Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: HDX-MS analysis of nsp7, nsp8, and nsp7:nsp8 complexes. Observed percent deuterium uptake of nsp7 (A) and nsp8 (B) during the 1h experiment time course. Secondary structure from PDB: 6YHU annotated in white and predicted secondary structure based on observed percent deuterium in gray.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Overlay of HDX-MS and XL-MS results on nsp7 and nsp8 sequences. Observed inter-nsp7 nsp8 crosslinks only identified in the nsp7:nsp8 1:1 complex are colored in orange and inter-nsp7 nsp8 crosslinks also identified in nsp7:nsp8 1:2 complex are colored in blue. Consolidated change in percent deuterium uptake from and are overlayed on the nsp7 and nsp8 sequences, respectively. Initial Met residue removed from both nsp7 and nsp8 sequences to maintain correct residue numbering.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Overlay of differential HDX-MS perturbation values onto PDB:6YHU. Perturbation values for nsp7 vs nsp7:nsp8 1:1 (A) , nsp8 vs nsp7:nsp8 1:1 (B) , nsp7 vs nsp7:nsp8 1:3 (C) , and nsp8 vs nsp7:nsp8 3:1 (D) colored according to change in percent deuterium levels shown in color bar and respective partner protein colored in pink. Residues not observed by HDX-MS are colored in white.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: XL-MS relative quantification results of nsp7, nsp8, and nsp7:nsp8 complex. (A) Volcano plot summarizing the relative quantification results of crosslinks in nsp8 versus nsp7:nsp8 1:1. Crosslinks enriched in nsp7:nsp8 1:1 complex shown in red box and crosslinks enriched in the nsp8 alone sample shown in blue box. (B) Volcano plot summarizing the relative quantification results of nsp8 versus nsp7:nsp8 1:2 (adjusted) crosslinking reactions. Crosslinks enriched in nsp7:nsp8 1:1 complex shown in red box. Intra-nsp8 crosslinks shown as squares and inter-nsp7 nsp8 crosslinks shown as circles.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7 nsp8 inter-protein crosslinks on the dimer (A) and heterotetramer (B) interaction surfaces of the SARS-CoV-2 nsp7 nsp8 heterotetramer crystal structure (PDB: 6YHU). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV-2 nsp7:nsp8 heterotetramer structure (PDB: 6YHU).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7 nsp8 inter-protein crosslinks on the dimer (A), heterotetramer (B), hexadecamer (C) interaction surfaces of the SARS-CoV nsp7 nsp8 hexadecamer cryo-EM structure (PDB: 2AHM). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV nsp7:nsp8 hexadecamer structure (PDB: 2AHM).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7-nsp8 inter-protein crosslinks to nsp8-I (A) or nsp8-II (B) of the SARS-CoV-2 replicating polymerase structure (PDB: 6YYT). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV-2 replicating polymerase structure (PDB: 6YYT).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to available SARS-CoV-2 replicating polymerase structures composed of nsp7, nsp8, nsp12, and RNA.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to available SARS-CoV-2 replicating polymerase structures composed of nsp7, nsp8, and nsp12.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: