Structural Biology – a concept
In vivo
function
structure & dynamics

PDB Statistics – by method
Where the structural information comes from ….


In vivo
function
X
buffered solutions, crystalline state, or deposited isolated molecules on the surface
Complex environment of living cells

approximation
vs.
Complex heterogenous aqueous env.
Simple homogenous anhydrous env.
-180 ºC
37 ºC
X-ray diffraction

approximation
vs.
Complex heterogenous aqueous env.
Simple homogenous aqueous env.
20 ºC
37 ºC
NMR spectroscopy

approximation
vs.
Complex heterogenous aqueous env.
-180 ºC
37 ºC
Electron microscopy
Simple homogenous aqueous env.

… underlying assumptions in STRUCTURAL BIOLOGY
The use of these techniques imposes …
Øbiologically active specie = lowest (free) energy structure
Øthermodynamically NOT kinetically preferred conformation is responsible for biological activity.
Østructure is independent of environmental conditions

…not entirely true, particularly for DNA
(D pH, D ion strengh, ion type, MC, hydration)
Østructure is independent of environmental conditions

…monocrystal production
screenig_crystv02.jpg crystal-screen example.tiff
X-ray – particularly prone to env. artifacts
monoc.jpg

… not generally true
Øthermodynamically NOT kinetically preferred conformation is responsible for biological activity.
Biological processes (folding, interactions) t < sec, min
Only H2 would be observed in conventional X-ray, NMR experiment
Bessi et al. Angew Chem Int Ed Engl. 2015 Jul 13;54(29):8444-8.

…not entirely true
Øbiologically active specie = lowest (free) energy structure
 …number of processes happens via conformational selection
    (from excited/non-visible states)
Folding coordinate
U
Lowest energy state
Kinetically preferred state
Thermodynamically preferred state
Exited state

…X-ray, NMR, cryoEM structures
•Towards thermodynamically preferred (lowest energy) conformation, which
     is not necessarily biologically active; the bias is a function of T [ºC]
•Towards conformation specific to selected environmental conditions
•
•Parameters used to assess structure quality refers
    only to precision not accuracy
ØSummary
 …are  biased
accuracy_precissionv02.jpg

…in addition, NMR
ØSummary
 …is biased by averaging
Exited state
Lowest energy state
Multiple states populated
at physiological temperatures
We observe ensemble average

Bias from external information on local structure
noestr.tiff
cryoEM
NMR
X-ray

Bias from external information on local structure
Example: Implicitly used assumption of planarity of nucleic acid bases
Data from ultra-high resolution structures of nucleotides
Nucleic Acids Res, Volume 37, Issue 21, 1 November 2009, Pages 7321–7331
During structure determination we presume planarity
However, NA bases are planar only when isolated and
when at 0 K.
… source of the artefact in NMR – affects interpretation of NOEs, Js, RDCs

… which can be easily fixed
Historically based artifacts
potassium
sodium
magnesium
calcium
ion not specified
Ionic composition of:
Intracellular space
Extracellular space
Ionic composition of buffers used for NMR studies of:
DNA
RNA
Statistics based on PDB data

How to recognize physiologically relevant (accurate) structure?
Structural Biology  – an issue
… assessment of structural accuracy presumes knowledge of reference structure
accuracy_precissionv02.jpg

buffered solutions or crystalline state
… characterization of structures at physiologically relevant env. conditions
Cellular Structural Biology – a concept
In vivo
function
X
Complex environment of living cells

in vivo function
in cell structure
DNA
Cell nucleus
Cellular structural biology
RNA
cytosol
analysis
protein
cryoEM
NMR
Homework – mandatory self-reading
Plitzko et al. Structural Biology Outside the Box-Inside the Cell.
Curr Opin Struct Biol. 2017 Oct;46:110-121

Conservation of repetitive sequences suggest their importance for function. Their distribution in
genome then provide hints on the function.

EXAM by homework
Model a 3D complex between TBA aptamer and FGF2 presuming that the
TBA binds to the heparin binding site via electrostatic interactions.
Consider visualization of charge distribution on structural models of FGF2 and TBA.
e.g., Raček et al. Atomic Charge Calculator II: Web-Based Tool for the  Calculation of
Partial Atomic Charges. Nucleic Acids Res. 2020 May 13;gkaa367. doi: 10.1093/nar/gkaa367

Conservation of repetitive sequences suggest their importance for function. Their distribution in
genome then provide hints on the function.