Advanced use of databases in the hybrid structural research: PDB - PowerPoint PPT Presentation

Kristina Djinovi ć -Carugo Advanced use of databases in the hybrid structural research: PDB Department of Structural and Computational Biology Max Perutz Labs University of Vienna Austria EMBO Global WS: Structural and biophysical methods for biological macromolecules in solution Santiago, 20 th October 2019

Structural databases • PDB • 156954 • SASDB • 1848 • EM • 4026

Statistics by method

PDB Growth of Released Structures Per Year

Pathway for generation of 3D structure

Pathway o Exp xperiments : Crystallisation, X-ray diffraction o Co Computatio ional : Structure determination, refinement, analysis

Why a crystal • X-ray scattering from a single molecule would be very weak and could not be detected above the noise level • A crystal arranges large numbers of molecules in the same orientation • Scattered waves can add up in phase and raise the signal to a measurable level • Crystal acts as an amplifier

Why crystal • The waves add up in phase in some directions and have to cancel out in other directions • X-rays diffracted from a crystal and detected on a flat 2D-detector

Image of molecule: electron density distribution • Electromagnetic radiation interacts with matter through its fluctuating electric field • The result of an X-ray crystallographic experiment is the distribution of electrons in the molecule

Image of molecule – electron density

MIR – trace an electron density map

Final result of structure determination (is none of what you see)

Final result: atomic 3D coordinates

Crystallographic terms

Reflection - Intensity • Intensities of diffracted beams are measured • Reflections, I – intensities ! " = |F o | - structure factor amplitudes •

Resolution • RES = ½ [ λ /sin( θ )] • Detail that can be resolved in electron density maps • C α -C β : 1.54 Å

Resolution of electron density map and consequently of the 3D model 1.0 Å 2.5 Å 3.0 Å 4.0 Å

Resolution of electron density map • 1 Å resolution individual atoms can be fitted

Resolution of electron density map • Alpha-helices are clear at 6 Å resolution, but beta-strands are not. • At lower resolutions than about 8 Å, only whole molecules can be placed.

RES in PDB

RES in PDB

R factor å - Fobs Fcalc h h = R h å Fobs h h • |F o | | - from measu sured diffraction intensity • Experimental data • |F |Fc| | - cal calcul culat ated ed from coordinates • Global measure of agreement between experiment and the model • Surface residues can be less recognizable

R free å - Fobs Fcalc h h = R h å Fobs h h • Calculated for 5-10% of reflections not used in refinement • How well the model agrees with the data that it has not been fit to

R/R free • For most structures refined at RES=2.5 Å, R is less than 0.2 and R free less than 0.25

R free in PDB

RES and R factor for ranking • Ranking of quality of structures # Q = $%& − R • Higher RES and lower R are associated with higher Q • RES(1) = 2.0; R(1) = 0.18; Q(1) = 0.32 • RES(1) = 1.5; R(1) = 0.16; Q(1) = 0.51

Thermal motion • B iso = isotropic thermal factor • B iso = 8 π 2 <u> 2 • u = mean amplitude of displacement from the mean position • B iso = 0 Å 2 at T = 0 K

Thermal motion B iso B aniso 3 coordinates + 1B 3 coordinates + 6B

Thermal motion

Atomic displacement parameters (B) • B main < B side • B absorb lattice defects, large scale movements, disorder

Atomic displacement parameters (B) • B abso sorb lattice defects, large scale movements, disorder • I nf nform orm on on funct unction on • Access to internal cavities, substrate channels • Correlation between thermal stability and thermal motions/flexibility

T of experiment • X-tal structures @ T = 100 K • NMR @ RT

T of experiment • 160K – 200K proteins undergo a phase transition • Conformational disorder goes from dynamic to static • Lower T reduces conformational distribution of sidechains: • à smaller and more packed and unique modes

Flowchart/Crystallographic terms Protein Solution Heavy Atom Derivative Crystals a, b, c, a , b , g , symmetry, |F o | h, k, l, |F P | h, k, l, |F PH | solvent content, # mol./a.u. a P Electron density r (x,y,z) h, k, l, |F calc |, a calc Structure (x i ,y i ,z i ,B i ) R-factor Publication, …

Folds and new folds

Redundancy of PDB • 135787 Biological Macromolecular Structures

Why redundancy… • Cove ver a limited sp space of biological ma macro romo molecular r unive verse se • Same or similar proteins, e.g. • Lysozyme > 500 entries • Membrane proteins : 2-3% PDB entries • 15% - 35% of human proteome • Intrinsically disordered proteins…

Non redundant PDB subsets • PDBse select : reject proteins with aa sequence identity > threshold • http://bioinfo.tg.fh-giessen.de/pdbselect/ • PI PISC SCES ES : download precompiled datasets • dunbrack.fccc.edu/PISCES.php • “ Adva vanced se search utility ” in PDB • Ski kip-Re Redundant of EMBOSS • Cd Cd-hi hit • http://weizhongli-lab.org/cd-hit/

… but sequence is not all • Same sequence can adopt 2 different structures • e.g.: Calmodulin

… but sequence is not all • Same sequence can adopt 2 different structures • e.g.: Calmodulin-like domain from alpha-actinin-1 Drmota et al, Sci Rep, 2016)

CHECK FIGURES OF MERIT, STEREOCHEMISTRY

Missing residues • Interpretation of electron density ( ρ ) allows positioning of atoms • Sometimes ρ is elusive and thus positioning of atoms uncertain or impossible

Treatments of invisible residues/atoms • Omit the atoms/ s/aa resi sidues • Amino acid residues ‘torsos’ • … molecular graphics not always warns about missign atoms

Treatments of invisible residues/atoms • Leave ve the atom in the model with occupancy y 0 • No alert by molecular graphics

Example “Torso”

Treatments of invisible residues/atoms • Leave ve the atom in the model à larg rge B B factors rs • Easily visualized by molecular graphics

Occurrence of invisible residues/atoms • 20% of structures at atomic RES contain invisible residues • 80% of structures 1.5 Å RES contain 80% of invisible residues • At atomic RES 2-3% residues are invisible • At 2.0 Å RES 7% residues are invisible • At 3.0 Å RES 10% residues are invisible

Reasons for invisible residues/atoms • Proteolysis Ltd • Quality/quantity of diffraction data • Conformational disorder

…caveat • Invi visi sible resi sidues s are often on su surfaces • Caution if surface properties are investigated: • Electrostatic potential K43

…caveat • Invisible residues are often on surfaces • Caution if surface properties are investigated: • Electrostatic potential calculation is affected! “Torso”

Conformational disorder / Occupancy • Residues/atoms do not reside on the same position in all residue/atoms in all molecules in the crystal/ensemble • à weak electron density à invisible • At medium/high RES observe multiple conformations • Static disorder • Dynamic disorder

Static/dynamic disorder • St Static : two or more conformations exist • Dyn ynamic (at higher T): shuffling from one conformation to another • Crystal structure determination gives time and space averaged structural information • à cannot distinguish between static and dynamic disorder

Alternative conformations The su sum of occupancies s of both posi sitions s conformations s is s 1

Disorder continued… • Occupancy y of atoms/ s/resi sidues s is s < 1 • Ligand is not bound to a fraction of molecules in the crystal • Weak binding, suboptimal binding conditions • Misplaced ligand • Partial disorder • X-ray induced radiation damage • loss of carboxylates, methyl groups…

Partial disorder of the ligand • Weichenberger et al. • Volume 73 | Part 3 | March 2017 | Pages 211–222 | 10.1107/S205979831601620X

Radiation damage at work • Garman • Volume 66 | Part 4 | April 2010 | Pages 339–351 | 10.1107/S0907444910008656

…caveat • Molecular graphics shows all alternative conformations • Surface properties calculation with all conformations!

Stereochemistry • Is the protein stereo-chemically sound? • Covalent distances, angles, torsion angles, backbone conformation, group planarity, chirality, H-bonds, electrostatic interactions

Ramachandran plot

Ideal stereochemical parameters Peptide bond Average Single Bond Average Cα - C 1.53 (Å) C - C 1.54 (Å) C - N 1.33 (Å) C - N 1.48 (Å) N - Cα 1.46 (Å) C - O 1.43 (Å) Hydrogen Bond Average ( � 0.3) O-H --- O-H 2.8 (Å) N-H --- O=C 2.9 (Å) O-H --- O=C 2.8 (Å) Rms deviations from ideal geometry: bond length 0.01 - 0.02 Å bond angles: 1.2 - 1.5 deg

Tools to check stereochemistry • Procheck • What_Check • MolProbity • ProSA • PDB – validation protocol, which examines also fit of experimental data