(A) Barcoded competition strategy to analyze the growth effects of all single codon variants of Hsp90 in a single bulk culture. Hsp82 is the stress-inducible gene that encodes for Hsp90 (B) …
Sequencing counts and selection coefficients for each individual amino acid change across amino acids 2–709 of Hsp90 in both replicates of standard conditions.
Average selection coefficient (excluding stops) at each position of Hsp90 in Standard replicate 1.
The blue dashed line indicates the line of best fit.
(A) The average initial reads measured per codon for each amino acid. (B) The difference in the selection coefficients between stops in the two standard replicates compared to the initial reads for …
(A) The average selection coefficient at each position in standard (30°C) conditions correlates with distance to ATP (R2 = 0.49). (B) The average selection coefficient at each position at 37°C does …
(A) Growth rate of yeast with normal and reduced expression of Hsp90 protein in standard and stress conditions based on individual growth curves. Growth rates are normalized to growth in standard …
Sequencing counts and selection coefficients for each individual amino acid change across amino acids 2–709 of Hsp90 in Nitrogen Depletion, Salt, Ethanol, Diamide and 37°C.
Mutations were categorized as beneficial (light blue shading), wild-type-like (white shading), intermediate (light pink shading) or deleterious (dark pink shading) based on the distribution of …
(A) Distribution of selection coefficients for the wild-type synonyms in each condition. The standard of deviation of wild-type synonyms in each condition relative to standard conditions (x) is …
(A) The average selection coefficient (s) at each position relative to standard conditions was mapped onto Hsp90 structure (Ali et al., 2006) (See Figure 3—source data 1). (B) Structural images …
Average selection coefficient (excluding stops) at each position of Hsp90 in each environmental condition relative to the average selection coefficient in standard conditions.
(A) The fraction of Hsp90 positions at interfaces that were categorized as environmentally responsive. (B) The average selection coefficient in each environment relative to standard at all the Hsp90 …
(A) Ts variants were identified that supported WT-like growth at 30°C, but were null-like at 37°C in bulk competitions. WT synonyms are shown in green and stops in red. The horizonal dashed line …
List of all temperature-sensitive mutants and associated selection coefficients.
Ts mutants were defined as variants with selection coefficients within the distribution of wild-type synonyms in standard conditions and that of stop codons at 37°C.
(A) Number of beneficial mutations identified in each condition based on selection coefficients more than two standard deviations greater than wild-type synonyms. Beneficial mutants at 37°C and in …
List of all beneficial mutants and associated selection coefficients at 37°C and in diamide.
Beneficial mutants were defined as variants with selection coefficients two standard deviations greater than wild-type synonyms.
(A) Selection coefficients of synonymous codon variants for four amino acid mutants with beneficial selection coefficients at 37°C show high correlation. (B) The same individual variants analyzed in …
Independent expectations were calculated as the probability of the stated number of mutations occurring at the same position by chance.
(A) Selection coefficients for all codon variants of synonymous mutations at each position of Hsp90 (black) at 37°C compared to the average selection coefficient of all mutations at each position …
(A) The distribution of selection coefficients of natural variants compared to all variants in each environmental condition (see Figure 6—source data 1). (B) Across all environments, the fraction of …
List of selection coefficients of all natural variants of Hsp90 in all environmental conditions.
Cells were grown for 12 hr in dextrose media at 30°C and then were transferred into the individual environmental conditions and grown for eight additional hours. Hsp90 levels were monitored by …
Euclidean clustering along rows is based on the Spearman’s rank correlation coefficients. See Figure 6—figure supplement 2—source data 1.
The Spearman’s rank correlation coefficients (r) and associated p-values between molecular features and selection coefficients for each environment.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Saccharomyces cerevisiae) | hsp82 | Saccharomyces Genome Database | SGD: S000006161 | Hsp90 chaperone |
Antibody | anti-Hsp90 α/β (Mouse monoclonal) | Cayman chemical; Cat# 10011439 | RRID:AB_10349777 | WB (1:3000) |
Recombinant DNA reagent | Barcoded Hsp90 plasmid library | This paper | See Materials and methods for library construction | |
Recombinant DNA reagent | p414ADH Δter plasmid | PMID: 23825969 | ||
Recombinant DNA reagent | p414GPD plasmid | PMID: 23825969 | ||
Commercial assay or kit | BCA Protein Assay Kit | Pierce | Cat #23227 | |
Commercial assay or kit | KAPA SYBR FAST qPCR Master Mix | Kapa Biosystems | KK4600 | |
Chemical compound, drug | Diamide | Sigma Aldrich | D3648 | |
Software, algorithm | Barcode – Hsp90 ORF assembly | This paper | https://github.com/JuliaFlynn/Barcode_ORF_assembly | Associates barcodes with open reading frame mutations from paired end sequencing data (Flynn, 2020a; copy archived at https://github.com/elifesciences-publications/Barcode_ORF_assembly) |
Software, algorithm | Tabulate Hsp90 counts | This paper | https://github.com/JuliaFlynn/Tabulate_counts | Counts Hsp90 alleles from raw fastq files and barcode_orf assembly file (Flynn, 2020b; copy archived at https://github.com/JuliaFlynn/Tabulate_counts) |
Software, algorithm | EmpiricIST | PMID: 30127529 | https://github.com/Matu2083/empiricIST | Estimates selection coefficients based on the MCMC approach |
Sequenced-based reagent | Sequencing primers | This paper | See Supplementary file 1 | |
Sequenced-based reagent | Site-directed mutagenesis primers | This paper | See Supplementary file 1 | |
Sequenced-based reagent | Library construction oligomers | This paper | Available upon request |
List of oligomers used in this study.