ModularNucleoid

Software for Generating Models of JCVI-Syn3A Nucleoids May 2023, David S. Goodsell, provided as is for unrestricted use.

ModularNucleoid is used to generate models of the minimal genome bacterium JCVI-Syn3A, producing a series of models that test hypotheses about transcription, condensation, and overall distribution of the genome.

Input Files

1. syn3Asmall_ribosomes.pdb: Centroids of ribosomes from cryoEM in nm, should be centered on the origin.
2. 4v6k_mask.pdb: Mask file for ribosome (lattice coordinates=3.4nm).
3. 6c6u_mask.pdb: Mask file for RNA polymerase.
4. 7nyx_mask.pdb: Mask file for SMC.
5. syn3A.genome: RNA polymerase positions and genomic information position of RNApol (bp), length of mRNA (bases), length of segment between pol and previous pol (bp), direction of transcription (1 or -1), transcribed (1) or noncoding (0), gene ID, common name of gene product

Compiling Source Code

gfortran generate_connect.f -o generate_connect
gfortran generate_pol.f subroutines.f -o generate_pol
gfortran modularlattice.f subroutines.f -o modularlattice
gfortran relax.f -o relax

Scripts

• syn3A.script: Includes the entire pipeline. The script includes a for loop for generating multiple instances of a particular model. In tests, the modularlattice step fails roughly half of the time due to crowding, so repeat with a different random seed.
• jmol.script: Includes a short script for Jmol for visualizing lattice models and relaxed models.

Pipeline

1. Generate connectivity information with generate_connect.f
   • Generates a ".connect" file used by ModularLattice.
   • This version is hardwired for a syn3A genome with 54300 beads, lattice units of 10 bp/bead.
   • Reads genome information from an input file with RNApol positions and nascent mRNA length.
   • SMC loops are placed between RNApol positions greater than a user defined minimum DNA segment length.
   • Mature mRNA is chosen randomly from nascent mRNA length, and one ribosome is placed at the center.
   • Input parameters:
     • syn3A.genome #genome file with RNApol positions and mRNA length (bp units)
     • syn3A.connect #output .connect file
     • 503,187,187 #number of ribosomes, RNApol, and SMC
     • 120 #minimum DNA length to add SMC (lattice units)

./generate_connect syn3A.genome syn3A.connect 503,187,187 120

2. Generate positions of RNA polymerase, ribosomes, and SMC with generate_pol.f
   • Reads ribosome positions and generates RNApol positions using a random walk.
   • SMC is placed between RNApol positions.
   • Hardwired for lattice units of 10bp/bead.
   • Input parameters:
     • syn3Asmall_ribosomes.pdb: Ribosome positions (nm, should be centered on origin)
     • 6c6u_mask.pdb: Mask file for RNApol
     • 4v6k_mask.pdb: Mask file for ribosome
     • 7nyx_mask.pdb: Mask file for SMC
     • syn3A.pdb: Output coordinate file (lattice units)
     • 187: Number of RNApol and SMC
     • 201.26: Radius of cell (nm)
     • 1234: Random seed
     • 20.: Max distance between successive RNApol
     • RANDOM: RANDOM or CIRCLE placement of RNApol

./generate_pol syn3Asmall_ribosomes.pdb 6c6u_mask.pdb 4v6k_mask.pdb 7nyx_mask.pdb syn3A.pdb 187 201.26 1234 20. RANDOM

Note: Uses a wasteful approach that repeats RNApol generation until a closed circular genome is obtained.

3. Generate lattice model of genome with modularlattice.f
   • Reads coordinates of RNApol, ribosomes and SMC from generate_pol.f, and DNA/RNA chain lengths and connectivity from generate_connect.f.
   • Creates lattice model of DNA, nascent/mature mRNA, and nascent protein.
   • Input parameters: syn3A.pdb, syn3A.connect, syn3A_lattice.pdb, 0, 1234, 60.
     • syn3A.pdb: input coordinate file with RNApol, ribosome, SMC templates
     • syn3A.connect: input file that defines DNA/RNA chain lengths and connections
     • syn3A_lattice.pdb: output lattice coordinates
     • 0: supercoiling number of DNA beads per turn
     • 1234: random seed
     • 60.: cell radius (lattice units)

./modularlattice syn3A.pdb syn3A.connect syn3A_lattice.pdb 0 1234 60.

Note: Largely hardwired for syn3A with simple RNApol, ribosomes, and SMC. In tests with syn3A models, fails ~half of the time due to crowding! Try again with a different random seed.

4. Optimize the model with relax.f
   • Relaxes a set of lattice coordinates using a simple force field.
   • Molecule positions and control points from generate_pol.f are held rigid.
   • Input parameters:
     • syn3A_lattice.pdb: input lattice coordinates
     • syn3A_relax.pdb: output relaxed coordinates
     • 1500: optimization steps

./relax syn3A_lattice.pdb syn3A_relax.pdb 1500