (Table S2). The gray arrows denote the direction and distance (multiplied by a factor of 2) for the Cα(EC)-Cα(hisPEC) changes that are > 2 Å. The average ΔCα is 1.8 Å (± 1.7 Å standard deviation). The minimum and maximum ΔCα’s are 0.084 and 16 Å, with the largest ΔCα’s occurring in SI2.

Article Snippet: We observed that the his PEC escaped the pause site 10 times slower than the ePEC ( Figure S1A ); that CHAPSO modestly reduced pause lifetime (by a factor of ~2; Figure S1B ); that neither the cryoEM scaffold nor the deletion of αCTD altered pause lifetimes ( Figure S1C ); that his PECs formed by nucleotide addition (C28→U29; Figure S1D ) were kinetically indistinguishable from those formed by direct reconstitution (U29, Figure S1C ; see also Kyzer et al., 2007 ); and that a 1-nt extension of the spacer between the PH and the RNA-DNA hybrid increased pause lifetime (~2-fold, Figure S1E ).

Techniques: Standard Deviation

Journal: Molecular cell

Article Title: RNA polymerase accommodates a pause RNA hairpin by global conformational rearrangements that prolong pausing

doi: 10.1016/j.molcel.2018.01.018

Figure Lengend Snippet: (A) Shown is the 5.5 Å resolution (4.3 Å resolution around the active site and RNA-DNA hybrid) cryo-EM density map (blue mesh) with the superimposed model of the hisPEC-minus-PH nucleic acids. Shown for reference are key RNAP structural elements (Sw3, lid, BH) and the RNAP active-site Mg2+-ion (yellow sphere). Like the hisPEC (Figure 2A), the half-translocated RNA:DNA (shown), but not a pre-translocated or fully translocated RNA:DNA, fit the density map.

Article Snippet: We observed that the his PEC escaped the pause site 10 times slower than the ePEC ( Figure S1A ); that CHAPSO modestly reduced pause lifetime (by a factor of ~2; Figure S1B ); that neither the cryoEM scaffold nor the deletion of αCTD altered pause lifetimes ( Figure S1C ); that his PECs formed by nucleotide addition (C28→U29; Figure S1D ) were kinetically indistinguishable from those formed by direct reconstitution (U29, Figure S1C ; see also Kyzer et al., 2007 ); and that a 1-nt extension of the spacer between the PH and the RNA-DNA hybrid increased pause lifetime (~2-fold, Figure S1E ).

Techniques: Cryo-EM Sample Prep