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 Perutz Labs University of Vienna

Austria

EMBO Global WS: Structural and biophysical methods for biological macromolecules in solution Santiago, 20th October 2019

Structural databases

• PDB
• 156954
• SASDB
• 1848
• EM
• 4026

Statistics by method

PDB Growth of Released Structures Per Year

Pathway for generation

• f 3D structure

Pathway

• Exp

xperiments: Crystallisation, X-ray diffraction

• 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
• ! " = |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

RES in PDB

Rfactor

• |Fo|

| - 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

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

• RES(1) = 2.0; R(1) = 0.18; Q(1) = 0.32
• RES(1) = 1.5; R(1) = 0.16; Q(1) = 0.51

Q =

# $%& − 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 abso

sorb lattice defects, large scale movements, disorder

• Inf

nform

• rm on
• n funct

unction

• n
• 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

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

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: PDBREDO

• 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

Structural and biophysical methods for biological macromolecules in solution

EMBO Global Exchange Lecture Course 14 – 20 October 2019 | Santiago, Chile