Journal: Microbiology

Article Title: Elucidating population-wide mycobacterial replication dynamics at the single-cell level

doi: 10.1099/mic.0.000288

Figure Lengend Snippet: Plasmids and strains

Article Snippet: pST5552 , hsp60(ribo)-egfp (inducible EGFP under control of theophylline-inducible riboswitch), Kan R , episomal , Seeliger et al. (2012 ), Addgene plasmid number 36255 .

Techniques: Control, Plasmid Preparation, Cloning

Journal: bioRxiv

Article Title: The BAF A12T mutation reduces BAF affinity to lamin A/C, preventing its recruitment to nuclear ruptures in Nestor Guillermo Progeria Syndrome cells

doi: 10.1101/2022.02.25.481780

Figure Lengend Snippet: a) Representative immunofluorescence images showing endogenous BAF staining in Control, NGPS patient cells and NGPS2 WT clone1. Scale bar, 20 μm. b) Quantification of the mean BAF nuclear to cytoplasmic ratio in 425, 495, 401 and 363 cells for Control, NGPS1, NGPS2 and NGPS2 WTclone1 respectively. Data was obtained from 3 independent experiments and quantified using a one-way ANOVA analysis with Šídák’s multiple comparisons test (****P<0.0001). c) Representative immunoblot showing the expression level of BAF in whole cell lysates from the indicated cell lines. Tubulin was used as a loading control. d-e) Using Nuclear Magnetic Resonance (NMR), 2D 1 H- 15 N Heteronuclear Single Quantum Coherence (HSQC) spectra were recorded on purified BAF WT (in shades of blue) and A12T (in shades of red) upon phosphorylation by the VRK1 kinase in vitro . Six spectra are superimposed, corresponding to: non-phosphorylated BAF WT and A12T (t = 0 min), in dark and light grey respectively, mono-phosphorylated BAF WT and A12T (t = 15 min), in light blue and pink respectively, and di-phosphorylated BAF WT and A12T (t = 8 h), in dark blue and red respectively. The zoom in (e) shows the spectral regions of the 1 H- 15 N HSQC of BAF WT and A12T spectra where signals of phosphorylated serines and threonines are commonly observed. The signals of the two BAF residues phosphorylated by VRK1 (Ser4 and Thr3) are annotated and the arrow shows the chemical shift perturbation of the phosphorylated Ser4 upon Thr3 phosphorylation.

Article Snippet: The VRK1 expression vector is a gift from John Chodera, Nicholas Levinson and Markus Seeliger (Addgene plasmid #79684 ( )).

Techniques: Immunofluorescence, Staining, Control, Western Blot, Expressing, Nuclear Magnetic Resonance, Purification, Phospho-proteomics, In Vitro

Journal: bioRxiv

Article Title: Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

doi: 10.64898/2026.03.24.713564

Figure Lengend Snippet: ( a ) Bound AMP-PNP in the active site of CDK11 (green). Residues involved in interactions with the nucleotide are shown as sticks, hydrogen bonds are indicated as black dashed lines, and magnesium ions are shown as green spheres. ( b ) The pseudo-substrate density occupying the substrate binding site of CDK11 is shown in light green (CDK11 green, T-loop blue, SAP30BP yellow, cyclin L purple). ( c ) Cryo-EM map shown as grey mesh and semi-transparent surface, with fitted model coloured as in b. ( d ) Molecular model of the CDK11 pseudo-substrate segment (residues 751-762, light green) occluding the substrate binding site. ( e ) CDK2-cyclin A (cyan and yellow) in complex with a substrate peptide (sand) in the same view as panel C (PDB ID 3QHR) . ( f ) Kinase assay using an SF3B1 1-463 substrate and trimeric kinase complexes containing CDK11B p110 wild type (WT) and the 1-743, S752E, and S752A variants. SF3B1 phospho-T313 (P-T313) was detected using Western blot. A loading control stained with Ponceau S is provided. ( g ) Quantification of Western blot band intensities, presented as the mean ± standard deviation and individual data points of N = 4 kinase assay technical replicates. The relative activity of the wild-type complex at 60 min was set to 1. The significance level for activity comparisons between complexes, determined by 2-way ANOVA and Tukey’s multiple comparisons test, is provided (*: p < 0.05; **: p < 0.01). ( h ) Schematic illustration of the effect of pseudo-substrate phosphorylation on the efficiency of binding of a simple model substrate to CDK11. Hypothetical models for effects on substrate specificity in the context of more complex substrates are shown in Supplementary Fig. 4e, f. The substrate binding site is schematically represented as a cleft, and the active site is indicated by a stylised ATP molecule.

Article Snippet: CDK2 was expressed with an N-terminal His 10 -tag (Addgene plasmid #79726; http://n2t.net/addgene:79726 ; RRID:Addgene_79726, provided by John Chodera, Nicholas Levinson, and Markus Seeliger) , and cyclin A2 was expressed with an N-terminal His 6 -tag.

Techniques: Binding Assay, Cryo-EM Sample Prep, Kinase Assay, Western Blot, Control, Staining, Standard Deviation, Activity Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

doi: 10.64898/2026.03.24.713564

Figure Lengend Snippet: ( a ) Rendering of CDK11-bound OTS964 (blue) in the cryo-EM map (shown as semi-transparent grey mesh and surface). ( b , c ) Active site views of the CDK11-cyclin L-SAP30BP-OTS964 complex in the two alternative conformations observed in the cryo-EM reconstruction. Hydrogen bonds (donor-acceptor distance 3.5 Å or less) are indicated by black dashed lines. The shortest distance between inhibitor and gatekeeper residue (M516 in CDK11) is indicated and shown by a white dashed line. The change in position of Y449 between panels b and c is indicated with a dashed arrow in panel b. ( d ) Comparison of the structure of CDK11-cyclin L-SAP30BP-OTS964 (CDK11 in green, cyclin L in purple with residues 173-181 in pink, OTS964 in blue) to the structure of isolated CDK11B-OTS964 (PDB ID 7UKZ ; light green and light blue, respectively). The site of steric incompatibility of the isolated CDK11B conformation with the presence of cyclin L is indicated by a black triangle. Shifts in adjacent loops because of cyclin binding are indicated by arrows. ( e ) Active site view of the CAK-OTS964 complex (CDK7 is shown in grey, water molecules as red spheres). Only the hydrogen bonds to the hinge region are preserved because several hydrogen bonding partners are absent or beyond hydrogen bonding distance (distance to D97 indicated by a yellow dashed line). The distance to the gatekeeper residue (F91 in CDK7) is enlarged (yellow dashed line). ( f ) Active site view of the CDK2-cyclin A2-OTS964 complex (CDK2 is shown in cyan). OTS964 forms three hydrogen bonds to CDK2, as it is within hydrogen bonding distance of CDK2 D86. Y15 is tucked under the dimethylamino-propyl group of OTS964.

Article Snippet: CDK2 was expressed with an N-terminal His 10 -tag (Addgene plasmid #79726; http://n2t.net/addgene:79726 ; RRID:Addgene_79726, provided by John Chodera, Nicholas Levinson, and Markus Seeliger) , and cyclin A2 was expressed with an N-terminal His 6 -tag.

Techniques: Cryo-EM Sample Prep, Residue, Comparison, Isolation, Binding Assay