Journal: The Journal of Cell Biology

Article Title: Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

doi: 10.1083/jcb.201201161

Figure Lengend Snippet: Cryo-EM structure of a GMPCPP microtubule. (A) A low-pass–filtered GMPCPP microtubule seen from the outside with a 10-Å cutoff for the fair comparison with the GDP-taxol microtubule ( Fig. S1 ). The plus end is up. α and β show the positions of α- and β-tubulins, respectively. The dashed orange circles and squares show the holes in the microtubule wall. The purple and cyan planes show sectioning planes for views in B and , respectively. Arrowheads indicate OCs. (B) Contour plot of a cross-section at 10-Å resolution observed from the plus end. Most of the α helices are well resolved as the local maxima of the densities. The arrowheads show OCs, and the dashed circle shows the low-density region at the middle of the tubulin monomers. Also see Figs. S1 and S2 and Video 1 for the comparison between GMPCPP and GDP-taxol microtubules. Bars, 2 nm.

Article Snippet: After division into these four subdomains, the atomic models of both α- and β-tubulins fitted well to the cryo-EM density map (AMV = 168.5 and 170.8, respectively; , S2, and S3 and Video 2).

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: SARS-CoV-2 nsp13 Helicase Forms a Stable Complex with the Holo-RdRp and an RNA Scaffold (A) The RNA scaffold used for biochemistry, native mass spectrometry (nMS), and cryo-EM. (B) A native gel electrophoretic mobility shift assay reveals that nsp13 forms a stable complex with holo-RdRp:RNA. The 4.5% polyacrylamide gel was visualized with Gel Red to stain the RNA. (C) nMS spectra and the corresponding deconvolved spectra for the holo-RdRp containing the RNA scaffold (A) with and without nsp13. The measured mass for the holo-RdRp:RNA complex corroborates the established stoichiometry of 1:2:1:1 for nsp7:nsp8:nsp12:RNA ( Hillen et al., 2020 ; ; Wang et al., 2020 ; Yin et al., 2020 ), respectively (bottom). Addition of the 67.5-kDa nsp13 helicase to the RNA-bound holo-RdRp sample forms a transcription complex/helicase assembly with 1:1 stoichiometry (top). See also and .

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Mass Spectrometry, Cryo-EM Sample Prep, Electrophoretic Mobility Shift Assay, Staining

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Cryo-EM Processing Pipeline and Analysis for the nsp13-RTC (No Detergent) Dataset, Related to Figure 2 A. Cryo-EM processing pipeline. B. Nominal 3.6 Å-resolution cryo-EM reconstruction of nsp13 2 -RTC (no detergent) filtered by local resolution ( Cardone et al., 2013 ) and colored by subunit according to the key on the right. C. Directional 3D Fourier shell correlation (FSC) for nsp13 2 -RTC (no detergent) calculated by 3DFSC ( Tan et al., 2017 ). D. Angular distribution plot for reported nsp13 2 -RTC (no detergent) calculated in cryoSPARC. Scale shows the number of particles assigned to a particular angular bin. Blue, a low number of particles; red, a high number of particles.

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Cryo-EM Processing Pipeline and Analysis for the nsp13-RTC (CHAPSO) Dataset, Related to Figure 2 A. Cryo-EM processing pipeline. B. Nominal 3.5 Å-resolution cryo-EM reconstruction of nsp13 2 -RTC (CHAPSO) filtered by local resolution ( Cardone et al., 2013 ). The view on the right is a cross-section. (top) Colored by subunit. (bottom) Color by local resolution (key on the bottom). C. Directional 3D Fourier shell correlation (FSC) for nsp13 2 -RTC (CHAPSO) calculated by 3DFSC ( Tan et al., 2017 ). D. Angular distribution plot for reported nsp13 2 -RTC (CHAPSO) calculated in cryoSPARC. Scale shows the number of particles assigned to a particular angular bin. Blue, a low number of particles; red, a high number of particles. E. Gold-standard FSC plot for nsp13 2 -RTC (CHAPSO), calculated by comparing two independently determined half-maps from cryoSPARC. The dotted line represents the 0.143 FSC cutoff which indicates a nominal resolution of 3.5 Å. F. FSC calculated between the refined structure and the half map used for refinement (work, red), the other half map (free, blue), and the full map (black).

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Cryo-EM Density Maps, Related to Figure 2 A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13. The color-coding corresponds to the figures throughout this manuscript unless otherwise specified. B-E. Orthogonal views showing the overall architecture of the nsp13 2 -RTC. Shown is the transparent cryo-EM density (local-resolution filtered) with the nsp13 2 -RTC model superimposed. Same views as B–2E. F. View of the nsp12 (RdRp) active site (refined model superimposed onto the cryo-EM density, shown as blue mesh), showing the post-translocated state of the RNA. G. View of the nsp8b-extension:nsp12-thumb:nsp13-ZBD tripartite interaction (refined model superimposed onto the cryo-EM density, shown as blue mesh). Similar view as Figure 3 A.

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Overall Structure of the SARS-CoV2 nsp13 Helicase with the Holo-RdRp:RNA Replication-Transcription Complex (RTC) (A) Schematic illustrating the domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, and nsp12) and nsp13. Structural domains discussed in the text are labeled. The color coding corresponds to the figures throughout this manuscript unless otherwise specified. (B–E) Orthogonal views showing the overall architecture of the nsp13 2 -RTC. Proteins are shown as molecular surfaces (except nsp13.1 in D) and RNA as atomic spheres. Adventitious CHAPSO detergent molecules are shown as atomic spheres and colored dark gray. The path of downstream tRNA through the nsp13.1 helicase, shown as cyan spheres, is revealed with low-pass-filtered (6 Å) difference density (shown in D). (B) Two copies of the nsp13 helicase bind to the RTC. Nsp13.1 forms a tripartite interaction with the nsp8b extension and the nsp12 thumb via the nsp13.1-ZBD. The 5′ end of the tRNA extrudes through the nucleic acid binding channel of nsp13.1. The two helicases interact via the nsp13.1-1B domain and the nsp13.2-RecA1 domain. (C) In addition to the nsp13.1-ZBD:nsp8b extension:nsp12 thumb tripartite interaction, nsp13.1-RecA1 interacts with nsp7 and the nsp8b head. (D) ADP-AlF 3 is modeled in the NTP binding site of each helicase. The low-pass-filtered (6 Å) cryo-EM difference density revealing the path of the downstream t-RNA is shown (dark blue mesh). (E) The nsp13.2-ZBD interacts with the nsp8a extension. ADP-Mg 2+ is bound to the NiRAN domain. See also Figure S3 , Figure S4 , Figure S5 , Figure S6 , , and .

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

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

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Comparison of the nsp13 2 -RTC (CHAPSO) Structure with nsp13 1 -RTC (CHAPSO) and nsp13 2 -RTC (No Detergent), Related to and A. Structure of nsp13 2 -RTC (CHAPSO) colored according to key in b and shown as a molecular surface except nsp13.1, which is shown as cartoon tubes. Superimposed on the overall structure is nsp13 (marine) modeled from the nsp13 1 -RTC (CHAPSO). Overall RMSD (calculated using ‘rms_cur’ in PyMOL) between the two nsp13.1 structures is 8.1 Å over 596 Cα atoms. (left) overall structure. (middle) overall structure rotated 90°. (right) zoom-in of boxed region in middle panel, showing region around nsp13.1-ZBD.s RMSD (calculated using ‘rms_cur’ in PyMOL) between the two nsp13.1 ZBDs is 3.6 Å over 100 Cα atoms. B. Structure of nsp13 2 -RTC (CHAPSO) is shown in cartoon tubes, colored based on key, and superimposed onto the cryo-EM map from the nsp13 2 -RTC (no detergent) dataset (shown as light blue transparent surface). Density map is locally filtered by resolution and difference density for nsp13 is highlighted using ‘isosurf’ command in PyMOL with 10 Å carve buffer. (left) overall structure. (right) zoom-in of boxed region in left panel, showing region around nsp13-ZBDs. C. Structure of nsp13 2 -RTC (CHAPSO) colored according to key in (b), the view is similar to the view of Figure S6A( left ). Protein is shown as pale, transparent backbone worms. The surface shows a cryo-EM different density for the RNA (t-RNA, cyan; p-RNA, red) low-pass filtered to 6 Å resolution. The difference map was generated by calculating a map from the nsp13 2 -RTC coordinates with the RNA removed using the molmap command in Chimera ( Pettersen et al., 2004 ), subtracting this map from the experimental map (Chimera vop command), then low-pass filtering this difference map at 6 Å resolution).

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Cryo-EM Sample Prep, Generated

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: The SARS-CoV-2 nsp12-NiRAN Domain, Pseudokinase SelO, and ADP Binding (A) The colored histograms denote identity in a sequence alignment of 45 α- and β-CoV nsp12 sequences (red bar, 100% identity; dark blue bar, 20% or less) in the N-terminal signature motifs A N , B N , and C N ( Lehmann et al., 2015a ) of the NiRAN domain. The consensus sequence is shown below. The SARS-CoV-2 nsp12 and the pseudokinase P. syringae ( Psy ) SelO ( Sreelatha et al., 2018 ) are aligned below. Residues that are 100% identical in the nsp12 alignment and conserved in SelO are highlighted by a red dot underneath. (B) Left: structures of the SARS-CoV-2 NiRAN domain (cyan ribbon) with ADP-Mg 2+ (spheres) and Psy SelO (orange) with AMP-PNP-Mg 2+ (PDB: 6EAC ; Sreelatha et al., 2018 ). The A N , B N , and C N regions are highlighted. Right: structure-based alignment via α-carbons of the A N , B N , and C N regions, with side chains of conserved residues shown. The α- and β-phosphates of the NiRAN domain ADP-Mg 2+ (lime carbon atoms and yellow sphere, respectively) superimpose almost exactly with the β- and γ-phosphates of the SelO AMP-PNP-Mg 2+ (dark gray), whereas the nucleoside moieties diverge. (C) Two views of the ADP-Mg 2+ -bound pocket of the SARS-CoV-2 NiRAN domain. Side chains interacting with the ADP-Mg 2+ are shown (polar interactions are denoted by gray dashed lines). D208 likely makes a water-mediated interaction with Mg 2+ ( Sreelatha et al., 2018 ). The cryo-EM difference density for ADP-Mg 2+ is shown (light gray mesh). See also .

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

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

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

Figure Lengend Snippet: Structural Basis for Possible nsp13 Helicase Functions during Viral Genome Replication-Transcription Structural models are shown as cartoons (holo-RdRp, light blue; nsp13.1 helicase, orange shades; RNA strands, colored tubes). The nsp13.2 helicase is not shown for clarity (all models are compatible with the presence of nsp13.2). With each structural diagram is a schematic cartoon illustrating the arrangement of RNA strands. Additional proteins involved in these processes are omitted. The product RNA (p-RNA) being elongated by the RdRp is shown in red. (A) The SARS-CoV-2 nsp13-RdRp cryo-EM structure likely represents an equilibrium between two states. (B) During RNA synthesis on a single-stranded RNA template (cyan), nsp13 could function distributively to clear downstream RNA secondary structure (or RNA binding proteins). Similarly, on a duplex RNA template (cyan and green), nsp13 could processively unwind downstream duplex RNA. (C) Proposed helicase function during template switching associated with sub-genomic transcription (sg-transcription) ( Enjuanes et al., 2006 ; Lehmann et al., 2015b ; Pasternak et al., 2001 ; Snijder et al., 2016 ; Sola et al., 2015 ). (i) Negative-strand RNA synthesis proceeds from the genomic 3′ poly(A)-tail until a transcription-regulating sequence (TRS-R, orange) ( Alonso et al., 2002 ) is transcribed (cTRS, yellow). (ii) The TRS causes transcription complex stalling. (iii) Helicase function acting on the + strand RNA (cyan) causes backtracking of the transcription complex, freeing the pRNA 3′ end. (iv) The p-RNA 3′-end cTRS (yellow) hybridizes with the complementary TRS-L (orange) following the genomic 5′ leader sequence (magenta) ( Alonso et al., 2002 ; Pasternak et al., 2001 ; Zúñiga et al., 2004 ). (v) Processive helicase function backtracks the RdRp complex and unwinds the p-RNA from the genomic 3′ end. A second RdRp complex (holo-RdRp2) can load into the p-RNA 3′ end and continue transcription using the 5′ leader as a template.

Article Snippet: Figure S5 Cryo-EM Density Maps, Related to A. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13.

Techniques: Cryo-EM Sample Prep, RNA Binding Assay, Sequencing

Journal: eLife

Article Title: Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

doi: 10.7554/eLife.59560

Figure Lengend Snippet: ( A ) EMSA comparing the ability of Scc2 and Scc2-Scc1 150-298 complexes to bind dsDNA. ( B ) S. cerevisiae Scc2 from the cryo-EM structure with the four resides within the putative DNA binding surface labelled that were mutated to glutamate (Scc2-4E). ( C ) EMSA comparing the ability of Scc2 and Scc2-4E complexes to bind dsDNA. ( D ) Entrapment of DNA in S-K rings in the presence of Scc3 and either Scc2 or Scc2-4E. Entrapment assay incubated for 40 min with time points taken every 10 min (*=damaged open circular DNA; I = input DNA).

Article Snippet: Crystal structures of yeast Smc1 head (PDB 1W1W; ) and Smc3 head (PDB:4U × 3; ) and the Scc2 homology model were docked into the tetramer:40 bp DNA cryo-EM density map using UCSF Chimera X ( ).

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

Journal: eLife

Article Title: Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

doi: 10.7554/eLife.59560

Figure Lengend Snippet: ( A ) Cryo-EM map of 40 bp DNA clamped by Scc2- and ATP-bound cohesin EQEQ trimer at 3.4 Å resolution. Both front and side views are coloured by subunit. ( B ) Same complex as shown in A but bound to ~1.8 Kbp relaxed circular DNA as a cryo-EM field view (using Volta phase plate, left) and a selection of 2D class averages (right) clearly showing DNA emanating from cohesin/Scc2 complexes. ( C ) 7.3 Å resolution cryo-EM map of the complex shown in B, coloured by subunit, demonstrating that the same conformation of the complex has been obtained as with linear DNA (panel A). Same orientations and colours as in A. ( D ) 2D class averages obtained by reprocessing of the same data set as used for A with an enlarged box size show the position of the coiled coils and the hinge. ( E ) ~ 10 Å resolution cryo-EM map of the entire tetramer complex as shown in D. Since we used the same complex as used in the in vitro entrapment reactions, we can deduce that the DNA within the clamped structure depicted in A, C and E must be entrapped in both the E-S and E-K compartments.

Article Snippet: Crystal structures of yeast Smc1 head (PDB 1W1W; ) and Smc3 head (PDB:4U × 3; ) and the Scc2 homology model were docked into the tetramer:40 bp DNA cryo-EM density map using UCSF Chimera X ( ).

Techniques: Cryo-EM Sample Prep, Selection, In Vitro

Journal: eLife

Article Title: Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

doi: 10.7554/eLife.59560

Figure Lengend Snippet: ( A ) Cartoon representation of the refined atomic model of cohesin’s clamped (E-S/E-K) state based on the 3.4 Å resolution cryo-EM map ( , same orientation and colours, ). ( B ) Basic and polar residues of Scc2 involved in the interaction with DNA. Scc2 interacts only with the backbone. Residues in its vicinity are labelled in black while those mutated in Scc2-4E in purple. ( C ) Scc2 makes extensive contacts with both Smc1 and Smc3 heads: (i) Scc2 binds Smc1 through its HEAT repeats 18–24 (residues 1127–1493) that dock onto the F-loop on Smc1 (residues 1095–1118) and the emerging coiled coils above it. (ii) Smc3’s K112 K113, whose acetylation reduces loading efficiency, are in the vicinity of a negatively charged patch on Scc2 (819-EDEED-823 and 781-DD-782). (iii) Scc2 binds to Smc3 through a β-strand (part of the otherwise disordered loop 1178–1203) that complements the central β-sheet of Smc3. (iv) The N-terminal section of Scc2 contacts parts of Smc3’s coiled coil arm/neck, close to where the last ordered region of Scc1’s N-terminal domain is bound to the Smc3 coiled coil. ( D ) DNA binding to the SMC head domains is pseudo-symmetrical. Top: the pseudo two-fold axis of the DNA neatly aligns with that of the head domains underneath. Bottom: The head domains interact with the DNA almost exactly two full DNA turns apart, utilising pseudo symmetry-related surfaces (Smc1: K63, S112, R113, and K124; Smc3: K57, K112, K113, and K125). ( E ) The two lysines K112 K113 are in contact with a negatively charged patch on Scc2 (see panel C iii), but are also in the vicinity of the DNA backbone. ( F ) The N-and C-terminal domains of the kleisin Scc1 bind canonically to Smc3 and Smc1, linking the heads and topologically closing the tripartite Smc1/Smc3/Scc1 (S–K) cohesin ring. A tentative path of the disordered regions of Scc1, not visible in our cryo-EM map is shown to demonstrate the topology as deduced from the loading reactions and subsequent crosslinking that show that the DNA must be outside the tripartite S-K ring.

Article Snippet: Crystal structures of yeast Smc1 head (PDB 1W1W; ) and Smc3 head (PDB:4U × 3; ) and the Scc2 homology model were docked into the tetramer:40 bp DNA cryo-EM density map using UCSF Chimera X ( ).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: a , Phylogenetic tree of the canonical human NHE1–9 (SLC9A1–9) cluster compared to human NHA1 and NHA2 (SLC9B1–2) and bacterial members NapA ( Thermus thermophilus ), NhaP1 ( Methanococcus janashi ), NhaP ( Pyrococcus abyssi ) and NhaA ( Escherichia coli ). b , Cryo-EM density map of NHA2 ΔN in detergent, showing the 6-TM core ion transport domains (colored in pink), the dimer domain (colored in green) and the N-terminal domain-swapped helix, TM –1 (colored in blue). c , Cartoon representation of dimeric NHA2 from the side (left) and a top view from the extracellular side (right). The ion-translocation 6-TM domain (transport) is colored in pink, the dimerization domain in green and the N-terminal domain-swapped transmembrane helix (TM –1) in blue, with the respective transmembrane helices enumerated. Inset (dashed box): cartoon representation from the extracellular side, with each monomer colored individually. d , Cartoon representation of the 14-TM NHA2 monomer from the extracellular side and colored as in c , superimposed onto the 13-TM outward-facing structure of NapA (PDB 4BWZ ) in gray. e , Cartoon representation of the NapA homodimer, colored as for NHA2 in c to highlight that, in the absence of the additional N-terminal helix TM –1, an extensive and more compact oligomer is formed compared to NHA2.

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

Techniques: Cryo-EM Sample Prep, Translocation Assay

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: a . Cryo-EM density map of NHA2 ΔN after focused refinement with the 6-TM core transport domain (colored in pink), the dimer domain (colored in green) and the N-terminal domain-swapped helix TM –1 from the neighboring protomer (blue and circled). b . Cryo-EM density map for protomer A before (left) and after (right) focused refinement. The protomer is made up from TM 1 to 13 of one monomer and TM -1 of the other monomer; as such oligomerization contacts are retained in the focused refined map. In TM6 the aspartate residues D277 and D278 (encircled) in TM6 were modeled based on the rotomer position in NapA at pH 8.0 (PDB id: 4BWZ).

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

Techniques: Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: a , The native high-resolution mass spectrum of purified NHA2 ΔN reveals a homodimer with multiple lipid adducts, as well as a small amount of lipid-free monomer (left inset). The masses of the first lipid adduct shown for the 19+ monomer are consistent with retention of a PI (829 ± 14.4 Da) or a PIP 2 (1,036 ± 14.5 Da) molecule (right inset). Peaks shown as inserts are highlighted by grey bars in the full spectrum. b , Thermal stabilization of DDM-purified dimeric NHA2 ΔN -GFP (blue bars) and NHA2 ΔTM−1 (green bars) by lipids. Normalized mean fluorescence (r.f.u., relative fluorescence units) is shown after heating ( T M + 5 °C, for the respective forms) and centrifugation in the presence of either the detergent DDM or DDM-solubilized lipids. Error bars represent the mean ± s.e.m. of n = 3 independent experiments . c , Thermal shift stabilization of purified dimeric NHA2 ΔN -GFP in the presence of DDM addition (black) compared to PIP 2 in DDM (blue), POPC in DDM (cyan) and PI in DDM (red). The data are normalized fluorescence mean ± s.e.m. of n = 5 independent experiments for DDM, n = 3 independent experiments for PI and PC and n = 2 independent experiments for PIP 2 . The apparent melting temperature T M was calculated with a sigmoidal four-parameter logistic regression function . d , Representative FSEC traces of DDM/CHS-purified NHA2 ΔN after heating at 40 °C for 10 min in the presence of DDM addition (black) compared to PIP 2 in DDM (blue) or PI in DDM (red). Inset: as in the main panel, but prior to heating. e , Cryo-EM density map of NHA2 ΔN in nanodiscs with the 6-TM core ion-transport domains (colored in pink), the dimer domain (colored in green), cholesterol (gray) and the N-terminal domain-swapped helix TM −1 (blue).

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

Techniques: Purification, Fluorescence, Centrifugation, Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: Cryo-EM density map of the NHA2 ΔN helices in nanodiscs. The respective helices are shown in cartoon representation for the 6-TM core transport (pink), the dimer domain (green), the N-terminal domain-swapped helix TM –1 (blue), with their cryo-EM map density in blue mesh. Density for several lipids is also shown in blue mesh, and modeled lipids as sticks with CHS (yellow), PI-headgroup (green) and PI tail (gray); other lipids and map density is shown in Extended Data Fig. .

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

Techniques: Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: a , Left: cartoon representation of the 14-TM NHA2 ΔN monomer in nanodiscs, from the intracellular side (colored as in Fig. ), superimposed onto the NHA2 monomer in detergent (in gray) to highlight the movement of TM −1, as indicated by the arrow. Right: as in the left panel, but from the side and including the highly conserved proline residues in yellow stick form (Extended Data Fig. ). b , Surface representation of the NHA2 ΔN structure from the cytoplasmic side in detergent (left) and in nanodiscs mixed with PI lipids (right). c , Left: cartoon representation of the NHA2 homodimer in nanodiscs, from the extracellular side, highlighting the bound PI lipids (red sticks, cryo-EM map in gray mesh) coordinated by tryptophan residues in TM3 at the protomer interface and cholesterol interacting with tryptophan residues in TM −1. Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface. Middle bottom: zoomed view showing the cryo-EM map density for one of the PI lipids (stick form, red) interacting with W171 in TM3 and potentially E107 in TM −1. More extensively phosphorylated forms of the inositol moiety, at either C4 or C4/C5 for PI 4 P and PIP 2 respectively, could be accommodated and enable additional interaction to the K168 and K170 residues. A salt bridge (dashed line) is also formed between residues R176 in TM3 and E406 in TM10. Right: cartoon representation of the NHA2 homodimer in nanodiscs, from the cytoplasmic side, highlighting the bound PI lipids (red, gray) coordinated by tryptophan residues, in TM8 at the protomer interface, that have come closer together by the movement of TM −1, which retains its interactions with the TM7–TM8 loop. Selected TM numbering are circled.

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

Techniques: Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Structure, mechanism and lipid-mediated remodeling of the mammalian Na + /H + exchanger NHA2

doi: 10.1038/s41594-022-00738-2

Figure Lengend Snippet: a . Electrostatic surface representation of the side view of the outward-facing NHA2 homodimer highlighting the large, intracellular gap between protomers and intracellular positively-charged surface in detergent ( left ) that is closed upon TM –1 rearrangement in nanodiscs incorporated with PI lipids ( right ). b . The cryo EM density for the nanodisc surrounding NHA2 ΔN from the side and top, and the manually placed bison NHA2 ΔN structure determined in detergent. c . Cartoon representation on the dimer interface showing the polar contacts between the K168, H169, K170 and W171 residues, in the end of TM3 of one protomer (green sticks, labeled), and the identical residues from the neighboring protomer (green sticks, labeled with ‘), with the PI lipids located in between these residues at the center of the dimer interface. Also interacting with R176 of T3 is E406 (TM10), which might stabilize the outward-facing cavity leading the ion-binding, interaction encircled. Modelled lipids are shown as sticks for, PI lipid headgroups (red), cholesterol located on extracellular half of the protein (yellow), and PI tails (gray). Cryo-EM density is shown as blue mesh around the mentioned residues and ligands.

Article Snippet: Middle top: zoomed view showing the cryo-EM map density (gray mesh) for the cholesterol lipids (stick form, yellow) interacting with TM −1 at the dimerization interface.

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