Gold-50

Unless otherwise stated, 25 μL PCR reactions were performed using Phusion® High-Fidelity DNA polymerase (NEB) with 0.1 μM primers and 1 ng template DNAs (0.5 ng of each for the multi-fragment assembly), according to the following protocol: 30 sec at 95 °C, 18 cycles of 10 sec at 95 °C, 30 sec at 60 °C, 4 min at 72 °C, and a final 5 min extension at 72 °C. Addition of 1 μL FastDigest DpnI enzyme (Thermo Fisher Scientific) was followed by 15 min incubation at 37 °C prior to transformation. For multi-site/multi-fragment procedures, PCR extension time was reduced to 3 minutes, yielding a 1.5 hr PCR reaction.
For insertions, deletions and mutagenesis 2 μL PCR reactions were transformed into 100 μL of RbCl competent cells. For sub-cloning and double modifications 2 μL of DpnI digested sample were transformed in 30 μL of commercial XL10-Gold cells. For multi-site reactions and multi-fragment assemblies 4 μL and 6 μL of PCR mix were transformed into 30 μL and 50 μL of commercial XL10-Gold cells respectively.
All mixtures were incubated for 15 min at 4 °C, followed by a heat shock at 42 °C for 30 sec and recovery at 37 °C for 45 min with 200 μL Super Optimal Broth with Catabolite repression (SOC) medium added. The entire volume was plated onto LB-agar plates with corresponding antibiotics for overnight incubation at 37 °C. Colonies were manually counted (number of colonies reported as Colony-Forming Units per plate (CFU/plate)) and successful plasmid construction was assessed by restriction digestion and/or Sanger sequencing (Beckman Coulter) of colony DNA.
Mutagenesis time courses were performed in multiple 50 μL reactions with 5 ng of DNA template for improved signal at early time points. Whole reactions were removed from the thermocycler every 2 cycles.

The primers for CLIVA optimization studies are listed in Table S1 and for DXP pathway assembling are listed in Table S2. The design details for all the 16 constructed plasmids are listed in Table S3. The modules containing various DXP pathway genes (dxs, dxr, ispD, ispE, ispF, ispG, ispG, idi, ispA or iron-sulfur (Fe-S) biosynthesis pathway (Isc operon, Suf operon), Figure 1B) were amplified from the source plasmids constructed by placing those genes between T7 promoter and T7 terminator in pET-11a plasmid from Stratagene. The genomic DNA purified from MG1655 DE3 (ATCC) strain was used as original source for E. coli genes. The ADS from Artemisia annua was codon optimized for bacteria expression (Figure S3). All the genes inside each module have their own ribosome binging sites (RBS). The PAC vector was amplified from pAC-Lyc plasmid from previous study [32 (link)]. The amplified DNA fragments were purified and treated with 20 U DpnI at 37 °C for one hour. After that, 100 mM Tris-HCL at pH 9, 0.3% (v/v) iodine and 10% (v/v) ethanol were supplied to the reactions and the mixtures were heated at 70 °C for 5 min. If the mixture turned out to be colorless, additional 0.3% (v/v) iodine and 10% (v/v) ethanol would be supplied and the mixture would be heated at 70 °C for another 5 min. The DNA fragments treated with iodine and ethanol were then purified by ethanol precipitation. For CLIVA optimization experiments, 0.15 pmol of every pieces together with different kinds and concentrations of salts were heated at 80 °C for 1 min, cooled down to the temperature which was 3 degree lower than the melting temperature of the overlapped sequences, kept for 10 min and then cooled down to 20 °C at 0.1 °C/s. 0.5 µl of the assembling mixture was mixed with 50 µl of XL10-Gold competent cell for electroporation. For DXP pathway assembling experiments, all the DNA fragments were prepared at 0.25 µM and equal amount of every pieces were mixed with MgCl₂ at 2.5 mM. The mixture were heated at 80 °C for 1 min, cooled down to 68 °C, kept for 10min and then cooled down to 20 °C at 0.1 °C/s. 0.5 µl of the assembling mixture was mixed with 50 µl of XL10-Gold competent cell for electroporation.

The gold standard CNVs utilized in this study were a subset of those CNVs released by the 1000 Genomes Project from 8-fold coverage Illumina paired-end population-scale sequencing data (available at 1000genomes.org) and analysis of the genomes of 2,504 individuals [4 ]. CNV discovery was conducted using the following tools: Delly, VariationHunter, BreakDancer, CNVnator, GenomeStrip, Pindel, and SSF. CNVs were merged by taking into account the confidence intervals around estimated boundaries. The merged set was genotyped across the entire population by GenomeStrip and filtered to remove redundancy and low quality sites based on genotype information. Genotypes were then updated though the integrated imputation of all variants (CNVs, SNPs, indels, etc.) with the MNV tool. The resulting genotypes were estimated to have a very low 3.1% false positive rate. Array-based experimental validation of CNV calls and PCR-based validation of CNV breakpoints were carried out by different contributors to the 1000 Genomes Project. CNVs genotyped as existing in NA12878 and not within 1000 Genomes Project described regions of VDJ recombination were selected to comprise our NA12878 gold standard CNV call set [Additional file 5: Spreadsheet 1].
A gold standard set of CNVs was similarly generated for the genome of NA10851, the aCGH control genome used in this study. 876 CNVs are common to the genomes of both NA12878 and NA10851 and are therefore are not expected to be detectable by aCGH platforms in this study.
Additionally, we generated an unfiltered CNV call set for NA12878 using1000 Genome Project deep sequencing data (2×250 bp paired-end sequencing to 60× coverage on Illumina HiSeq available at ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/phase3/data/NA12878/high_coverage_alignment) and read-depth analysis by the CNVnator algorithm [14 (link)]. This set contained 4293 total CNV calls and 4047 autosomal CNV calls [Additional file 5: Spreadsheet 1]. Of these CNVnator calls, 609 total and 587 autosomal calls overlapped a gold standard call by 50% reciprocally. A further 137 autosomal CNVnator calls overlapped a gold standard call by less than 50% reciprocally.

All glassware used in the synthesis of gold nanosphere particles was washed thoroughly with deionized water followed by ultrapure water before use. Citrate-capped spherical gold nanoparticles (AuNPs) were synthesized following a method previously described (27 (link)). Briefly, 50 mL of 0.02% gold (III) chloride solution (Sigma, USA) was boiled in distilled water and 1.2 mL of 1% sodium citrate solution (Merck, USA) was added immediately with constant stirring. A color change from grey to blue and purple-violet occurred within 50 - 60 seconds. After the color change, the heat was turned off, the solution was stirred for 2 - 3 minutes, and allowed to cool at room temperature. The final colloidal solution suspension was characterized by ultraviolet-visible spectroscopy (UV-Vis) by scanning between 400 and 800 nm. The batch having λ-max between 525 and 535 nm was used to prepare antibody conjugate. Enhanced colloidal stability was measured using NaCl solution.The gold particles were stable in normal water solution but started aggregating in different concentration of NaCl (125mM, 250mM, 500mM and 1M). (Supplementary Material Figure S1).

The anti-spike mAb (SpMA-01) of IgG1 isotype was selected for conjugation with a colloidal gold nanoparticle. The conjugation was optimized as described (28 (link)). Briefly, 6 to 10 µL of 1% K₂CO₃ (to adjust the pH to 6.5) was added to 1ml of colloidal gold solution in a glass tube, followed by 5 to 6 µg of anti-S SpMA-01 mAb and vortexed. The mAb-colloidal gold solution was incubated for 2 to 5 minutes at room temperature, followed by the addition of 20% BSA (final concentration approximately 0.1%), and the contents were transferred to an Eppendorf tube and centrifuged for 5 minutes in an Eppendorf centrifuge at 5000 RPM. The supernatant was removed, the pellet dissolved with 50 µL gold conjugation buffer.

SEQ-9-bound A2296-methylated Mtb 70S was prepared by incubating 800 nM methylated 70S with 16 μM SEQ-9 on ice for 30 min. 3 μl of the sample was applied to a glow-discharged Quantifoil R2/2 holey carbon grid (300 mesh), and vitrified using a Vitrobot Mark III (FEI company, The Netherlands) at 22°C with 100% relative humidity. Cryo-EM data were collected on a Titan Krios electron microscope (Thermo Fisher, USA) operating at 300 kV, at a nominal magnification of 130,000, which yields a pixel size of 1.06 Å/pixel. Image stacks were recorded on a Gatan K2 Summit (Gatan, Pleasanton CA, USA) direct detection camera in the electron counting mode. A total exposure time of 8.0 s with 0.2 s intervals and dose rate of ∼6.0 electrons/ Ų/s were used, resulting in 40 frames per image stack and accumulated total dose of ∼48 electrons per Ų.
Drift correction of collected image stacks was done by MotionCor2 with dose weighting.⁴⁴ (link) Aligned and summed image stacks were subjected to CTF estimation using gCTF.⁴⁵ (link) Images showing ice contamination and low resolution according to the estimation from gCTF were discarded, resulting in a total of 6,481 selected micrographs. Particle picking and reference-free 2D class average were done by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and RELION-3.0,⁴⁹ (link) respectively. A total of 1,065,544 clean particles were selected and refined into one consensus map. Then 3D classification was used with the option ‘--skip_align’ to classify different states of ribosomes, and each state was processed according to the pipeline of RELION-3.0. The overall resolution was estimated according to the gold-standard Fourier shell correlation.⁵⁰ (link)
SEQ-9-bound unmethylated Mtb 70S was prepared by incubating 700 nM unmethylated 70S with 16 μM SEQ-9 on ice for 30 min. 3 μl of the sample was applied to a glow-discharged Quantifoil R2/1 holey carbon grid (300 mesh) with 2nm carbon film, and vitrified using a Vitrobot Mark III (FEI company, The Netherlands) at 22°C with 100% relative humidity. Cryo-EM data were collected under a Titan Krios electron microscope (Thermo Fisher, USA) operating at 300 kV, at a nominal magnification of 165,000, which yields a pixel size of 0.83 Å/pixel. Image stacks were recorded on a Gatan K2 Summit (Gatan, Pleasanton CA, USA) direct detection camera in the electron counting mode. A total exposure time of 7.0 s with 0.2 s intervals and dose rate of ∼6.0 electrons/ Ų/s was used, resulting in 35 frames per image stack and accumulated total dose of ∼42 electrons per Ų.
Drift correction of collected image stacks was done by MotionCor2 with dose weighting.⁴⁴ (link) Aligned and summed image stacks were subjected to CTF estimation using Cryosparc Patch CTF estimation function.³⁹ (link) Images showing ice contamination and low resolution according to the estimation from Patch CTF were discarded, resulting in a total of 11,621 selected micrographs. Particle picking and reference-free 2D class average were done by Cryosparc.³⁹ (link) A total of 318,826 Mtb 70S particles were selected and refined into one consensus unmethylated Mtb 70S ribosome map at 2.8 Å (Figure S3A). To improve the resolution of the drug-binding site, we combined another 400,424 Mtb 50S particles in the same dataset from the same purification, yielding a total number of 719,250 particles of 70S and 50S. The drug-binding site was far away from the ribosomal subunit interface and was conserved in both the 70S and 50S, justifying the strategy to combine the 70S and 50S data to improve the resolution of the drug-binding site (Figure S3B). By applying a mask around the 50S subunit of the data mixing 70S and 50S, we obtained an unmethylated Mtb 50S ribosome map at 2.6 Å. The overall resolutions were estimated according to the gold-standard Fourier shell correlation (Relion 0.143 criterion).⁵⁰ (link) Final Fourier Shell Correlation plots for the density maps were generated using MATLAB R2021a.