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This page shows three representative examples to run MDFF:

• 6c54A: a 3-domain protein using an experimental density map at resolution of 5.8Å. The initial model has a high quality with TM-score=0.90 and RMSD=3.0Å to the native structure. After the MDFF domain assembly and refinement, the model was significantly improved with TM-score increased to 0.97 and RMSD decreased to 1.4Å.
  
• 2piaA: a 2-domain protein using a synthesized density map at resolution of 4.3Å. The initial model obtains a high clash-score because all domain models were superposed to the same position by Situs. After the MDFF domain assembly and refinement, the Clashscore (defined by Molprobity) was significantly reduced and the model obtains better fit, but the model accuracy was reduced and the overall model geometry was compromised.
  
• 6nr9A: a 3-domain protein (2 discontinuous domain + 1 continuous domain) using an experimental density map at resolution of 8.5Å. The initial model also obtains a high clash-score, but it was significantly reduced after the MDFF domain assembly and refinement.
  

Explanations:

• Initial model---------------------the starting model created through Situs by superposing each domain model into the density map.
  
• Domain definition---------------the predicted domain definition, which should be assigned into the initial model before running the domain rigid-body assembly. In the domain definition,Domains are separated by semicolons, while segments of a discontinuous domain are separated by commas. For example, "1-104" in the domain definition of 2piaA means that residue 1 to residue 104 belong to the first domain. "1-100,496-521" in the domain definition of 6nr9A means that the first domain is a discontinuous domain, where residue 1 to residue 100 belong to the first segment, and residue 496 to residue 521 belong to the second segment.
  
• Cryo-EM density map-----------the segmented cryo-EM density map.
  
• Map resolution------------------the resolution of the cryo-EM density map.
  
• MDFF final model---------------the final model refined by MDFF.
  
• domain-step1_new.namd.log---the log file of the MDFF domain rigid-body assembly.
  
• adk-step1.namd.log-------------the log file of the first step of the MDFF flexible refinement using the original density map.
  
• adk-step2.namd.log-------------the log file of the second step of the MDFF flexible refinement using the original density map.
  
• adk-step3.namd.log-------------the log file of the third step of the MDFF flexible refinement using the original density map.
  The following log files are included in the folder for cases with resolution <5Å because cMDFF is employed:
  
• adk-step1.namd.logN-----------the log file of the first step of the MDFF flexible refinement using the N-th Gaussian blurred density map.
  
• adk-step2.namd.logN-----------the log file of the second step of the MDFF flexible refinement using the N-th Gaussian blurred density map.
  
• adk-step3.namd.logN-----------the log file of the third step of the MDFF flexible refinement using the N-th Gaussian blurred density map.
  Here, N=1,2,3,4,5, which corresponds to the half-width of 5, 4, 3, 2, 1, respectively.
  

1. 1. Generate the "*.psf", "*.dx", "*grid.pdb" and other files and assign the domain definition to the starting model using VMD based on the starting model and the cryo-EM density map (see MDFF tutorial )
   

2. 2. The following command was used to run the MDFF domain rigid-body assembly:
   
   mdff setup -o domain -psf xxx.psf
      -pdb xxx.pdb
      -griddx xxx.dx
      -gridpdb xxx-grid.pdb
      -extrab {xxx-extrabonds.txt xxx-extrabonds-cispt.txt xxx-extrabonds-chaira.txt}
      -gscale 1.0
      -minsteps 2000
      -numsteps 200000
   

3. 3. After the rigid-body assembly, the following command was employed to run the flexible refinement in the direct MDFF:
   
   mdff setup -o adk -psf xxx_autopsf.psf
      -pdb xxx_autopsf.pdb
      -griddx xxx_autopsf-grid.dx
      -gridpdb xxx_autopsf-grid.pdb
      -extrab {xxx-extrabonds.txt xxx-extrabonds-cispt.txt xxx-extrabonds-chaira.txt}
      -gscale 1.0
      -minsteps 2000
      -numsteps 500000
   mdff setup -o adk -psf xxx_autopsf.psf
      -pdb xxx_autopsf.pdb
      -griddx xxx_autopsf-grid.dx
      -gridpdb xxx_autopsf-grid.pdb
      -extrab {xxx-extrabonds.txt xxx-extrabonds-cispt.txt xxx-extrabonds-chaira.txt}
      -gscale 0.3
      -minsteps 2000
      -numsteps 500000
      -step 2
   mdff setup -o adk -psf xxx_autopsf.psf
      -pdb xxx_autopsf.pdb
      -griddx xxx_autopsf-grid.dx
      -gridpdb xxx_autopsf-grid.pdb
      -extrab {xxx-extrabonds.txt xxx-extrabonds-cispt.txt xxx-extrabonds-chaira.txt}
      -gscale 10
      -minsteps 2000
      -numsteps 0
      -step 3
   
   where "-numsteps" of the first two steps of the flexible fitting should set to 200000 in cMDFF (cases with map resolution < 5 Å).
   

1. Please remember to run the domain rigid-body assembly and flexible refinement in order using the above commands. Otherwise, you may fail to run the MD simulation of MDFF.
   
2. The domain definition should be assigned to the starting model "initial_model.pdb" before running the domain rigid-body assembly of MDFF. The method to assign the domain can be found in the MDFF tutorial.
   
3. Please read README.txt if you download the tarball file of each case.
   
   

• 6c54A:
  

  Input data
  Initial model	Domain definition: 1-107;108-201;202-347;	Cryo-EM density map	Map resolution: 5.8Å
  Output model
  MDFF final model
  MDFF log file
  Domain rigid-body assembly:	domain-step1_new.namd.log
  Flexible refinement using original map:	adk-step1.namd.log	adk-step2.namd.log	adk-step3.namd.log
  Click here to download all data of 6c54A

  
  
• 2piaA:
  

  Input data
  Initial model	Domain definition: 1-104;105-225;226-321;	Cryo-EM density map	Map resolution: 4.3Å
  Output model
  MDFF final model
  cMDFF log file
  Domain rigid-body assembly:	domain-step1_new.namd.log
  Flexible refinement using Gaussian blurred map 1:	adk-step1.namd.log1	adk-step2.namd.log1	adk-step3.namd.log1
  Flexible refinement using Gaussian blurred map 2:	adk-step1.namd.log2	adk-step2.namd.log2	adk-step3.namd.log2
  Flexible refinement using Gaussian blurred map 3:	adk-step1.namd.log3	adk-step2.namd.log3	adk-step3.namd.log3
  Flexible refinement using Gaussian blurred map 4:	adk-step1.namd.log4	adk-step2.namd.log4	adk-step3.namd.log4
  Flexible refinement using Gaussian blurred map 5:	adk-step1.namd.log5	adk-step2.namd.log5	adk-step3.namd.log5
  Flexible refinement using original map:	adk-step1.namd.log	adk-step2.namd.log	adk-step3.namd.log
  Click here to download all data of 2piaA

  
  

• 6nr9A:
  

  Input data
  Initial model	Domain definition: 1-100,496-521;101-135,394-495;136-393;	Cryo-EM density map	Map resolution: 8.5Å
  Output model
  MDFF final model
  MDFF log file
  Domain rigid-body assembly:	domain-step1_new.namd.log
  Flexible refinement using original map:	adk-step1.namd.log	adk-step2.namd.log	adk-step3.namd.log
  Click here to download all data of 6nr9A

  
  

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