Molecular biophysics

Last updated

A ribosome is a biological machine that utilizes protein dynamics Protein translation.gif
A ribosome is a biological machine that utilizes protein dynamics

Molecular biophysics is a rapidly evolving interdisciplinary area of research that combines concepts in physics, chemistry, engineering, mathematics and biology. [1] It seeks to understand biomolecular systems and explain biological function in terms of molecular structure, structural organization, and dynamic behaviour at various levels of complexity (from single molecules to supramolecular structures, viruses and small living systems). This discipline covers topics such as the measurement of molecular forces, molecular associations, allosteric interactions, Brownian motion, and cable theory. [2] Additional areas of study can be found on Outline of Biophysics. The discipline has required development of specialized equipment and procedures capable of imaging and manipulating minute living structures, as well as novel experimental approaches.

Contents

Overview

Molecular biophysics typically addresses biological questions similar to those in biochemistry and molecular biology, seeking to find the physical underpinnings of biomolecular phenomena. Scientists in this field conduct research concerned with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis, as well as how these interactions are regulated. A great variety of techniques are used to answer these questions.

Fluorescent imaging techniques, as well as electron microscopy, X-ray crystallography, NMR spectroscopy, atomic force microscopy (AFM) and small-angle scattering (SAS) both with X-rays and neutrons (SAXS/SANS) are often used to visualize structures of biological significance. Protein dynamics can be observed by neutron spin echo spectroscopy. Conformational change in structure can be measured using techniques such as dual polarisation interferometry, circular dichroism, SAXS and SANS. Direct manipulation of molecules using optical tweezers or AFM, can also be used to monitor biological events where forces and distances are at the nanoscale. Molecular biophysicists often consider complex biological events as systems of interacting entities which can be understood e.g. through statistical mechanics, thermodynamics and chemical kinetics. By drawing knowledge and experimental techniques from a wide variety of disciplines, biophysicists are often able to directly observe, model or even manipulate the structures and interactions of individual molecules or complexes of molecules.

Areas of Research

Computational biology

Computational biology involves the development and application of data-analytical and theoretical methods, mathematical modeling and computational simulation techniques to the study of biological, ecological, behavioral, and social systems. The field is broadly defined and includes foundations in biology, applied mathematics, statistics, biochemistry, chemistry, biophysics, molecular biology, genetics, genomics, computer science and evolution. Computational biology has become an important part of developing emerging technologies for the field of biology. [3] Molecular modelling encompasses all methods, theoretical and computational, used to model or mimic the behaviour of molecules. The methods are used in the fields of computational chemistry, drug design, computational biology and materials science to study molecular systems ranging from small chemical systems to large biological molecules and material assemblies. [4] [5]

Membrane biophysics

Membrane biophysics is the study of biological membrane structure and function using physical, computational, mathematical, and biophysical methods. A combination of these methods can be used to create phase diagrams of different types of membranes, which yields information on thermodynamic behavior of a membrane and its components. As opposed to membrane biology, membrane biophysics focuses on quantitative information and modeling of various membrane phenomena, such as lipid raft formation, rates of lipid and cholesterol flip-flop, protein-lipid coupling, and the effect of bending and elasticity functions of membranes on inter-cell connections. [6]

Motor proteins

Kinesin walking on a microtubule is a molecular biological machine using protein domain dynamics on nanoscales Kinesin walking.gif
Kinesin walking on a microtubule is a molecular biological machine using protein domain dynamics on nanoscales

Motor proteins are a class of molecular motors that can move along the cytoplasm of animal cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. A good example is the muscle protein myosin which "motors" the contraction of muscle fibers in animals. Motor proteins are the driving force behind most active transport of proteins and vesicles in the cytoplasm. Kinesins and cytoplasmic dyneins play essential roles in intracellular transport such as axonal transport and in the formation of the spindle apparatus and the separation of the chromosomes during mitosis and meiosis. Axonemal dynein, found in cilia and flagella, is crucial to cell motility, for example in spermatozoa, and fluid transport, for example in trachea. Some biological machines are motor proteins, such as myosin, which is responsible for muscle contraction, kinesin, which moves cargo inside cells away from the nucleus along microtubules, and dynein, which moves cargo inside cells towards the nucleus and produces the axonemal beating of motile cilia and flagella. "[I]n effect, the [motile cilium] is a nanomachine composed of perhaps over 600 proteins in molecular complexes, many of which also function independently as nanomachines...Flexible linkers allow the mobile protein domains connected by them to recruit their binding partners and induce long-range allostery via protein domain dynamics. [7] Other biological machines are responsible for energy production, for example ATP synthase which harnesses energy from proton gradients across membranes to drive a turbine-like motion used to synthesise ATP, the energy currency of a cell. [8] Still other machines are responsible for gene expression, including DNA polymerases for replicating DNA, RNA polymerases for producing mRNA, the spliceosome for removing introns, and the ribosome for synthesising proteins. These machines and their nanoscale dynamics are far more complex than any molecular machines that have yet been artificially constructed. [9]

These molecular motors are the essential agents of movement in living organisms. In general terms, a motor is a device that consumes energy in one form and converts it into motion or mechanical work; for example, many protein-based molecular motors harness the chemical free energy released by the hydrolysis of ATP in order to perform mechanical work. [10] In terms of energetic efficiency, this type of motor can be superior to currently available man-made motors.

Richard Feynman theorized about the future of nanomedicine. He wrote about the idea of a medical use for biological machines. Feynman and Albert Hibbs suggested that certain repair machines might one day be reduced in size to the point that it would be possible to (as Feynman put it) "swallow the doctor". The idea was discussed in Feynman's 1959 essay "There's Plenty of Room at the Bottom". [11]

These biological machines might have applications in nanomedicine. For example, [12] they could be used to identify and destroy cancer cells. [13] [14] Molecular nanotechnology is a speculative subfield of nanotechnology regarding the possibility of engineering molecular assemblers, biological machines which could re-order matter at a molecular or atomic scale. Nanomedicine would make use of these nanorobots, introduced into the body, to repair or detect damages and infections. Molecular nanotechnology is highly theoretical, seeking to anticipate what inventions nanotechnology might yield and to propose an agenda for future inquiry. The proposed elements of molecular nanotechnology, such as molecular assemblers and nanorobots are far beyond current capabilities. [15] [16]

Protein folding

Constituent amino-acids can be analyzed to predict secondary, tertiary and quaternary protein structure. Protein-structure.png
Constituent amino-acids can be analyzed to predict secondary, tertiary and quaternary protein structure.

Protein folding is the physical process by which a protein chain acquires its native 3-dimensional structure, a conformation that is usually biologically functional, in an expeditious and reproducible manner. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from a random coil. [17] Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids. This polypeptide lacks any stable (long-lasting) three-dimensional structure (the left hand side of the first figure). As the polypeptide chain is being synthesized by a ribosome, the linear chain begins to fold into its three-dimensional structure. Folding begins to occur even during the translation of the polypeptide chain. Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the right-hand side of the figure), known as the native state. The resulting three-dimensional structure is determined by the amino acid sequence or primary structure (Anfinsen's dogma). [18]

Protein structure determination

As the three-dimensional structure of proteins brings with it an understanding of its function and biological context, there is great effort placed in observing the structures of proteins. X-ray crystallography was the primary method used in the 20th century to solve the structures of proteins in their crystalline form. Ever since the early 2000s, cryogenic electron microscopy has been used to solve the structures of proteins closer to their native state, as well as observing cellular structures. [19]

Protein structure prediction

Protein structure prediction is the inference of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its folding and its secondary and tertiary structure from its primary structure. Structure prediction is fundamentally different from the inverse problem of protein design. Protein structure prediction is one of the most important goals pursued by bioinformatics and theoretical chemistry; it is highly important in medicine, in drug design, biotechnology and in the design of novel enzymes). Every two years, the performance of current methods is assessed in the CASP experiment (Critical Assessment of Techniques for Protein Structure Prediction). A continuous evaluation of protein structure prediction web servers is performed by the community project CAMEO3D.

The challenge in predicting protein structures is that there lacks a physical model that can fully predict protein tertiary structures from their amino acid sequence. This problem is known as the de novo protein structure prediction problem and is one of the great problems of modern science. [20] AlphaFold, an artificial intelligence program, is able to accurately predict the structures of proteins with genetic homology to other proteins that have been previously solved. [21] Though, this is not a solution to the de novo problem, as it relies on a database of prior data which results in it always being biased. [22] The solution to the de novo protein structure prediction problem must be a purely physical model that will simulate the protein folding in its native environment, resulting in the in silico observation of protein structures and dynamics that were never previously observed. [23]

Spectroscopy

Spectroscopic techniques like NMR, spin label electron spin resonance, Raman spectroscopy, infrared spectroscopy, circular dichroism, and so on have been widely used to understand structural dynamics of important biomolecules and intermolecular interactions.

See also

Related Research Articles

<span class="mw-page-title-main">Alpha helix</span> Type of secondary structure of proteins

An alpha helix is a sequence of amino acids in a protein that are twisted into a coil.

<span class="mw-page-title-main">Protein</span> Biomolecule consisting of chains of amino acid residues

Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.

<span class="mw-page-title-main">Structural biology</span> Study of molecular structures in biology

Structural biology, as defined by the Journal of Structural Biology, deals with structural analysis of living material at every level of organization.

<span class="mw-page-title-main">Protein folding</span> Change of a linear protein chain to a 3D structure

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

<span class="mw-page-title-main">Biophysics</span> Study of biological systems using methods from the physical sciences

Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. Biophysical research shares significant overlap with biochemistry, molecular biology, physical chemistry, physiology, nanotechnology, bioengineering, computational biology, biomechanics, developmental biology and systems biology.

<span class="mw-page-title-main">Integral membrane protein</span> Type of membrane protein that is permanently attached to the biological membrane

An integral, or intrinsic, membrane protein (IMP) is a type of membrane protein that is permanently attached to the biological membrane. All transmembrane proteins can be classified as IMPs, but not all IMPs are transmembrane proteins. IMPs comprise a significant fraction of the proteins encoded in an organism's genome. Proteins that cross the membrane are surrounded by annular lipids, which are defined as lipids that are in direct contact with a membrane protein. Such proteins can only be separated from the membranes by using detergents, nonpolar solvents, or sometimes denaturing agents.

<span class="mw-page-title-main">Protein structure prediction</span> Type of biological prediction

Protein structure prediction is the inference of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its secondary and tertiary structure from primary structure. Structure prediction is different from the inverse problem of protein design.

<span class="mw-page-title-main">Structural bioinformatics</span> Bioinformatics subfield

Structural bioinformatics is the branch of bioinformatics that is related to the analysis and prediction of the three-dimensional structure of biological macromolecules such as proteins, RNA, and DNA. It deals with generalizations about macromolecular 3D structures such as comparisons of overall folds and local motifs, principles of molecular folding, evolution, binding interactions, and structure/function relationships, working both from experimentally solved structures and from computational models. The term structural has the same meaning as in structural biology, and structural bioinformatics can be seen as a part of computational structural biology. The main objective of structural bioinformatics is the creation of new methods of analysing and manipulating biological macromolecular data in order to solve problems in biology and generate new knowledge.

<span class="mw-page-title-main">Protein structure</span> Three-dimensional arrangement of atoms in an amino acid-chain molecule

Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers – specifically polypeptides – formed from sequences of amino acids, which are the monomers of the polymer. A single amino acid monomer may also be called a residue, which indicates a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in which the amino acids lose one water molecule per reaction in order to attach to one another with a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions, such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, cryo-electron microscopy (cryo-EM) and dual polarisation interferometry, to determine the structure of proteins.

<span class="mw-page-title-main">Nanorobotics</span> Emerging technology field

Nanoid robotics, or for short, nanorobotics or nanobotics, is an emerging technology field creating machines or robots, which are called nanorobots or simply nanobots, whose components are at or near the scale of a nanometer. More specifically, nanorobotics refers to the nanotechnology engineering discipline of designing and building nanorobots with devices ranging in size from 0.1 to 10 micrometres and constructed of nanoscale or molecular components. The terms nanobot, nanoid, nanite, nanomachine and nanomite have also been used to describe such devices currently under research and development.

Site-directed spin labeling (SDSL) is a technique for investigating the structure and local dynamics of proteins using electron spin resonance. The theory of SDSL is based on the specific reaction of spin labels with amino acids. A spin label's built-in protein structure can be detected by EPR spectroscopy. SDSL is also a useful tool in examinations of the protein folding process.

<span class="mw-page-title-main">Max Planck Institute of Biochemistry</span> Research institute in Martinsried, Germany

The Max Planck Institute of Biochemistry is a research institute of the Max Planck Society located in Martinsried, a suburb of Munich. The institute was founded in 1973 by the merger of three formerly independent institutes: the Max Planck Institute of Biochemistry, the Max Planck Institute of Protein and Leather Research, and the Max Planck Institute of Cell Chemistry.

<span class="mw-page-title-main">Nanobiotechnology</span> Intersection of nanotechnology and biology

Nanobiotechnology, bionanotechnology, and nanobiology are terms that refer to the intersection of nanotechnology and biology. Given that the subject is one that has only emerged very recently, bionanotechnology and nanobiotechnology serve as blanket terms for various related technologies.

<span class="mw-page-title-main">Max Planck Institute for Biophysical Chemistry</span> Research institute

The Max Planck Institute for Biophysical Chemistry, also known as the Karl-Friedrich Bonhoeffer Institute, was a research institute of the Max Planck Society, located in Göttingen, Germany. On January 1, 2022, the institute merged with the Max Planck Institute for Experimental Medicine in Göttingen to form the Max Planck Institute for Multidisciplinary Sciences.

<span class="mw-page-title-main">Intrinsically disordered proteins</span> Protein without a fixed 3D structure

In molecular biology, an intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure, typically in the absence of its macromolecular interaction partners, such as other proteins or RNA. IDPs range from fully unstructured to partially structured and include random coil, molten globule-like aggregates, or flexible linkers in large multi-domain proteins. They are sometimes considered as a separate class of proteins along with globular, fibrous and membrane proteins.

<span class="mw-page-title-main">Protein domain</span> Self-stable region of a proteins chain that folds independently from the rest

In molecular biology, a protein domain is a region of a protein's polypeptide chain that is self-stabilizing and that folds independently from the rest. Each domain forms a compact folded three-dimensional structure. Many proteins consist of several domains, and a domain may appear in a variety of different proteins. Molecular evolution uses domains as building blocks and these may be recombined in different arrangements to create proteins with different functions. In general, domains vary in length from between about 50 amino acids up to 250 amino acids in length. The shortest domains, such as zinc fingers, are stabilized by metal ions or disulfide bridges. Domains often form functional units, such as the calcium-binding EF hand domain of calmodulin. Because they are independently stable, domains can be "swapped" by genetic engineering between one protein and another to make chimeric proteins.

<span class="mw-page-title-main">DNA nanotechnology</span> The design and manufacture of artificial nucleic acid structures for technological uses

DNA nanotechnology is the design and manufacture of artificial nucleic acid structures for technological uses. In this field, nucleic acids are used as non-biological engineering materials for nanotechnology rather than as the carriers of genetic information in living cells. Researchers in the field have created static structures such as two- and three-dimensional crystal lattices, nanotubes, polyhedra, and arbitrary shapes, and functional devices such as molecular machines and DNA computers. The field is beginning to be used as a tool to solve basic science problems in structural biology and biophysics, including applications in X-ray crystallography and nuclear magnetic resonance spectroscopy of proteins to determine structures. Potential applications in molecular scale electronics and nanomedicine are also being investigated.

The following outline is provided as an overview of and topical guide to biophysics:

<span class="mw-page-title-main">Macromolecular assembly</span> Large chemical complexes composed of polymers and other macromolecules

In molecular biology, the term macromolecular assembly (MA) refers to massive chemical structures such as viruses and non-biologic nanoparticles, cellular organelles and membranes and ribosomes, etc. that are complex mixtures of polypeptide, polynucleotide, polysaccharide or other polymeric macromolecules. They are generally of more than one of these types, and the mixtures are defined spatially, and with regard to their underlying chemical composition and structure. Macromolecules are found in living and nonliving things, and are composed of many hundreds or thousands of atoms held together by covalent bonds; they are often characterized by repeating units. Assemblies of these can likewise be biologic or non-biologic, though the MA term is more commonly applied in biology, and the term supramolecular assembly is more often applied in non-biologic contexts. MAs of macromolecules are held in their defined forms by non-covalent intermolecular interactions, and can be in either non-repeating structures, or in repeating linear, circular, spiral, or other patterns. The process by which MAs are formed has been termed molecular self-assembly, a term especially applied in non-biologic contexts. A wide variety of physical/biophysical, chemical/biochemical, and computational methods exist for the study of MA; given the scale of MAs, efforts to elaborate their composition and structure and discern mechanisms underlying their functions are at the forefront of modern structure science.

References

  1. ↑ "The goal of Molecular Biophysics". Program in Molecular Biophysics. Johns Hopkins University. Archived from the original on 15 March 2012.
  2. ↑ Jackson MB (2006). Molecular and Cellular Biophysics. New York: Cambridge University Press. ISBN   978-1-139-44724-9.
  3. ↑ Ouzounis CA (2012). "Rise and demise of bioinformatics? Promise and progress". PLOS Computational Biology. 8 (4): e1002487. Bibcode:2012PLSCB...8E2487O. doi: 10.1371/journal.pcbi.1002487 . PMC   3343106 . PMID   22570600.
  4. ↑ "NIH working definition of bioinformatics and computational biology" (PDF). Biomedical Information Science and Technology Initiative. 17 July 2000. Archived from the original (PDF) on 5 September 2012. Retrieved 18 August 2012.
  5. ↑ "About the CCMB". Center for Computational Molecular Biology. Providence RI: Brown University. Retrieved 18 August 2012.
  6. ↑ Zimmerberg J (April 2006). "Membrane biophysics". Current Biology. 16 (8): R272–R276. Bibcode:2006CBio...16.R272Z. doi: 10.1016/j.cub.2006.03.050 . PMID   16631568.
  7. ↑ Satir P, Christensen ST (June 2008). "Structure and function of mammalian cilia". Histochemistry and Cell Biology. 129 (6): 687–693. doi:10.1007/s00418-008-0416-9. PMC   2386530 . PMID   18365235. 1432-119X.
  8. ↑ Kinbara K, Aida T (April 2005). "Toward intelligent molecular machines: directed motions of biological and artificial molecules and assemblies". Chemical Reviews. 105 (4): 1377–1400. doi:10.1021/cr030071r. PMID   15826015.
  9. ↑ Bu Z, Callaway DJ (2011). "Proteins MOVE! Protein dynamics and long-range allostery in cell signaling". Protein Structure and Diseases. Advances in Protein Chemistry and Structural Biology. Vol. 83. pp. 163–221. doi:10.1016/B978-0-12-381262-9.00005-7. ISBN   9780123812629. PMID   21570668.
  10. ↑ Bustamante C, Chemla YR, Forde NR, Izhaky D (2004). "Mechanical processes in biochemistry". Annual Review of Biochemistry. 73: 705–748. doi:10.1146/annurev.biochem.72.121801.161542. PMID   15189157.
  11. ↑ Feynman RP (December 1959). "There's Plenty of Room at the Bottom". Archived from the original on 2010-02-11. Retrieved 2017-01-01.
  12. ↑ Amrute-Nayak M, Diensthuber RP, Steffen W, Kathmann D, Hartmann FK, Fedorov R, et al. (2010). "Targeted optimization of a protein nanomachine for operation in biohybrid devices". Angewandte Chemie. 49 (2): 312–316. Bibcode:2010AngCh.122..322A. doi:10.1002/ange.200905200. PMID   19921669.
  13. ↑ Patel GM, Patel GC, Patel RB, Patel JK, Patel M (February 2006). "Nanorobot: a versatile tool in nanomedicine". Journal of Drug Targeting. 14 (2): 63–67. doi:10.1080/10611860600612862. PMID   16608733. S2CID   25551052.
  14. ↑ Balasubramanian S, Kagan D, Hu CM, Campuzano S, Lobo-Castañon MJ, Lim N, et al. (April 2011). "Micromachine-enabled capture and isolation of cancer cells in complex media". Angewandte Chemie. 50 (18): 4161–4164. doi:10.1002/anie.201100115. PMC   3119711 . PMID   21472835.
  15. ↑ Freitas Jr RA (December 2005). Kasabov N, Havukkala I (eds.). "Current Status of Nanomedicine and Medical Nanorobotics" (PDF). Journal of Computational and Theoretical Nanoscience. 2 (4): 471. Bibcode:2005JCTN....2..471K. doi:10.1166/jctn.2005.001.
  16. ↑ Freitas Jr RA, Merkle RC (2006). "Nanofactory Collaboration". Molecular Assembler.
  17. ↑ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walters P (2002). "The Shape and Structure of Proteins". Molecular Biology of the Cell; Fourth Edition. New York and London: Garland Science. ISBN   978-0-8153-3218-3.
  18. ↑ Anfinsen CB (July 1972). "The formation and stabilization of protein structure". The Biochemical Journal. 128 (4): 737–749. doi:10.1042/bj1280737. PMC   1173893 . PMID   4565129.
  19. ↑ Curry S (July 2015). "Structural Biology: A Century-long Journey into an Unseen World". Interdisciplinary Science Reviews. 40 (3): 308–328. Bibcode:2015ISRv...40..308C. doi:10.1179/0308018815Z.000000000120. PMC   4697198 . PMID   26740732.
  20. ↑ "So Much More to Know". Science. 309 (5731): 78–102. 2005-07-01. doi:10.1126/science.309.5731.78b. ISSN   0036-8075.
  21. ↑ Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. (2021-08-26). "Highly accurate protein structure prediction with AlphaFold". Nature. 596 (7873): 583–589. Bibcode:2021Natur.596..583J. doi:10.1038/s41586-021-03819-2. ISSN   0028-0836. PMC   8371605 . PMID   34265844.
  22. ↑ Terwilliger TC, Liebschner D, Croll TI, Williams CJ, McCoy AJ, Poon BK, et al. (2023-11-30). "AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination". Nature Methods. 21 (1): 110–116. doi:10.1038/s41592-023-02087-4. ISSN   1548-7091. PMC   10776388 . PMID   38036854.
  23. ↑ Dill KA, Ozkan SB, Weikl TR, Chodera JD, Voelz VA (2007-06-01). "The protein folding problem: when will it be solved?". Current Opinion in Structural Biology. Nucleic acids / Sequences and topology. 17 (3): 342–346. doi:10.1016/j.sbi.2007.06.001. ISSN   0959-440X. PMID   17572080.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.