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

Kristina Djinović-Carugo

Advanced use of databases in the hybrid structural research: PDB

Department of Structural and Computational Biology

Max F. Perutz Laboratories University of Vienna

Austria

EMBO Global WS: Structural and biophysical methods for biological macromolecules in solution Singapore, 12th December 2017

Structural databases

• PDB
• SASDB
• EM

Statistics by method

Pathway for generation

• f 3D structure

Pathway

• Experiments: Crystallisation, X-ray diffraction
• Computational: 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

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
• 𝐽

"

= |Fo| - structure factor amplitudes

Resolution

• RES = ½ [λ/sin(θ)]
• Detail that can be resolved in electron density

maps

• Cα-Cβ : 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

Rfactor

• |Fo| - from measured defecation intensity
• Experimental data
• |Fc| - calculated from coordinates
• Global measure of agreement between experiment and

the model

• Surface residues can be less recognizable

å å

• =

h h h h h

Fobs Fcalc Fobs R

Rfree

• 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

å å

• =

h h h h h

Fobs Fcalc Fobs R

R/Rfree

• For most structures refined at RES=2.5 Å, R is

less than 0.2 and Rfree less than 0.25

Rfree in PDB

RES and Rfactor for ranking

• Ranking of quality of structures
• Higher RES and lower R are associated with

higher Q

Q =

1 RES − R

Thermal motion

• B iso = isotropic thermal factor
• B iso = 8π2<u>2
• u = mean amplitude of displacement from the

mean position

• B iso = 0 Å2 at T = 0 K

Thermal motion

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

Thermal motion

Atomic displacement parameters (B)

• Bmain < Bside
• B absorb lattice defects, large scale

movements, disorder

Atomic displacement parameters (B)

• B absorb lattice defects, large scale movements,

disorder

• Inform on function
• 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 Publication, … Structure (xi,yi,zi,Bi) Electron density r(x,y,z) aP

a, b, c, a, b, g, symmetry, solvent content, # mol./a.u.

h, k, l, |FP|

Heavy Atom Derivative

Crystals h, k, l, |FPH| h, k, l, |Fcalc|, acalc |Fo|

R-factor

PDB statistics – depositions per year and cumulative growth

Folds

Redundancy of PDB

• 135787 Biological Macromolecular Structures
• 12.12.2017

Why redundancy…

• Cover a limited space of biological

macromolecular universe

• 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

• PDBselect: reject proteins with aa sequence

identity > threshold

• http://bioinfo.tg.fh-giessen.de/pdbselect/
• PISCES: download precompiled datasets
• dunbrack.fccc.edu/PISCES.php
• “Advanced search utility” in PDB
• Skip-Redundant of EMBOSS
• Cd-hit
• http://weizhongli-lab.org/cd-hit/

… but sequence is not all

• Same sequence can adopt 2 different structures
• e.g.: Calmodulin
• Procedure taking in account also topology
• Bioinformatics (2008), 24, 2632

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/aa residues
• Amino acid residues ‘torsos’
• … molecular graphics not always warns

Example

“Torso”

Treatments of invisible residues/atoms

• Leave the atom in the model à large B

factors

• Easily visualized by molecular graphics

Treatments of invisible residues/atoms

• Leave the atom in the model with occupancy 0
• No alert 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

• Invisible residues are often on 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

• Static: two or more conformations

exist

• Dynamic (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 sum of occupancies of both positions conformations is 1

Disorder continued…

• Occupancy of atoms/residues is < 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

PDB – validation report

10 20 30 40 50 60 70 80 90 100

15% of the PDB files of similar resolution are worse than this

• ne (and 85% are better).

35% of the PDB files than this one (and 65% are better).

WORSE BETTER

READ the REPORT

Re-refined structures

• Database of automatically re-refined structures

Carugo O, & Djinovic Carugo, K. Methods Mol Biol 2016 Criteria to extract high quality protein databank subsets from PDB And references therein