Macromolecular complexes and interactions

Oligomers in crystals

RCSB PDBhttps://www.rcsb.org/pdb/home/home.do MakeMultimer script (Python2)../downloads/makemultimer_pymol.pym MakeMultimer script (Python3)../downloads/makemultimer_pymol_python3.pym 3D COMPLEXhttps://shmoo.weizmann.ac.il/elevy/3dcomplexV6/Home.cgi PiQSihttps://shmoo.weizmann.ac.il/elevy/piqsiV6/piqsi_home.cgi PISAhttp://www.pdbe.org/pisa

The crystal unit and crystallization artifacts

  • Display the prepared crystal unit Crystal _unit_4mdh.pdb:
    • show → as → cartoon
    • color → by chains
  • align monomers A and B and explore the effect of crystallization on the conformation of the surface loop composed of residues 93-99
    • display loops in distinct conformations differently
      • change Selecting Residues to Selecting Chains
      • select monomer A
      • action → copy to object
      • name new object „monomer A“
    • repeat similarly for monomer B
    • align monomer B to A: action → align → to molecule
  • How do the conformations of the studied loop differ and why?
  • use symmetry operators to obtain information about crystal environment of the structure of cytoplasmic malate dehydrogenase; PDB ID: 4MDH
    • action → generate → symmetry mates → within 12 Å
    • try to find the crystal unit in this part of the crystal

Assymetric and biological unit

  • Compare three PDB entries representing the structure of hemoglobin; PDB ID: 2HHB, 1HHO, and 1HV4
  • subsequently, download, and display their biological units from RSCB PDB
    • before loading the PDB structure, set the following setting:
      • set assembly, 1
    • after loading the PDB structure, show the whole biological unit:
      • movie → show all states
    • in which state are their assymetric and biological units?
    • Inspect the REMARK 350 fields of the PDB files. What do the rows and columns represent?
    • download and view the biological unit of the FMD virus capsid; PDB ID: 1QQP
      • show → as → cartoon
      • movie → show all states

    Obtaining and assessment of biological units

    • compare the structure of the biological unit of D-Xylose isomerase; PDB ID: 2GLK, obtained from RSCB PDB, and prepared by the MakeMultimer script MakeMultimer_pymol.pym or MakeMultimer_pymol_python3.pym
        • run makemultimer_pymol_python3.pym
        • make_multimer 2glk
      • show → as → cartoon
      • color → by chains
    • verify the correctness of the quarternary structure in databases
    • verify the correctness of the quarternary structure using the following tools
      • PISA
        • for PDB ID: 2GLK analyse the possible „assemblies“
        • save and display in PyMOL
        • for some browsers, PISA does not work properly. In that case, use Google Chrome or the following files:
        • assembly 1, assembly 2, assembly 3, assembly 4

    Nucleic acid-binding proteins

    DNA-binding proteins

    • explore which parts of the protein are interacting with DNA specifically, and whether nonspecific stabilization by charged amino acids is also present
      • explore the ZIF268 protein, PDB ID: 1A1F, utilizing the „zinc finger“ motif
      • explore the Cro protein, PDB ID: 6CRO, utilizing the „helix-turn-helix“ motif. The protein is a dimer. Therefore, prepare its structure using the MakeMultimer script, or download the structure of its biological unit and display it:
        • movie → show all states

    RNA-binding proteins

    • explore which parts of the protein are interacting with RNA specifically, and whether nonspecific stabilization by charged amino acids is also present
      • study the Fox-1 protein; PDB ID: 2ERR, utilizing the „RNA recognition motif“
      • study the heterogeneous nuclear ribonucleoprotein K protein; PDB ID: 1ZZI, utilizing the „K-homology domain“ motif
      • study the Pumilo1 protein; PDB ID: 1M8Y, utilizing the „Pumilio repeat domain“ motif
        • sele resn ARG+HIS+LYS

      Database of macromolecular interactions

      STRINGhttp://string-db.org

      STRING database

      • Based on the information from the database STRING, analyse the potential interactions of the protein fibroblast growth factor 2 (FGF2) from Homo sapiens
      • study the evidence for particular interaction and involvement of FGF2 to signal cascades:
        • Databases → KEGG

      Prediction of 3D structures of macromolecular complexes

      ClusProhttps://cluspro.bu.edu AlphaFold3https://alphafoldserver.com

      Macromolecular docking

      • perform macromolecular docking of molecules FGFR1 (receptor) and FGF2 (ligand) using ClusPro server
      • analyse the obtained complexes in PyMOL
      • compare the results with the experimental 3D structure of the FGF2-FGFR1 complex; PDB ID: 1FQ9 - which one of the obtained binding modes matches the experimental structure the most?

      Prediction using AlphaFold3

      • model the FGFR1 and FGF2 complex using AlphaFold3
      • compare the obtained complexes with the previous ones using PyMOL - which of the AlphaFold3 models matches the experimental structure the most? Is the orientation of the structure identical with the „most accurate“ mode (i.e. the mode most similar to the experimental structure) from the ClusPro server?

      Analyses of macromolecular complexes

      PDBsumhttp://www.ebi.ac.uk/pdbsum/

      Analysis of macromolecular interfaces – PDBsum database

      • study the schematic representation of the interface between FGF2; PDB ID: 1FQ9, chain A, a FGFR1; PDB ID: 1FQ9, chain C, in the PDBsum database
      • which type of interactions give rise to the binding of FGF2 to FGFR1?
      • how large is the interaction and how many residues form it?
      • are there any highly conserved residues on FGF2 (conservation degrees 8 and 9)? Mark the highly conserved residues and study them in PyMOL

      Interaction hot spots

      Robettahttp://robetta.bakerlab.org/alascansubmit.jsp

      In silico alanine scanning

      • conduct in silico alanine scanning of the residues forming the interface between FGF2; PDB ID: 1FQ9, chain A with FGFR1; PDB ID: 1FQ9, chain C, using the server Robetta
      • which residues from FGF2 were predicted as potential hot spots for the interaction with FGFR1 (mutations with ∆∆G ≥ 1kcal/mol)?
      • compare the predictions further with the previous residue lists.
        • Were some of them highly conserved?
        • Which residues with a large number of interactions were predicted as a hot spot?
      • Take all previous analyses together and guess which residues are the most probable hot spots
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