Golden Gate assembly with CAM insert and pSEVA backbone

Purpose:

To create our pSEVA3411 plasmid, which will be the backbone for the final plasmid coding our system

Procedure:

1. We performed golden gate assembly using BsaI-HFv2 and T4 ligase to combine our CAM insert and our pSEVA backbone.
2. Remember that these steps need to be detailed enough that someone can reproduce our work

Results:

• Final concentration: 23.15 ng/µL
• pSEVA3411 plasmid backbone is available on the iGEM parts registry as BBa_K4215000

Golden Gate Assembly with Linkers and Binders

Purpose:

To create our “A” and “C” cassettes, which functioned as the binder+linker portion of our complex.

Procedure:

1. We ran a golden gate reaction with all binders and all linkers using BBSI. We then ran PCR to amplify final product with primers iGEM_GG_FWD/REV

Results:

• Very low concentrations were achieved, so a repeat of the Golden Gate assembly was warranted.

Pictures:

Golden Gate Assembly with Complementation systems and Ribo J

Purpose:

To create our “B” cassette, which functioned as the complementing portion of our complex.

Procedure:

1. We ran a golden gate reaction with comp system I, Ribo_complex , comp system II using BBSI. Then we ran PCR to amplify primer sequences in both ends.

Results:

• After running the products on a gel, the lengths did not correspond to the expected length of a fully assembled complex. At best, we only got partial assembly, of just the RiboJ and one half of the complementation system.

Pictures:

HRP, 100kb ladder, Rluciferase, Beta lactamase, TEV protease(left to right). Highest bands all represent lengths that correspond to one fragment of the complementation system. This gel helped us to recognize that an error was made in the initial design of the riboJ complex in which the 5’ BbsI cut site left an incompatible overhang, which prevented the assembly from joining with the N-terminal split proteins.

Golden Gate Assembly with 3411 and D-promoter

Purpose:

To assemble the backbone of our final cassette, in which the D-promoter is crucial.

Procedure:

1. We performed golden gate assembly using BbsI-HFv2 and T4 ligase to combine our 3411 plasmid with our D-Promoter

Pictures:

Adapted from iGEM Distribution Handbook

Materials

1. iGEM Distribution kit

Procedure

1. With a pipette tip, punch a hole through the foil cover into the well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross-contamination between the wells.
2. Pipette 10 µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1 µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic, and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic); grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock. We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Materials

1. 1 or 2 1 L bottles with caps
2. 1 or 2 magnetic stir bars
3. Autoclave tape
4. 25 g LB broth powder
5. 15 g Agar powder (omit if making liquid media)
6. Appropriate concentrations of antibiotic(s)
7. Autoclave safe tray (if using 1 bottle)

Procedure

1. Weigh 25 g of LB Broth in weigh boat
• Folded weigh boat can be used to funnel broth powder into bottle
2. Slowly pour broth powder into bottle
3. Fill bottle to 1000 mL mark with DI water & shake to combine
4. If using two bottles, pour half of the broth solution into each and add half of the agar and antibiotic to each in steps 5 and 10
• If only one bottle is used, the mixture will boil over in the autoclave
5. Add stir bar & 15 g agar powder
6. Stir until no dry clumps are present
7. Loosely screw on cap & secure lid with autoclave tape
• Autoclave tape will show black stripes after heating
8. Autoclave on cycle 3 (liquid) until complete (≈1 hour)
9. While the solution is being autoclaved, prepare the petri dishes
• Arrange dishes on lab bench with the tops (larger half) covering about 1⁄3 of the plate with the rest resting on the bench
• Be careful when opening the petri dish package, you will put your completed plates in there
10. When mixture reaches 55–60°C, pipette 100 μL (green box) of antibiotic into bottle
• Liquid media is complete at this point, and can be stored at ambient temperature (covered) or in walk-in fridge
11. Return antibiotic to −80°C freezer
12. Stir to combine on magnetic hotplate
13. Pour solution into petri dishes, each plate should be about 1⁄3 full of media
• A good rule of thumb is to pour enough solution into the plate so that the bottom is 3⁄4 covered, then swirl the plate
• Rotating the bottle while pouring minimizes dripping
• Clean floor spills as soon as possible, any solution spilled on the lab bench will be easier to clean after it has hardened
14. Once the plates have cooled (they will change color to a light yellow), put the lids on and stack the completed plates, upside-down in their original packaging
15. Label the package with the name(s) of whoever poured them, iGEM, the date, as well as the concentration and identity of antibiotic (“100 μg mL⁻¹ carb”)

Materials

1. Suspended DNA
2. Cells to be transformed
3. Cuvette
4. Transformation media
5. Glass beads

Procedure

1. Combine 1 µL of your resuspended DNA with 25 µL of your cells and place on ice
2. LABEL TUBE
3. Mix gently and transfer to cuvette (between metal plates), place in cuvette holder and hit “pulse”
4. Immediately add transformation media (SOB) to the cuvette and transfer recovered cells to a test tube
5. Leave to shake in the shaking incubator for 45 minutes
6. Remove test tubes from incubator and rest on a tube rack
7. Label plates to correspond to the tubes
8. Transfer 100 µL of your transformation to its respective plate, pour 10–15 glass beads onto the plate, close the lid, and shake the plate to cover all the afar with the cells (not the lid, just side to side shaking)
9. Pour beads into a used beads bin
10. Replace lid on plate and place in the incubator upside-down (agar-side up)
11. Leave plates in the incubator for 12–16 hours
12. Bleach tubes after plating cells

Materials

1. Transformed E. coli colonies
2. Cellular growth medium

Procedure

1. Circle colony and label it with a number/letter
2. Transfer 5 ml of media into a test tube
3. Use a pipette tip to very gently tap on selected colony
4. Eject pipette tip into test tube
5. Re-parafilm plate
6. Place tubes in 37°C shaking incubator for 16 hours

Adapted from NEB Protocol

Materials

1. 4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2
2. Culture to be miniprepped

Procedure

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2–5 minutes at 16,000 RCF.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
10. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Materials

1. 2.5 μL Primer 1 (10 μM concentration)
2. 2.5 μL Primer 2 (10 μM concentration)
3. 25 μL Q5 2X Mastermix
4. 0.5 μL pSEVA1411 plasmid template (1 ng/μL concentration)
5. 19.5 μL nuclease free Water
6. 1 μL Dpn1

Procedure

Do the following in 2 PCR tubes, 50 μL each, 100 μL total

1. Make sure to dilute primers appropriately
2. Mix gently by pipetting up and down, then spin to ensure a homogeneous mixture
3. Place in thermocycler and run PCR cycling
1. 98°C for 1 min
2. 98°C for 10 sec
3. __°C for 20 sec (determine using NEB Tm calculator)
4. 72°C for __ sec (depends on length, 10-15sec/kb for simple template like plasmid, for very long amplicons >6kbp use 40– 50 seconds/kb)
5. Go to step 2, 30X
6. 72°C for 5 min
7. 12°C forever
4. Add 0.5 µL DpnI to each reaction
5. Incubate at 37°C for 1 hour
6. Column clean with Monarch PCR clean up kit, follow protocol
7. Elute 15 μL
8. Record Nanodrop concentration, 260/280 ratio, 260/230 ratio from nanophotometer

Adapted from kit insert of New England Biolabs #T1030, all centrifuging should be carried out at 16,000 RCF

Materials

1. 50 μL DNA sample from PCR
2. 100 or 250 μL DNA Cleanup Binding Buffer with 0.36 volumes isopropanol added
3. 200 μL DNA Wash Buffer with 4 volumes of 95%+ ethanol added

Procedure

1. Dilute sample with correct volume of DNA Cleanup Binding Buffer
• The volume of DNA Cleanup Binding Buffer needed depends on the size of your target DNA. Use 100 μL for samples smaller than 2kb (Chloramphenicol & Kanamycin resistance), and 250 μL for samples larger than 2kb (pSEVA & PEV backbones)
2. Mix by flicking the tube or pipetting do not vortex
3. Insert column into collection tube and load sample onto column, spin for 1 minute, then discard flow-through
4. Re-insert column into collection tube, add 200 μL DNA Wash Buffer, and spin for 1 minute
5. Repeat previous step, discarding flow through if necessary
6. Transfer column to a clean 1.5 ml microcentrifuge tube, if the tip of the column contacts the flow-through, re-spin for 1 minute
7. Add DNA Elution Buffer to the center of the matrix, wait 1 minute and spin for 1 minute
8. Store at -20°C for later use

Materials

1. PCR samples
2. 240 mg agarose
3. 30 mL TAEr
4. 3 μL SYBR Safe
5. 5 μL dye for each ladder and sample

Procedure

1. Combine 300 mg agarose and 30 mL TAE
2. Microwave solution in 20 seconds increments until agarose is fully dissolved, making sure to avoid boiling (a lower intensity setting can help with this)
3. Add 3 μL SYBR Safe into solution
4. Insert comb into tray
5. Cast gel into tray, gel should be less than 1 cm thick
6. Cover gel with tinfoil, wait 10–15 minutes to harden
7. Add 5 μL of dye to each 5 μL DNA sample
8. Prep ladders (1 kb and 100 bp) (4 μL H20, 1 μL ladder, 5 μL dye)
9. Transfer the full 10 μL volume of each dyed dna sample into a lane (if possible, avoid using the edge lanes and adjacent lanes)
10. Run gels at 100V
11. Weigh eppendorf tube and write empty mass on tube
12. Use razor blade to precisely cut out the band of the sample fragment
13. Store in marked eppendorf tube, & weigh again. Record full mass on tube as well.
14. Store in -20℃ overnight

Adapted from New England Biolabs kit #T1020L insert, all centrifuging should be carried out at 16,000 RCF

Materials

1. Extracted agarose gel containing DNA sample in a 1.5 ml microcentrifuge tube
2. 4 Times the agarose gel’s volume of Gel Dissolving Buffer
3. 200 μL DNA Wash buffer diluted with 4 times its volume of 95%+ ethanol
4. 6+ μL DNA Elution Buffer

Procedure

1. Add 4 volumes Gel Dissolving Buffer to the gel slice in microcentrifuge tube
2. Incubate sample sample between 37–55℃ (typically 50℃), vortexing periodically until the gel slice is completely dissolved (no visible pieces), about 5–10 minutes
3. Insert column into collection tube and load sample onto column
4. Spin for 1 minute, discard flow-through
5. Re-insert column into collection tube, add 200 μL DNA Wash Buffer, and spin for 1 minute
6. Repeat previous step, discarding flow through if necessary
7. Transfer column to a clean 1.5 ml microcentrifuge tube, if the tip of the column contacts the flow-through, re-spin for 1 minute
8. Add DNA Elution Buffer to the center of the matrix, wait 1 minute and spin for 1 minute
9. Store at -20℃ for later use

Use online calculator to determine total pmol and pmol/µL

Materials

1. 0.06 pmol plasmid backbone (calculate required volume with pmol/µL from above link)
2. 0.12 pmol insert (calculate required volume with pmol/µL from above link). If using multiple inserts, make sure the combined amount is 0.12 pmol
3. 2.5 µL 10X T4 DNA Ligase Buffer
4. 0.25 µL T4 DNA Ligase (NEB #M0202), 2000 U/µL
5. 0.75 µL BbsI-HF (Neb #R3539)
6. to 25 µL nuclease free water

Procedure

1. Combine all components in PCR tube, mixing gently by pipetting up and down after each addition
2. Program the PCR Cycle
1. 37°C for 5 min
2. 16°C for 5 min
3. go to step 1, 30X
4. 65°C for 20 min
5. 12°C forever
3. Column clean and elute to 13 µL
4. Determine concentration using Qubit fluorometer and record final concentration in ng/µL

Calculate using link to determine total pmol and pmol/µL

Materials

1. 0.06 pmol plasmid backbone (calculate required volume with pmol/µL from above link)
2. 0.12 pmol insert (calculate required volume with pmol/µL from above link)
3. 2.5 µL 10X T4 DNA Ligase Buffer
4. 0.25 µL T4 DNA Ligase (NEB #M0202), 2000 U/µL
5. 0.75 µL BsaI-HFv2 (Neb #R3733)
6. To 25 µL nuclease free water

Procedure

1. Combine all components in PCR tube on ice (enzyme should be added last), mixing gently by pipetting up and down after each addition
2. Microfuge briefly to combine
3. Program the PCR Cycle
1. 37°C for 5 min
2. 16°C for 5 min
3. go to step 1, 30X
4. 80°C for 20 min
5. 12°C forever
4. PCR sample
5. Column clean and elute to 13 µL
6. Determine concentration using Qubit fluorometer and record final concentration in ng/µL

In-Fusion assembly works best with ~50ng of insert and ~200ng of vector. Determine the required volumes of backbone and insert to get a 2:1 molar ratio of backbone to insert with those approximate final masses of DNA. This procedure in adapted from the manufacturer instructions.

Materials

1. 2 μl 5X In-Fusion HD Enzyme Premix
2. ≈200 ng backbone plasmid
3. ≈50 ng vector
4. To 10 μl nuclease free water

Procedure

1. Mix the reaction by pipetting up and down
2. Incubate for 15 minutes at 50°C
3. Place on ice
4. Store at -20°C for future use

Adapted from IDT protocol

Materials

1. gBlocks to resuspend (DNA fragment in 25 µL of nuclease-free water at 10 ng/µL)

Procedure

1. Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds
2. Add nuclease free water to 10 ng/µL
3. Vortex briefly
4. Incubate at 50°C for 15–20 min
5. Briefly vortex and centrifuge
6. Use suspended gBlock fragments for BbsI cassette assembly

Materials

1. Suspended gBlocks

Procedure

Cassettes A&C

Do the following separately for each biomarker, i.e. all GFAP binders together, but UCHL1 and GFAP binders separately

1. Perform Bbs1 golden gate protocol with all binders and linkers
2. Label all reactions appropriately
3. Run PCR to amplify final product with appropriate primers
4. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

Cassette B

Separate these reactions by complementation system, only paired complementation systems will interact with each other (n-term GFP only interacts with c-term GFP, etc.). No need to separate by biomarker, cassette B will contain both parts of a complementation system and the riboJ-RBS complex, so it’s binder-agnostic.

1. Depending on how much RiboJ+RBS complex is needed, it may need to be amplified using appropriate primers
2. Perform Bbs1 golden gate protocol with comp system I, Ribo_complex, and comp system II
3. Label all reactions appropriately
4. Run PCR to amplify final product with appropriate primers
5. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

Cassette D (backbone)

pSEVA3411 already contains chloramphenicol resistance, this step only adds a single promoter g block (and a BsaI site) to turn it into Cassette D.

1. Perform Bbs1 golden gate protocol with pSEVA3411 and iGEM_promoter-RBS gBlock
2. Label all reactions appropriately
3. Run PCR to amplify final product with appropriate primers
4. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

Combining Cassettes A, B, C and D

1. Perform BsaI golden gate assembly process with all 4 cassettes
2. Label appropriately
3. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL
4. Store plasmids at -20°C for later transformation

Requirements

1. Working PyMOL installation
2. .pdb files
3. InterfaceResidues.py script

Procedure

1. File → open all .pdb files
2. On the highest ranked file, click A → align → all to this
3. Record executive RMSD values
4. Hide all structures other than rank 1
5. Run the InterfaceResidues.py python script
6. Run: interfaceResidues <object-name>, chain A, chain B in the PyMOL console
7. After step 7, the interface residues should be already selected. Then run these two lines to have each chain as a separated object:

   select mol1, chain A
   select mol2, chain B

8. Color it with this style: bg_color white
9. For rank 1 object: color → gray90
10. For interface object:

    show → sticks 
    color → spectrum → b factor

11. You can get the number of residues in the interface with this command: count_atoms byca <interface-object>
12. Look at the sequence (set seq_view, 1)
• [if the color of residues is not quite visible, you can change the color by set seq_view_color, <your-color>]
13. Color the first 10 residues (N-terminal) of each “chain” with “marine blue” and the last 10 residues (C-terminal) with “deep salmon red.”
14. Click on your interface selection and then show hydrogen bonds by A → find → polar contacts → between chains
15. Count the number of hydrogen bonds manually

AlphaFold is heavily biased by examples of natural binding and folding, so its predictions will probably not be accurate for non-native binding (such as nanobodies) or for point mutations. For these applications, (py)Rosetta or docking would be a better choice.

Requirements

1. Working Google account
2. Computer with sleep settings disabled

Procedure

1. Open this colab notebook while logged into your google account, it should be named "AlphaFold2_advanced.ipynb"
2. Make sure you're on your personal account, then click "copy to drive"
3. Complete the following steps on the copied version in your drive (the "copy to drive" option will not be visible)
4. Press the "Play" button at the left of the first cell, this will install required software on the colab GPUs<. This should take around a minute to complete.
5. When the software is installed and the green progress bar at the bottom is full, proceed to the next cell
6. Enter your desired amino acid sequence in the field labelled "sequence"
7. If you're interested in the interactions between multiple sequences (such as a biomarker and a binder), enter both sequences in this field separated by :
8. Enter a meaningful name in the "jobname" field
9. Enter the homo-oligomeric assembly state in the "homooligomer" cell. This will very depending on your input in the "sequence" field.
• If you are folding a single protein, leave the value as 1
• If you are simulating the binding of two monomeric proteins, set the value to 1:1
• If you are simulating the binding of a dimeric binder and a monomeric biomarker (such as s1000β dimer and GFAP), set the value to 1:2. The position of these numbers depends on how the original sequences were input, this example assumes that the input was <GFAP Sequence>:<S100β Sequence> and tells AlphaFold to calculate the interaction between one GFAP and one s100β dimer.
10. Run the "Search against genetic databases" cell by clicking the play button and leaving the default settings
11. Skip the "filter options", and run the "run AlphaFold" cell with default settings
• The free tier of google colab will discard your data if your computer goes to sleep or the browser tab is closed, which is why your sleep settings should be disabled
12. As the AlphaFold cell runs, it will generate five images with different configurations. The colored picture on the right is colored by pLDDT, the more blue a chain is the more confident AlphaFold is in its prediciton.
13. When the cell completes, skip all the optional cells and jump to "Download predictions" at the bottom
14. This will download a .zip file containing several files
• msa.pickle & msa_coverage.png : These are the results of the MSA (multiple sequence alignment) from the "Search against genetic databases" cell. You can upload these to that cell during future runs to avoid calculating it from scratch again, but it often isn't worth it since the calculation is so quick.
• settings.txt These are the settings you chose during the run
• rank_#_model_#_ptm_seed_#_unrelaxed.pdb These are the files you want to analyze in pyMOL later. The most important number is the rank (first number), the higher it is the more confident AlphaFold is in that structure.
• rank_#_model_#_ptm_seed_#_unrelaxed.png These are the images that the AlphaFold cell produced while working. They're helpful for a quick overview, but the information is redundant with the .pdb files.
15. Upload the zip file to the computational team drive in the appropriate subfolder for archiving

This is only technically necessary for HDOCK, ZDOCK is smart enough to avoid confusing chains for simple settings. For consistency, we recommend keeping as many variables constant as possible, which means adjusting the chain numbers for all docking software, not just those for which it’s absolutely necessary. Note that chain [#] refers to a chain identifier, which is usually a letter, not a number.

Requirements

1. Working pyMOL installation
2. .pdb file of the receptor protein or PDB ID
3. .pdb file of the ligand protein or PDB ID

Procedure

1. Enable sequence view by executing set seq_view, 1 in the pyMOL console
2. Load the receptor and ligand into pyMOL by opening the .pdb files or getting the structures from PDB by executing fetch [PDB ID] in the console
3. Examine the structures to determine which chains you’re interested in in each object
4. Extract receptor and ligand structures as pyMOL objects lig and rec
1. extract rec, chain [#] and [RECEPTOR_OBJECT]
2. extract lig, chain [#] and [LIGAND_OBJECT]
3. delete [RECEPTOR_OBJECT]
4. delete [LIGAND_OBJECT]
5. To dock dimers (receptor or ligand) in HDOCK or ZDOCK, the dimer must be a single chain, it doesn’t matter if the chains aren’t connected via amino acids
6. The above steps still work for dimer chain(s), but you need to select all the chains associated with the dimer, i.e. extract rec, chain A and chain B and [RECEPTOR_OBJECT]
5. To avoid chain identifier collisions when docking, change the chain identifier of the ligand to B by executing alter lig, chain = ‘B’
6. To avoid residue number collisions when docking, change the ligand residue numbering to start after the last residue of the receptor
• You can do this manually by looking at the sequence in pyMOL’s viewer, but to be sure you can have pymol tell you the residue numbers present in your object by executing iterate [OBJECT_NAME], resi. The highest value will be the end of the chain.
• Set the starting residue number of the ligand (chain B), to be one greater than the highest residue number of the receptor by executing alter lig, resi = int(resi) + [#] , where [#] is one greater than the highest residue number of the receptor
7. Save the receptor object as a .pdb file by executingsave rec.pdb, rec
8. Save the receptor object as a .pdb file by executingsave lig.pdb, lig

HDOCK and ZDOCK work very similarly (for example, they both perform energy minimization), but they use different processes “behind the scenes”. If the outputs of both systems look very similar, that means the protein is more likely to work that way in vitro and the reverse is also true.

Requirements

1. Working email address
2. .pdb files of the proteins you want to dock or PDB ID and chain number

Procedure

1. Navigate to the HDOCK server website
2. Upload your AlphaFold .pdb file of the receptor (biomarker). If the receptor is a dimer, make sure to adjust the numbering before proceeding or HDOCK may not work properly.
3. If using a PDB identifier, remember to add the chain separated by a colon, i.e. 3kw5:A after the identifier so you only dock that specific part of the structure
4. Repeat the above steps for the ligand protein
5. Remember to exclude non-binding residues (such as the insides of a ligand) under “Advanced Options” to get the most accurate model possible
6. Enter a meaningful jobname and your email, and HDOCK will email you a link to your results in a few hours
7. The output from HDOCK is a .tar.gz file, all of the predictions in .pdb format are in there
8. Upload the output tarball to the computational drive, analyze the docking effectiveness with pyMOL, and compare them to the ZDOCK predictions

HDOCK and ZDOCK work very similarly (for example, they both perform energy minimization), but they use different processes “behind the scenes”. If the outputs of both systems look very similar, that means the protein is more likely to work that way in vitro and the reverse is also true. Unlike HDOCK, ZDOCK is only able to dock the entirety of PDB structures, and there is no option to specify docking with only a particular chain.

Requirements

1. Working email address
2. .pdb files of the proteins you want to dock or PDB ID

Procedure

1. Navigate to the ZDOCK server website
2. Upload your AlphaFold .pdb file of the receptor (biomarker). If the receptor is a dimer, make sure to adjust the numbering before proceeding or ZDOCK may not work properly.
3. Repeat the above steps for the ligand protein
4. Enter your email, and ZDOCK will email you a link to your results in a few hours
5. Remember to exclude non-binding residues (such as the insides of a ligand) in the next screen
6. The output from ZDOCK is a .zip file, all of the predictions in .pdb format are in there
7. Upload the output zip archive to the computational drive, analyze the docking effectiveness with pyMOL, and compare them to the HDOCK predictions