advances in structural biology · 15.10-15.30 nada Žnidaršič conventional transmission electron...
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MiniSimpozij 2017
ADVANCES IN STRUCTURAL
BIOLOGY
26th October 2017
Ljubljana
Department of Molecular Biology and Nanobiotechnology D11
National Institute of Chemistry
MiniSimpozij 2017
ADVANCES IN STRUCTURAL
BIOLOGY
CIP - Kataložni zapis o publikaciji
Narodna in univerzitetna knjižnica, Ljubljana
577.2(082)
ADVANCES in structural biology : mini simpozij 2017, [26th October 2017
Ljubljana] / [editors Gregor Anderluh & Marjetka Podobnik]. - Ljubljana : Department of
Molecular Biology and Nanobiotechnology D11, National Institute of Chemistry, 2017
ISBN 978-961-6104-37-1
1. Anderluh, Gregor, 1969-
292397056
6th
MiniSimpozij 2017
ADVANCES IN STRUCTURAL BIOLOGY
Organised by
Katja Pirc, Simon Žurga & Marjetka Podobnik
Department of Molecular Biology and Nanobiotechnology D11
National Institute of Chemistry
Editors
Gregor Anderluh & Marjetka Podobnik
Technical editors
Matic Kisovec, Katja Pirc & Simon Žurga
Issued by
Department of Molecular Biology and Nanobiotechnology D11
National Institute of Chemistry
Printed by
Infokart
Ljubljana, 2017
Contents
Program 4
Foreword 5
Department of Molecular Biology and Nanobiotechnology 6
Abstract of the plenary lecture 8
Abstracts of short lectures 10
4
Program
14.00-14.20 Marjetka Podobnik
& Gregor Anderluh
Welcome speech
14.20-14.50 Jiří Nováček Structure and genome delivery mechanism
of Staphylococcus aureus phage therapy
agent phi812-K1 determined by cryo-
electron microscopy
14.50-15.10 Andreja Šink Structural studies of flexible filamentous
virus
15.10-15.30 Nada Žnidaršič Conventional transmission electron
microscopy as a complementary tool in
imaging of macromolecules and
macromolecular assemblies
15.30-15.50 Magda Tušek
Žnidarič
Negative staining method for transmission
electron microscopy of biological samples –
the player in the team
15.50-16.10 Elena Tchernychova Advanced transmission electron microscopy
at NIC
16.10-16.30 Samo Hudoklin Electron tomography of cellular structures
16.30-16.50 Damijan Knez Structure-based drug design: from selective
butyrylcholinesterase inhibitors towards
multifunctional anti-Alzheimer ligands
16.50-17.10 Dušan Turk Rfree: a dinosaur marked for extinction?
17.10-17.40 Coffee break
17.40-18.00 Silvia Onesti Macromolecular machines involved in DNA
replication: an integrated structural biology
approach
18.00-18.20 Miha Pavšič Tail and intermodule linker flexibility of
testicans explored by SAXS
18.20-18.40 Ajasja Ljubetič Coiled-coil protein origami cages (capable of
in vivo self-assembly)
18.40-19.00 JEOL-SCAN Corporate presentation
19.00-19.20 Thermo Fischer
Scientific
Corporate presentation
19.20-19.25 Marjetka Podobnik Closing remarks
5
Foreword
Welcome,
to the traditional 6th
MiniSimpozij organized by Department of Molecular Biology and
Nanobiotechnology. We study molecular background of various biological processes at
different resolution levels, and in particular focus on interactions between biological
molecules and their mechanism of action. In addition, we attempt to translate the
potential of these molecules into various biotechnological applications.
The main goal of these mini-symposia is to get together a local scientific community as
well as colleagues from abroad to share our scientific knowledge, expertise and
experience, and to continue (or hopefully start new) collaborations, or to simply
educate us in various research topics and methodological approaches.
The title of this year’s mini-symposium is “ADVANCES IN STRUCTURAL BIOLOGY”.
Therefore, our program includes presentations by invited speakers who use different
approaches of biophysics and structural biology to elucidate structural properties of
various biological systems as well as those provided by synthetic biology, at different
resolution levels. Importantly, in the light of recent extraordinary advances is the field
as well as the Nobel Prize Award for Chemistry for 2017, we are paying a special
attention to different modes of electron microscopy, in particular cryo-electron
microscopy. We strongly believe that many Slovenian scientists are highly interested in
this powerful methodological approach and should have an opportunity to become
educated in the usage of this technique, and moreover, to actually have the state-of-
the-art equipment, which Slovenian scientists are lacking at the time being.
This year we have eleven invited speakers, including guests from aboard. In addition,
the representatives of two companies producing high-end (cryo)-electron microscopes
will give their presentations and have stands in front of the lecture room. The official
language of the meeting is English.
We wish you all a pleasant and fruitful meeting, with lively scientific discussions
between presentations, during the coffee break and after the meeting. Let us hope that
the meeting will bring along new collaborations and a positive outlook for the
Slovenian science, especially in the field of cryo-electron microscopy.
Assist. Prof. Marjetka Podobnik Prof. Gregor Anderluh
6
Department of Molecular Biology and Nanobiotechnology
D11
We are performing top level research of biological processes, focusing on
understanding the mechanism of action of proteins and molecular interactions. We
create new basic knowledge as well as introduce modern methodologies in the field of
Life Sciences. We develop applications for solving actual problems in biotechnology
and pharmaceutical industry, in particular in development of biological drugs. Head of
department is Assist. Prof. Marjetka Podobnik, PhD.
7
Past events organised by
Department of Molecular Biology and Nanobiotechnology
D11
MiniSimpozij 2012
PLANT-PATHOGEN INTERACTIONS
National Institute of Chemistry, Ljubljana, 16.2.2012
https://www.ki.si/odseki/d11-odsek-za-molekularno-biologijo-in-
nanobiotehnologijo/dogodki/mini-simpozij-piran-2013/
MiniSimpozij 2013
INTERAKCIJE MED PROTEINI IN MEMBRANAMI
Morska biološka postaja, Piran, 6.9.2013
https://www.ki.si/odseki/d11-odsek-za-molekularno-biologijo-in-
nanobiotehnologijo/dogodki/mini-simpozij-piran-2013/
MiniSimpozij 2014
MOLEKULSKE INTERAKCIJE
in Deset let Infrastrukturnega centra za raziskave molekulskih interakcij
Oddelek za agronomijo, Biotehniška fakulteta, Univerza v Ljubljani, 3.12.2014
https://www.ki.si/odseki/d11-odsek-za-molekularno-biologijo-in-
nanobiotehnologijo/dogodki/mini-simpozij-ljubljana-2014/
MiniSimpozij 2015
PROTEINSKE PORE, SEKVENCIRANJE IN BIOINFORMATIKA
Kemijski inštitut, Ljubljana, 5.11.2015
https://www.ki.si/odseki/d11-odsek-za-molekularno-biologijo-in-
nanobiotehnologijo/dogodki/mini-simpozij-2015/
MiniSimpozij 2016
ADVANCES IN MOLECULAR INTERACTION ANALYSIS
National Institute of Chemistry, Ljubljana, 22.11.2016
https://www.ki.si/odseki/d11-odsek-za-molekularno-biologijo-in-
nanobiotehnologijo/dogodki/mini-simpozij-2016/
8
Abstract of the plenary lecture
9
Structure and genome delivery mechanism of Staphylococcus
aureus phage therapy agent phi812-K1 determined by cryo-
electron microscopy
Jiří Nováček
Central European Institute of Technology, Masaryk University, Brno, Czech Republic
Worldwide occurrence of multidrug-resistant pathogenic bacteria has increased
interest in alternative treatments including bacteriophage-based therapy.
Bacteriophage phi812 belongs to genus Twort-like virus, subfamily Spounavirinae and
can infect at least 75% of Methicilin-resitant S. aureus strains (MRSA) and 95% of
Methicillin-sensitive S. aureus strains. We have employed cryo-electron microscopy to
determine structure and genome delivery mechanism for polyvalent staphylococcal
backteriophage phi812-K1. Phi812-K1 has a 90 nm diameter isometric head and 240
nm long contractile tail ended by a double layered baseplate. The tail and baseplate of
the native phage are dynamic. Therefore, a divide-and-conquer strategy was employed
to separately determine the cryo-EM reconstructions of the individual phage parts.
Similarly to other phages from the family Myoviridae, host recognition and infection is
accompanied with by tail sheath contraction with significant conformational change of
baseplate. However, the release of the phage dsDNA is not governed only by tail
sheath contraction. The data reveal presence of three species of phi812-K1 particles – a
native virion, particles with contracted tail and DNA in the head, and phages with
contracted tail and empty head. Structural analysis of these three species reveals that
additional structural changes in the neck region are required after the tail contraction
before the DNA is allowed to transfer to the host.
10
Abstracts of short lectures
11
Structural studies of flexible filamentous virus
Andreja Šink1, Martin Pólak2, Ion Gutiérrez-Aguirre3, Magda Tušek-Žnidarič3,
Maja Ravnikar3, Gregor Anderluh1, Jiří Nováček2, Marjetka Podobnik1
1Department of Molecular Biology and Nanobiotechnology, National Institute of
Chemistry, Ljubljana, Slovenia; 2Core Facility Cryo-electron Microscopy and
Tomography, Central European Institute of Technology, Brno, Czech Republic; 3Department of Biotechnology and Systems Biology, National Institute of Biology,
Ljubljana, Slovenia
Plant viruses exist in all kinds of shapes and sizes. They have great impact in agriculture
and can be used in applied science. We are studying a flexible filamentous plant virus,
which is responsible for a huge damage on crop fields, moreover, it has a high
potential for usage as a template in development of new pharmaceuticals or
nanomaterials. To enable further studies of viral infectivity and scientific potential, it is
important to determine its near-atomic three-dimensional structure.
Due to high flexibility, flexible filamentous viruses are not capable of forming protein
crystals, which would be suitable for high-resolution X-ray crystallography. Fortunately,
recent advances in cryo-electron microscopy can help us toward obtaining high-
resolution three-dimensional structures of flexible filamentous particles. We are
processing collected data of the virus using single particle analysis with helical
reconstruction.
Schematic representation of flexible filamentous virus (A); infected plant (B); micrograph showing
viral particles (C).
12
Conventional transmission electron microscopy as a
complementary tool in imaging of macromolecules and
macromolecular assemblies
Nada Žnidaršič1, Magda Tušek Žnidarič2, Jasna Štrus1, Saša Rezelj3, Marjetka
Podobnik3, Gregor Anderluh3
1Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana,
Slovenia; 2Department of Biotechnology and Systems Biology, National Institute of
Biology, Ljubljana, Slovenia; 3Department of Molecular Biology and
Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
Imaging is an integrative part of biological structures characterization and transmission
electron microscopy (TEM) enables imaging at the best resolution available. TEM is a
versatile method, offering different techniques for specimen preparation and
visualization. Concerning imaging of isolated macromolecules or macromolecular
assemblies, negative staining has been used for years and it is still a valuable tool, as
preparation procedure is quick, a small amount of the sample is needed and high
contrast imaging is achieved. Thus, it is advantageous for evaluating
isolation/purification processes, for visualization of smaller particles and is
indispensable in immunolocalization. However, staining and drying introduce artefacts
that we have to take into account at interpretation and some can be partially
prevented. State of the art TEM are cryo-electron microscopy methods, including single
particle analyses and tomography. Complementary imaging approaches should be
used in combination with quantitative biophysical/biochemical techniques to reveal
adequate structural and functional information on macromolecules.
Transmission electron microscopy image of listeriolysin O pores on liposome.
13
Negative staining method for transmission electron
microscopy of biological samples – the player in the team
Magda Tušek Žnidarič1, Polona Kogovšek1, Nada Žnidaršič2
1Department of Biotechnology and System Biology, National Institute of Biology,
Ljubljana, Slovenia; 2Department of Biology, Biotechnical Faculty, University of
Ljubljana, Ljubljana, Slovenia
A huge number of microscopic methods and their variations were developed in last 30
years. Negative staining method is simple, but very useful for visualisation of small
biological particles in the solution with transmission electron microscope (TEM). In
combination with other methods, it represents a powerful tool in different research
fields.
In parallel to biochemical methods, like different separation and spectroscopic
techniques, the changes in protein conformation could be followed and TEM gives a
great contribution to determine protein structure. Many diseases base on protein
misfolding, aggregation and fibrillation (Fig. A) and one of model proteins to the study
protein fibrillation is human stefin B1.
Adeno-associated viruses (Fig. B) represent promising vehicles for delivering genetic
material in gene therapy. During production of clinical grade biomolecules, it is of great
importance to check concentration and purity in each step and only TEM enable us to
detect everything present in the sample.
Biological samples prepared with negative staining method and observed with transmission
electron microscope. A. Fibrilated protein. B. Adeno associated viruses.
Reference:
(1) Žerovnik E. et al. (2006) FEBS J. 273, 4250–4263.
14
Advanced transmission electron microscopy at NIC
Elena Tchernychova, Goran Dražić, Francisco Ruiz Zepeda
Department for Materials Chemistry, National Institute of Chemistry, Ljubljana, Slovenia
“The devil is in the detail”. Transmission electron microscopy (TEM) equipped with
simultaneous visual and analytical capabilities represents one of the finest performers
in the field of investigation of structural and chemical details in materials. State of the
art scanning TEM (STEM) instruments fortified with probe spherical aberration
correctors allow these days a point-to-point resolution of 0.8 Å and less. This lies below
the lattice spacing of a large number of crystalline materials, allowing the investigation
of atomic species at almost “personal” level. In the past 4 years we used the
opportunities of such atomic resolution STEM (AR-STEM) instrument installed at our
institute to shed the light on the details of catalytic nanomaterials, Li-ion battery
cathode materials, polymers, various ceramic nanostructures, etc. Our aim is to connect
the chemical and structural peculiarities observed in the nano-world with the
macroscopic properties of the investigated material. The scientific problems related to
the life sciences tackled by our microscope will also be addressed in the presentation.
15
Electron tomography of cellular structures
Samo Hudoklin
Institute of cell biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
Biological processes ongoing in the organisms are critically depended on spatio-
temporal distribution and interactions of macromolecules, assembled into cellular
compartments. Microscopic methods, in particular transmission electron microscopy
(TEM), are the only methods that have resolving power required to provide information
about the presence and location of macromolecules and cellular compartments within
the cells. However, thickness (30-80 nm) of classical TEM sections reduces the
information about inherently three-dimensional cellular compartments into two-
dimensional images. Electron tomography is a microscopic method, which overcomes
this problem and provides three-dimensional spatial organization of cellular
compartments at nanometre resolution. Electron tomography is implemented at the
Institute of Cell Biology, Faculty of Medicine, together with the cryo-fixation methods
and other methods, which are crucially important for the complete and competent
preparation of biological samples closes to their in vivo conditions. In the talk, workflow
and results obtained by electron tomography on urothelial tissue will be presented.
Three-dimensional model of fusiform vesicles obtained by the method of electron tomography.
Fusiform vesicles are characteristic transport vesicles of differentiated urothelial cells, which
contribute to the permeability barrier of human urinary bladder. Bar: 250 nm.
16
Structure-based drug design: from selective
butyrylcholinesterase inhibitors towards multifunctional anti-
Alzheimer ligands
Damijan Knez,1 Urban Košak,1 Boris Brus,1 Anja Pišlar,1 Nicolas Coquelle,2 Jurij
Stojan,3 Janko Kos,1 Jacques-Philippe Colletier, 2 Stanislav Gobec1
1Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia;
2Institut de Biologie
Structurale (IBS), University Grenoble Alpes, Grenoble, France; 3Insitute of Biochemistry,
Faculty of medicine, University of Ljubljana, Ljubljana, Slovenia
Alzheimer’s disease (AD) is a complex disorder characterized by progressive and
chronic deterioration of the memory and other cognitive functions. Numerous factors
are involved in AD progression: oxidative stress, increased activity of monoamine
oxidase B (MAO-B), loss of metal homeostasis, and a severe decrease in levels of
acetylcholine.1 In the late stages of AD, enzyme butyrylcholinesterase (BChE) takes over
the acetylcholine breakdown, which makes it a promising target in the therapy of AD.2
Piperidine based selective BChE inhibitor identified in a structure-based virtual
screening3 was used as a starting point for on-target activity optimization and further
on to design two series of multifunctional ligands with metal chelating properties,
antioxidant activity and neuroprotective properties against amyloid β fibrils.4 Dual
BChE/MAO-B inhibitors with N-propargylpiperidine scaffold were also developed.5 The
resolved crystal structures of several inhibitors in complex with BChE reveal their
binding mode and explain their low micromolar to picomolar inhibitory potencies.
References:
(1) a) Querfurth H.W. et al. (2010) N. Engl. J. Med., 362, 329; b) Winbald B. et al. (2016) Lancet Neurol., 15, 455.
(2) a) Greig N.H. et al., (2001) Curr. Med. Res. Opin. 17, 159; b) Greig N.H. et al. (2005) Proc. Natl. Acad. Sci.
USA, 102, 17213; c) Mushtaq G. et al. (2014) CNS Neurol. Disord. Drug Targets, 13, 1432. (3) Brus B. et al.
(2014) J. Med. Chem., 57, 8167. (4) a) Košak U. et al. (2016) Sci. Rep., 6, 39495; b) Košak U. et al., submitted for
publication; c) Knez D. et al. (2015) Bioorg. Med. Chem., 23, 4442; d) Knez D. et al., manuscript in preparation.
(5) Košak U. et al. (2017) Bioorg. Med. Chem., 25, 633.
17
Rfree: a dinosaur marked for extinction?
Dušan Turk
Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute,
Ljubljana, Slovenia
Over fitting in refinement is in macromolecular crystallography controlled by R-free in a
cross validation procedure. However, the R-free concept has its limitations: it does not
allow the use of all data in refinement and map calculations, the presence of NCS
makes it impossible to decouple the independence of TEST set reflections from the rest
of the data, and the exchange of the TEST set can result in a considerably different gap
between R-work and R-free, the distribution of errors in model is not random but
correlated. To overcome the limitations of the R-free concept we developed an
approach that uses the WORK set to calculate the phase error estimates in the ML
refinement from simulating the model errors. We call it ML Free Kick refinement.
This approach of calculation of error estimates is superior to the cross validation
approach in accuracy and phase errors of resulting structures.
18
Macromolecular machines involved in DNA replication: an
integrated structural biology approach
Silvia Onesti
Elettra - Sincrotrone Trieste, Area Science Park, Trieste, Italy
The CMG (Cdc45–MCM–GINS) complex is the eukaryotic replicative helicase, the
enzyme that unwinds double-stranded DNA at replication forks. Over the last few years
we have carried out biochemical and structural studies on MCM, GINS and Cdc45
which provided important insights into the three dimensional architecture of the
complex, its function and evolution. We have recently focussed our attention to the
archaeal homologue of Cdc45: the eukaryotic Cdc45 shows sequence and structural
similarity to protein belonging to the RecJ family of exonucleases. We have obtained a
number of crystal structure of archaeal Cdc45-RecJ, with and without nucleotides and
oligonucleotides, which shed light on the exonuclease function and provide a
framework to understand the evolution of Cdc45 and the CMG complex.
19
Tail and intermodule linker flexibility of testicans explored by
SAXS
Miha Pavšič
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical
Technology, University of Ljubljana, Ljubljana, Slovenia
Testicans, modular proteoglycans of the extracellular matrix (ECM) of various tissues,
play important role in tissue formation and remodeling – they stimulate neurite
outgrowth, promote cell migration, and regulate activity of extracellular proteases.
Similarly to several other components of the ECM they are believed to contain flexible
regions making them notoriously difficult to study by conventional high-resolution
structural approaches like X-ray crystallography. Here, we employed small angle X-ray
scattering (SAXS) to gain insight into the flexibility of the testican core protein. Results
confirmed the hypothesis that both N- and C-terminal tails are disordered, and
indicated that short flexible regions are also found within the central part. Therefore,
the follistatin-like (FS), extracellular calcium-binding (EC), and thyroglobulin type-1
domain (TY) do not form a completely compact globule as previously speculated. The
observed disorder could thus provide the necessary structural plasticity for both ECM
organization as well as interaction with various proteins within different tissues.
20
Coiled-coil protein origami cages (capable of in vivo self-
assembly)
Ajasja Ljubetič1, Jana Aupič1, Igor Drobnak1, Tomaž Pisanski3, Fabio Lapenta1,
Žiga Strmšek1, Helena Gradišar1,2, Roman Jerala1,2
1Department of Synthetic Biology and Immunology, National Institute of Chemistry,
Ljubljana, Slovenia; 2EN-FIST Centre of Excellence, Ljubljana, Slovenia;
3Faculty of
Mathematics and Physics, University of Ljubljana, Slovenia
Polypeptides are nature’s most versatile nano-machines, capable of efficient self-
assembly. A similar strategy as in DNA nanotechnology can be applied to construct
protein origami cages using coiled-coil (CC) dimers as building modules. The
developing field of CC protein origami1,2
would benefit from software to ease the
creation of novel origamis.
We have created CoCoPOD, a coiled-coil protein origami design platform, for
automated design of arbitrary polyhedral CC cages. The end result of the design is a
single-chain amino acid sequence, as well as an ensemble of probable 3D models.
CoCoPOD enabled the design of tetrahedra, square pyramids and a triangular prism,
which contains more than 700 amino acid residues3.
The correct shape of the designs was confirmed by solution SAXS, TEM and biophysical
analysis. Cages were produced and self-assembled in bacteria, as well as in mammalian
cells and in animals, without causing inflammation or other adverse pathological
effects.
Next generation CC polyhedra. Tetrahedron, square pyramid and triangular prism designs are
presented. For each polyhedral the topology, i.e. the connectivity of segments, (upper left) a 3D
model (upper right) and the linear order of segments are shown.
References:
(1) Gradišar H. et al. (2013) Nat. Chem. Biol., 9, 362–366; (2) Drobnak I. et al. (2016) Designed Protein Origami,
in Protein-based Engineered Nanostructures (Springer) 7–27; (3) Ljubetič A. et al. (2017) Nat Biotech,
doi:10.1038/nbt.3994.
WEB PAGE OF THE DEPARTMENT
https://www.ki.si/en/departments/d11-department-of-molecular-biology-and-
nanobiotechnology/
LINK TO THE EVENT
https://www.ki.si/ms2017/
@D11_NIC & @KEMIJSKI
