METHOD FOR MAPPING THE SURFACE OF A MACROMOLECULE

Description

TECHNICAL AREA OF INVENTION

[0001] The invention relates to a method for mapping the surface of a macromolecule.

[0002] In this context, a macromolecule refers primarily, but not exclusively, to proteins and protein complexes. The surface of interest is an essential part of the so-called tertiary structure of proteins and the so-called quaternary structure of complexes. Surface mapping primarily involves determining its three-dimensional shape. This also includes identifying the location of regions with specific chemical and / or physical properties. Ultimately, the goal can be to identify and characterize binding sites for other molecules or macromolecules located on the surface of the macromolecule. STATE OF THE ART

[0003] A well-known method for determining the structure of macromolecules is X-ray crystallography. This method requires growing crystals of the macromolecules. Therefore, not only must a large number of macromolecules be present, but these macromolecules must also be arranged in a crystal lattice. Unfortunately, many macromolecules cannot be crystallized, or crystal growth is very time-consuming and expensive. Even if crystallization is successful, there is a risk that the structure of the macromolecules will be altered by the crystallization process. Furthermore, while X-ray crystallography provides information about the tertiary structure of proteins or the quaternary structure of complexes, a complete mapping of their surfaces is generally not possible.

[0004] Furthermore, it is known to determine the structures of macromolecules using nuclear magnetic resonance spectroscopy (NMR spectroscopy). This method does not require crystals of the macromolecules. However, it only provides information about the environments of the nuclei of individual chemical elements within the macromolecules and not direct information about the surface structure of the macromolecules. This method is also very complex.

[0005] The structure of proteins can also be determined using cryo-electron microscopy (cryo-EM). In this method, a large number of images are acquired from electrons scattered by many individual proteins or protein complexes, and these images are averaged using computer algorithms to reveal the structure. To achieve the desired structure, this method requires high-resolution electron microscopes and sample preparation at low temperatures. Furthermore, it is very expensive, time-consuming, and incompatible with the physiological environment of proteins. In addition, the physical and chemical properties of the surface of the macromolecules or complexes under investigation cannot be directly determined using this method.

[0006] Furthermore, it is known that the tertiary structure of proteins can be predicted using artificial intelligence based on their primary structure, i.e., their amino acid sequence, and structural information available for other macromolecules with known amino acid sequences. Such AI-based predictions are performed, for example, by the computer program AlphaFold from Google. However, the predicted tertiary structures are, at least so far, subject to significant errors. The tertiary structure is not determined directly, but rather estimated based on plausibility considerations.

[0007] Furthermore, none of these known methods are suitable for directly mapping the surface of a protein in a living cell. The information provided by these methods only indirectly relates to the surface of the macromolecules of interest. Directly, it relates to the atomic structure of the macromolecules, which alone does not allow for any conclusions about the actual binding capacity of the macromolecules in their native environment. TASK OF INVENTION

[0008] The invention is based on the objective of demonstrating a method for mapping the surface of a macromolecule that eliminates the aforementioned disadvantages. FURTHER STATE OF THE TECHNOLOGY

[0009] From EP 3 055 674 B1, a method for imaging a sample is known in which a fluorescent particle is positioned in the sample at a spatially limited minimum in the light intensity distribution of fluorescence excitation light, and movements of the particle in the sample are tracked using the minimum of the light intensity distribution. For this purpose, the light intensity distribution in the sample is manipulated such that the rate of fluorescence photons emitted by the particle remains minimal. The current position of the minimum in the light intensity distribution in the sample is equated with the current position of the particle in the sample. From this, a residence time of the particle is determined for each of a plurality of regions of the sample, and a distribution of residence times is imaged over the sample.This method can be performed with various fluorescent particles that can be excited to fluorescence by fluorescence excitation light of different wavelengths. The known method can also be carried out for each of a plurality of fluorescent particles with the same or different wavelengths of the fluorescence excitation light. Furthermore, photoactivatable fluorophores can be used as fluorescent particles.

[0010] From EP 3 372 989 A1, a method for the spatial measurement of a nanoscale structure is known. The structure is labeled at various locations with fluorescent markers. At a local minimum in the intensity distribution of fluorescence-preventing light, the fluorescent markers are excited to emit fluorescent light. The local minimum is positioned at various locations in a near-field within the sample, the dimensions of which are no larger than the diffraction limit at the wavelength of the excitation light and the wavelength of the fluorescent light. The fluorescent light emitted from the sample is recorded separately for each fluorescent marker and for the different positions of the minimum; and the positions of the individual fluorescent markers in the sample are determined from the intensities of the fluorescent light recorded for each fluorescent marker at the different positions of the minimum.

[0011] From EP 3 523 631 B1, methods designated MinFlux for spatially determining the high-resolution position of a single molecule in a sample, which can be excited to emit luminescence by excitation light, are also known. The excitation light is directed onto the sample with an intensity distribution that exhibits a local minimum. This minimum is positioned at various locations near the molecule. The molecule's position in the sample is determined from the intensities of the luminescence emitted by the molecule. Using these known methods, the molecule's position in the sample can be determined with a precision in the range of 0.5 to 20 nm. The known methods can also be used to track a molecule moving within the sample, i.e., to monitor its position over time.

[0012] GWOSCH KLAUS C ET AL, "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells", NATURE METHODS, NATURE PUBLISHING GROUP US, NEW YORK, Vol. 17, No. 2, pp. 217-224, January 13, 2020, disclose a MinFlux method comprising the steps of: introducing at least one fluorescent probe into a medium in which a macromolecule is embedded, determining a plurality of spatial positions of the at least one fluorescent probe relative to the macromolecule by localizing the at least one isolated fluorescent probe with a simple standard deviation of no more than 2 nm, registering fluorescence light photons emitted by the isolated fluorescent probe, and creating a three-dimensional image of the plurality of spatial positions of the at least one fluorescent probe as well as a contour projection onto the xy-plane.

[0013] From WEBER MICHAEL ET AL, "MINSTED nanoscopy enters the Angstrom localization range", https: / / www.biorxiv.org / content / 10.1101 / 2022.03.18.484906v1, posted on March 19, 2022, subsequently published in NAT BIOTECHNOL, Volume 41, pages 569-576, April 2023, a method called MinSTED is known in which the position of a single fluorescent molecule is determined using superimposed intensity distributions of fluorescence excitation light and fluorescence suppression light, as known from STED scanning fluorescence microscopy. However, the sample is not scanned with the zero point of the fluorescence suppression light and the superimposed focus of the fluorescence excitation light, but rather the zero point is positioned at individual, rapidly changing locations in the vicinity of the molecule to be localized. Based on 2.With 000 registered fluorescence light photons, the position of the fluorescent molecule can be determined with a precision whose simple standard deviation is less than 0.5 nm.

[0014] WEBER MICHAEL ET AL, "MINSTED fluorescence localization and nanoscopy", NATURE PHOTONICS, NATURE PUBLISHING GROUP UK, LONDON, Volume 15, No. 5, pages 361-366, March 15, 2021, already disclose a MinSTED method for localizing individual fluorescent probes with a simple standard deviation of 1-3 nm. As an application example, the mapping of the distribution of Mic60 proteins labeled with the fluorophore ONB-2SiR in the inner membrane of mitochondria in human cells is described. SOLUTION

[0015] The object of the invention is achieved by a method for mapping the surface of a macromolecule with the features of independent claim 1. Preferred embodiments of the method according to the invention are defined in the dependent claims. DESCRIPTION OF THE INVENTION

[0016] To map the surface of a macromolecule according to the invention, at least one fluorescent probe is introduced into a medium in which the macromolecule is already embedded, or is embedded together with the at least one fluorescent probe, or will be embedded later. A plurality of spatial positions of the at least one fluorescent probe relative to the macromolecule are determined by localizing the at least one isolated fluorescent probe with a simple standard deviation of no more than 2 nm, whereby fluorescence photons emitted by the isolated fluorescent probe are registered. Then, an interface delineating the determined spatial positions relative to the macromolecule is defined. From this interface, a three-dimensional map of at least a part of the surface of the macromolecule is created.

[0017] The inventive method can utilize the macromolecule's natural environment, for example, within a cell, including a living cell, as the medium in which the macromolecule is embedded. Alternatively, the medium can be one in which the macromolecule is specifically embedded for carrying out the inventive method. In any case, at least one fluorescent probe is introduced into the medium. The term "at least one fluorescent probe" refers to at least one single fluorescent unit. Typically, several such fluorescent units are introduced into the medium simultaneously or sequentially because creating the three-dimensional map requires determining a large number of the spatial positions of the at least one fluorescent probe relative to the macromolecule with high precision, which necessitates a large number of photons from the at least one fluorescent probe.Therefore, there is a risk that the fluorescent probe will be permanently photochemically bleached or at least rendered dark for an extended period and will need to be replaced by another fluorescent probe. Each fluorescent probe contains a fluorophore, i.e., a fluorescent component. In addition, the fluorescent probe may have other components related to the fluorescent component to adjust its properties as desired. Specific examples will be given later. A fluorophore, i.e., the fluorescent component of the fluorescent probe, typically has a diameter of about 1 to 2 nm. Determining the spatial position of the at least one fluorescent probe relative to the macromolecule involves determining the precise position of this fluorophore or fluorescent component of the fluorescent probe.The required precision, with a standard deviation of no more than 2 nm, can be achieved using various high-resolution localization techniques, which detect fluorescence photons emitted by the fluorescent probe. These include techniques known as PALM or STORM, where the precision in determining the position of each individual probe increases with the square root of the number of detected fluorescence photons. Preferred localization techniques are described below.

[0018] In the method according to the invention, the space in which the fluorescent probe is located is not probed using the at least one fluorescent probe, but rather the space in which the macromolecule is located and which is therefore inaccessible to the probe. In other words, the spatial positions of the at least one fluorescent probe form a three-dimensional negative image that omits the macromolecule. The surface of this negative image, i.e., the interface that delineates the specific spatial positions of the fluorescent probe from the macromolecule, essentially corresponds to the surface of interest of the macromolecule. The interface is not exactly identical to the surface of interest, but differs from it depending on the properties of the fluorescent probe. This will be explained in more detail below.The result of the method according to the invention is a three-dimensional map of at least a part of the surface of the macromolecule, which, compared to known methods of structural analysis, is obtained in a very direct manner and therefore maps the surface of the macromolecule very accurately. In particular, the method according to the invention not only detects pockets in the surface or other spatial structures associated with functionalities of the macromolecule, such as binding sites, but also describes them precisely.

[0019] The spatial resolution of the map created using the inventive method depends on the areal density of the spatial positions of the fluorescent probe determined on the surface of the macromolecule. The higher the areal density, the higher the spatial resolution. Preferably, enough spatial positions of the at least one fluorescent probe are determined such that the average areal density of the spatial positions determined on the surface of the macromolecule is at least 25 positions per 100 square nanometers (10 nm x 10 nm). Particularly preferably, at least 50 positions are determined, even more preferably at least 100, and most preferably at least 200 positions per 100 square nanometers of surface area.The method according to the invention is less concerned with improving or even necessarily achieving the spatial resolution of known methods of structural analysis when determining the tertiary structure of a macromolecule, but rather with obtaining additional information about the surface of the macromolecule, the course of which is determined with sufficiently high spatial resolution, which allows direct statements about possible bonds of other molecules and macromolecules to the macromolecule under investigation.

[0020] Preferably, not only is the interface defining the specific spatial positions of the at least one fluorescent probe relative to the macromolecule determined, but also a local areal density of the spatial positions along the interface is recorded and plotted on the three-dimensional map. If the density of the spatial positions outside the macromolecule is generally uniform, the local areal density of the spatial positions at the interface indicates an affinity of the fluorescent probe for a specific region of the macromolecule's surface. Thus, in addition to its spatial orientation, further information about the macromolecule's surface is obtained. This information depends on the properties of the at least one fluorescent probe.

[0021] In general, it is advantageous to consider the properties of the at least one fluorescent probe when creating the three-dimensional map. These properties include, for example, the dimensions of the at least one fluorescent probe, which determine the spatial distance between specific positions and the surface of the macromolecule of interest. Furthermore, these properties include the physical and / or chemical affinities of the at least one fluorescent probe to specific regions of the surface. These affinities allow the location of these specific surface regions to be determined.

[0022] Specifically, in the method according to the invention, binding affinities and / or binding constants for molecules with predetermined physical and / or chemical affinities can be derived from the specific spatial positions and properties, in particular of fluorescent probes that have been mobile relative to the macromolecule in the medium, and entered into the three-dimensional map. The determination of such binding affinities and / or binding constants using the fluorescent probes is a significant advantage of the method according to the invention compared to all methods of structural analysis in which such binding affinities and / or binding constants must be derived indirectly from the spatial and chemical structure of a macromolecule.

[0023] For the purposes described above, the at least one fluorescent probe can be specifically equipped with an affinity group selected to exhibit an exclusive or at least enhanced affinity for regions of interest on the surface of the macromolecule with specific chemical and / or physical properties. Specifically, these physical and / or chemical properties may include polarity, hydrophilicity, lipophilicity, bond strength for van der Waals forces or hydrogen bonds, electrostatic attraction, or the like. It is understood that the entire surface of the macromolecule cannot be mapped using only one probe with a single affinity group exhibiting an exclusive affinity for specific regions of the macromolecule's surface.Rather, for the complete mapping of the surface, either fluorescent probes with affinity groups that cover all areas of the surface of the macromolecule must be used, or at least a probe must be used that scans all areas of the surface of the macromolecule with their spatial positions.

[0024] It is understood that determining the spatial positions of the at least one fluorescent probe relative to the macromolecule requires knowing the macromolecule's orientation. However, it suffices that the macromolecule's orientation is the same when determining at least some of the positions of the at least one fluorescent probe. In this case, the low-resolution negative images of the macromolecule obtained for each orientation can be combined into a higher-resolution negative image using computer algorithms, such as those known from cryo-EM. Nevertheless, it is advantageous to know, or at least record, the macromolecule's orientation by determining all, or at least as many as possible, of the positions of the at least one fluorescent probe.The spatial orientation of the macromolecule can be predetermined by coupling it in a fixed orientation to a fixed coupling site in the medium and / or by fixing it to the medium in which it is or will be embedded. Alternatively or additionally, the spatial orientation can be determined by identifying at least one spatial marker position of at least one position marker attached to the macromolecule. Such position markers can also be tracked to continuously monitor the spatial orientation of the macromolecule. This can be useful if the macromolecule is mobile within the medium.

[0025] Preferably, the majority of the spatial positions of the at least one fluorescent probe relative to the macromolecule are determined by localization according to one of the methods known as MinFlux or MinSTED. These methods enable the determination of the spatial positions of the at least one fluorescent probe with the required precision, where the simple standard deviation is no greater than 2 nm, based on a relatively small number of detected fluorescence photons. In particular, a precision with a simple standard deviation of no more than 1 nm and even no more than 0.5 nm can advantageously be achieved. This allows the surface of interest of the macromolecule to be mapped with particularly high spatial resolution.As already stated, other high-resolution localization methods are also applicable in principle for determining the spatial positions of the at least one fluorescent probe based on the fluorescence light it emits.

[0026] All high-resolution localization methods that can determine the spatial positions of at least one fluorescent probe with the required high precision necessitate that the at least one fluorescent probe is isolated when its spatial position is determined; that is, its fluorescence photons must be distinguishable from the fluorescence photons of other fluorescent probes, particularly due to a sufficient spatial separation between the fluorescent probes. This sufficient spatial separation can be achieved, at least on average, by introducing the at least one fluorescent probe into the medium at a low spatial concentration. However, only a low spatial concentration of the actually fluorescent probes in the medium is relevant. Probes that are not currently in a fluorescent state are not considered.In this context, a fluorescent state of a probe is understood to mean that the probe can be excited to emit fluorescent photons by fluorescence excitation light. However, the term "fluorescent state" here does not refer to an electronic state of the probe that is already excited by fluorescence excitation light and emits a fluorescent photon upon relaxation to its ground state.

[0027] The spatial positions of the at least one fluorescent probe, which are determined in the method according to the invention, can be achieved by the at least one fluorescent probe through relative movements with respect to the macromolecule in the medium. The mobility of the fluorescent probe with respect to the macromolecule can be based entirely or substantially on diffusion. In any case, the diffusion rate of the at least one fluorescent probe in the medium can be controlled by the composition and / or temperature of the medium and / or the dimensions of the fluorescent probe. The composition of the medium, in particular, defines its viscosity, which is often temperature-dependent. The temperature of the medium also influences the thermal excitation of the diffusion of the at least one fluorescent probe. The dimensions of the fluorescent probe, in turn, affect its diffusion rate via the viscosity of the medium.The diffusion rate can also be influenced by the chemical or physical affinity of the fluorescent probe to the medium, such as its polarity or charge. Specifically, a glycerin-containing medium can be used as the embedding medium, and the diffusion rate can be adjusted within wide limits by modifying its temperature, glycerin content, and / or water content. A similar effect can be achieved with thiodiethanol (TDE), which is familiar to those skilled in both electron and fluorescence microscopy. For example, the formation of ice crystals in the medium can be prevented by using TDE when setting low temperatures, even if the medium is the natural environment of the macromolecule in a biological cell.When selecting the medium, its optical properties must also be considered to avoid introducing disruptive aberrations when fluorescence excitation light is irradiated into the medium and / or when fluorescent photons emitted from the medium are detected, as is necessary for determining the spatial positions of the at least one fluorescent probe by localization. Such aberrations reduce the precision achievable when determining the spatial positions of the at least one fluorescent probe.

[0028] Relative motions of the at least one fluorescent probe with respect to the macromolecule can also be caused by external excitation of the medium to vibrations and / or by a flow of the medium and / or by movement of the macromolecule relative to the medium and / or by an electric or magnetic field exerting forces on the at least one fluorescent probe in the medium. It is recommended to vary the direction of the vibrations, flow, motions, or electric or magnetic field in order to map the surface of interest of the macromolecule equally in all spatial directions. At a minimum, the direction of any flow of the medium or of an electric or magnetic field should be reversed when determining the spatial positions of the at least one fluorescent probe.

[0029] Alternatively or additionally to a relative movement of the at least one fluorescent probe with respect to the macromolecule in the medium, the spatial positions of the at least one fluorescent probe, which are determined in the method according to the invention, can be achieved by singulating varying subsets of a total number of identical fluorescent probes contained in the medium. With a sufficient spatial concentration of the total number of identical fluorescent probes, a sufficient number of spatial positions can also be achieved without any relative movement of the individual fluorescent probes with respect to the medium. The singulation of the fluorescent probes for determining their spatial positions, so that the fluorescence photons emitted by each singulated fluorescent probe can be registered separately, can be carried out in a generally known manner, e.g.,This is achieved by switching on and / or off switchable fluorescent probes. In principle, this switching can be triggered by any physical or chemical signal. The switching can occur stochastically, i.e., spontaneously, while the switching rates can be adjusted by chemical conditions such as pH, concentrations of specific buffers, etc., which are known to those skilled in the art from the literature on single-molecule switching microscopy.

[0030] Switching light is frequently used for this purpose. Thus, singulation can also be achieved by activating photoactivatable fluorescent probes with activation light, whereby the activation light can have the same wavelength as, or a different wavelength than, the fluorescence excitation light subsequently used to excite the fluorescent probes to emit fluorescence photons. Furthermore, fluorogenic fluorescent probes, which are converted into their fluorescent state by a binding process, can be used for singulation. In this case, it is particularly preferred if the bonds that convert the fluorogenic fluorescent probes into their fluorescent state are those to the surface of interest of the macromolecule.The fluorogenic fluorescent probes are then selective on the surface of the macromolecule in its fluorescent state, and no or at least fewer spatial positions of the fluorescent probe in the volume of the medium are determined that do not contain information about the surface of the macromolecule of actual interest.

[0031] In general terms, determining the spatial positions of the at least one fluorescent probe can be concentrated on the region of the expected surface of the macromolecule. To this end, the surface profile can be estimated from initially determined spatial positions of the at least one fluorescent probe, and this estimate can be improved with each further determined spatial position of the at least one fluorescent probe. Alternatively or additionally, one can start with a spatial orientation of the macromolecule's center point determined by other means and search for spatial positions of the fluorescent probe in all directions at increasing distances from this center point. In this way, at most only a few spatial positions of the fluorescent probe within the volume of the medium will be determined, which contain no information about the surface of interest of the macromolecule.

[0032] As previously mentioned, various fluorescent probes with different affinities for different regions of the macromolecule's surface can be introduced into the medium, with the determination of the majority of the spatial positions of these individual fluorescent probes being performed separately. This separation can be achieved by using different wavelengths of fluorescence excitation light or fluorescence light, or temporally, by introducing the different fluorescent probes into the medium sequentially. Similarly, the determination of the interface defining the spatial positions can be performed separately for each fluorescent probe, for example, to account for the different properties of the probes with respect to potential differences between the interface and the surface of interest.

[0033] The inventive method for mapping the surface of the macromolecule can be parallelized. This means that several copies of the macromolecule can be used and scanned in parallel with each other using at least one fluorescent probe. The information obtained for each copy of the macromolecule is then compiled to provide overall information about the surface of interest. Specifically, several copies of the macromolecule can be embedded in the medium, each with at least one fluorescent probe, and this medium can be continuous across all copies of the macromolecule.Then, a plurality of spatial positions of each of the at least one isolated fluorescent probes can be determined relative to the multiple copies of the macromolecule, and the interface can be defined such that it delineates the determined spatial positions of all isolated fluorescent probes relative to all copies of the macromolecule. It is understood that a fundamental requirement for parallelizing the method according to the invention is that the spatial positions of the individual copies of the macromolecule not only remain constant over time but are also known for all copies in order to summarize the information obtained from the individual copies about the surface of the macromolecule. This can be achieved using computer programs such as those known from structure determination by cryo-EM.

[0034] In practice, individual copies of the macromolecule can be coupled to coupling sites arranged in a fixed grid. The spatial positions of the individual fluorescent probes relative to their respective copies can then be determined using MinFlux or MinSTED, whereby the local minima or zeros of the light intensity distributions used to determine the spatial positions can be arranged in the same grid and shifted together relative to the copies of the macromolecule.

[0035] When creating a three-dimensional map of at least part of the macromolecule's surface from the interface, existing structural information about the macromolecule can be taken into account. This structural information may have been obtained, for example, through X-ray crystallography, NMR, cryo-EM, or prediction using artificial intelligence.

[0036] The method according to the invention can also be carried out repeatedly in order to detect changes in the surface of interest of the macromolecule, in particular after a structural change in the macromolecule has been caused by changes in physical and / or chemical environmental conditions and / or another macromolecule has been added to the macromolecule and / or a complex comprising the macromolecule has been at least partially dissolved.

[0037] In the inventive method, all common fluorophores are suitable as fluorescent probes or fluorophores of such fluorescent probes, in particular those that exhibit low transition probabilities to their triplet state or other dark states, so that their spatial positions can be tracked over a longer period. Specific examples of suitable fluorophores are Atto647N and Abberior Star 635.

[0038] By coupling copies of the macromolecule to coupling sites with known spatial orientations, the spatial orientations of the copies can be determined with an accuracy of a few nanometers. For typical macromolecule sizes of a few to a few tens of nanometers, the zero point of the light intensity distribution, used to determine the spatial positions of the fluorescent probes on the surface of interest of the macromolecule copies, can therefore be shifted from the outset within a very small region whose spatial orientation is defined by the respective coupling site. The grid of zero points of the intensity distributions can be formed by a so-called donut array, by standing waves, or by crossed standing waves. Specifically, two opposing lenses in a so-called 4π arrangement can be used. Standing waves in the z-direction can be generated via a substrate with a mirror surface.Standing waves can also be arranged three-dimensionally, as is generally known, such as in a stack of wood. The zero point can be limited three-dimensionally by higher intensities of the light intensity distribution. The detection of the fluorescence light from the fluorescent probe can be confocal or using a confocal detector array. Alternatively, the fluorescence light can be captured with a camera. The fluorescence light can be polarization-selective or polarization-sensitive to detect the spatial orientation of the fluorescent probe on the surface of interest of the macromolecule. For better background suppression, the use of time-resolved fluorescence detection, also known as time-gating, can be advantageous. When mapping its surface, the macromolecule can be selectively deformed, even by direct force application, to make specific areas of its surface accessible to the fluorescent probe.

[0039] Advantageous further developments of the invention result from the patent claims, the description and the drawings.

[0040] The advantages of features and combinations of features mentioned in the description are merely exemplary and can have an effect alternatively or cumulatively, without the advantages necessarily having to be achieved by embodiments according to the invention.

[0041] The features mentioned in the claims and the description are to be understood, with regard to their number, as meaning that exactly that number or a greater number than the stated number is present, without the need for the explicit use of the adverb "at least". Thus, for example, if a card is mentioned, this is to be understood as meaning that exactly one card, two cards, or more cards are produced. The features listed in the claims may be supplemented by further features or may be the only features that the method of the respective claim possesses.

[0042] The reference numerals contained in the patent claims do not constitute a limitation of the scope of the subject matter protected by the patent claims. They merely serve the purpose of making the patent claims easier to understand. BRIEF DESCRIPTION OF THE FIGURES

[0043] The invention will now be further explained and described with reference to preferred embodiments shown in the figures. Fig. 1 schematically shows a first macromolecule embedded in a medium, with fluorescent probes introduced into the medium. Fig. 2 The figure schematically shows a negative image of the first macromolecule resulting from a multitude of spatial positions of the fluorescent probes determined by localization. Fig. 3 shows different regions of a second macromolecule embedded in a medium with different fluorescent probes embedded in the medium, which have different affinities to different regions of a surface of the macromolecule. Fig. 4 shows an area of ​​the surface of a third macromolecule embedded in a medium, wherein probes introduced into the medium enter a fluorescent state upon binding to this area of ​​the surface. Fig. 5schematically shows the structure of a fluorescent probe consisting of a fluorophore and an affinity group. Fig. 6 shows a fourth macromolecule embedded in a medium, wherein a spatial position of the macromolecule is defined by coupling the macromolecule to a coupling site. Fig. 7 shows multiple copies of a fifth macromolecule coupled to coupling sites in a fixed grid spacing, and a light intensity distribution with an arrangement of local intensity minima in the same fixed grid spacing. Fig. 8 illustrates the application of an electric field to move fluorescent probes relative to a sixth macromolecule embedded in a medium. Fig. 9 illustrates a flow of a medium around a seventh stationary macromolecule. Fig. 10 is a block diagram of an embodiment of the inventive method for mapping the surface of a macromolecule; and Fig. 11Figure 1 schematically shows a map of the first macromolecule obtained as a result of the inventive method according to the inventive method. Fig. 1 . FIGURE DESCRIPTION

[0044] Fig. 1Figure 1 schematically depicts a macromolecule 1, for example a protein, embedded in a medium 2. The medium 2 can represent the natural environment of the macromolecule 1, for example, in a biological cell. The medium 2 may be modified from this natural environment. Alternatively, the medium 2 may be a special medium designed to facilitate the following steps for mapping a surface 3 of the macromolecule 1 with particular efficiency. Fluorescent probes 4 are introduced into the medium 2. The spatial positions of the fluorescent probes 4 relative to the macromolecule 1 are determined by localization, i.e.,The location of each fluorescent probe 4 is determined with such high precision by registering fluorescence photons emitted by the fluorescent probes 4 that the simple standard deviation is no greater than 2 nm, preferably no greater than 1 nm, and even more preferably no greater than 0.5 nm. For this localization, each fluorescent probe 4 must be isolated at its respective spatial position so that the fluorescence photons emitted by it can be registered separately, i.e., independently of fluorescence photons from other fluorescent probes 4, for their respective spatial positions. This can be achieved by introducing the fluorescent probes 4 into the medium 2 at a very low spatial concentration, with the different spatial positions being reached by relative motions of the fluorescent probes 4 with respect to the macromolecule 1.Alternatively or additionally, the fluorescent probes 4 can be isolated by either converting only a subset into a truly fluorescent state or leaving them in the truly fluorescent state, with each subset exhibiting a low spatial concentration. This allows for the determination of a multitude of spatial positions of the fluorescent probes 4 without requiring them to move relative to the macromolecule 1. In any case, the multitude of spatial positions of the fluorescent probes 4 results in a three-dimensional negative image of the macromolecule 1, from which the surface profile 3 can be derived. Even if... Fig. 1 It is only a two-dimensional representation and only shows the fluorescent probes insofar as they are arranged in a layer of medium 2 that is perpendicular to the viewing direction. Fig. 1As the process progresses, the spatial positions of the fluorescent probes 4 are determined in all three dimensions in order to capture the course of the surface 3 of the macromolecule 1 in all three dimensions.

[0045] The in Fig. 2 The negative image 28 shown, in which the spatial positions 27 of the fluorescent probes 4 determined by localization omit a space 29 filled by the macromolecule 1, also only considers the one layer of the medium 2 perpendicular to the viewing direction. Fig. 1 The representation of Fig. 2 is particularly schematic insofar as the spatial positions 27 of the fluorescent probes 4 determined by localization are shown in a regular arrangement for the sake of simplicity and not in their actual and, without affinities of the fluorescent probes 4 to the macromolecule 1, basically stochastic arrangement.

[0046] Fig. 3Figure 1 schematically shows two different fluorescent probes 4' and 4" with crosses and circles. The different probes 4' and 4" exhibit different affinities for different regions of the surface 3 of the macromolecule 1 embedded in the medium 2. Fig. 3 For example, nonpolar regions 6 of the surface 3, for which the fluorescent probes 4' have an increased affinity, are indicated by an additional solid line 5. Conversely, a polar region 8, for which the fluorescent probes 4" have an increased affinity, is indicated by an additional dashed line 7. By determining the spatial positions of the different fluorescent probes 4' and 4" separately, the different regions 6 and 8 can be distinguished when determining the profile of the surface 3.

[0047] Fig. 4This illustrates the case where fluorogenic fluorescent probes 4‴ in the medium 2 only reach their fluorescent state 9 and their spatial positions can only be determined if they bind to a specific region 6 of the surface 3. On the one hand, this allows only spatial positions of the fluorescent probes 4‴ that contain information about the surface 3 to be determined, and on the other hand, it specifically identifies the region 6 of the surface 3 to which the fluorogenic fluorescent probes 4‴ bind.

[0048] Fig. 5Figure 1 schematically shows the structure of a fluorescent probe 4 consisting of a fluorophore 10 and an activity and / or affinity group 11. The activity and / or affinity group 11 can define the affinity of the fluorescent probe 4 to specific regions 6, 8 of the surface 3 of the macromolecule 1, or impart fluorogenic properties to the fluorescent group 4, or make the fluorescent probe 4 photoactivatable, switchable on or off, or increase the dimensions of the fluorescent probe 4 to reduce its diffusion rate in the medium 2, or the like.

[0049] Fig. 6This illustrates that the macromolecule 1 in the medium 2 is coupled to a coupling site 12. As a result, the macromolecule 1 in the medium 2 has a defined position and spatial orientation relative to a base 13 and thus relative to external reference points. The spatial positions of the fluorescent probes 4 relative to the base or the external reference points can therefore be transformed into spatial positions relative to the macromolecule 1. Fig. 6 It is further indicated that only a few fluorescent probes 4 are introduced into the medium 2, which move along undirected diffusion paths 14 due to diffusion in the medium 2. In this way, the fluorescent probes 4 reach different spatial positions relative to the macromolecule 1, which can be determined in order to record the orientation of the surface 3 of the macromolecule 1. Fig. 6Furthermore, a double arrow 15 indicates that the macromolecule 1 can be moved relative to the medium 2, for example via its coupling site 12, or set into vibration, in order to provoke additional relative motions between the fluorescent probes 4 and the macromolecule 1. However, care must be taken to ensure that forced movements of the macromolecule 1 relative to the medium 2 do not lead to motion inaccuracies when determining the spatial positions of the fluorescent probes 4 relative to the macromolecule 1.

[0050] In Fig. 7The diagram schematically depicts several copies of the macromolecule 1 being arranged in a fixed grid by coupling to coupling sites 12, which are themselves arranged in a fixed grid relative to a base 4 and aligned with respect to their spatial positions. Simultaneously, a light intensity distribution 16 across the arrangement of coupling sites 12 is indicated (very schematically), exhibiting local minima or zeros 17 spaced within the same grid. If the spatial positions of the fluorescent probes 4 are determined, for example, by the method known as MinFlux, the zeros 17 of the light intensity distribution 16 are those of fluorescence excitation light. If the fluorescent probes 4 are localized by the method known as MinSTED, the zeros 17 are those of fluorescence suppression or STED light, which is directed onto the copies of the macromolecule 1 together with fluorescence excitation light.In any case, the light intensity distribution 16 can be shifted overall relative to the totality of the coupling sites 12 and the coupled copies of the macromolecule 1 in order to determine parallel spatial positions of fluorescent probes 4 relative to each of the copies of the macromolecule 1.

[0051] Fig. 8 Figure 1 illustrates how fluorescent probes 4"" with a net charge are moved by an electric field between an external anode 18 and an external cathode 19 relative to the macromolecule 1 embedded in the medium 2. Since the fluorescent probes 4"" do not reach spatial positions on the back side of the macromolecule 1 opposite their direction of movement, it is useful to vary the direction of the electric field, or at least to reverse it once. Furthermore, in Fig. 8It is indicated that position markers 20 are attached to the macromolecule 1. By determining the spatial positions of these at least three position markers 20, which do not lie on a straight line, the spatial orientation of the macromolecule 1 can be determined and, in particular, tracked if the macromolecule 1 can move in the medium 2. A spatial orientation of the macromolecule 1 that remains constant or is known across at least some spatial positions of the fluorescent probes 4"" is a prerequisite for determining the spatial positions of the fluorescent probes 4"" relative to the macromolecule 1.

[0052] In Fig. 9It is indicated that the macromolecule 1 is arranged in a flow 21 of the medium 2 with the fluorescent probes 4 introduced therein, the macromolecule 1 being held in place by coupling to a coupling site 12 relative to a base 13. Even with the resulting relative motion of the fluorescent probes 4 with respect to the macromolecule 1, the fluorescent probes 4 do not reach spatial positions on the back side of the macromolecule 1 opposite their direction of motion, and a variation of the direction of the flow 21 relative to the macromolecule 1, or at least a reversal of the direction of the flow 21, is advisable.

[0053] Fig. 10Figure 1 is a block diagram of a method according to the invention for mapping the surface 3 of the macromolecule 1. Embedding 22 of the macromolecule 1 in the medium 2 is optional insofar as the natural environment of the macromolecule 1, for example in a biological cell, can be used as the medium 2. This is followed by the introduction 23 of the fluorescent probe 4 into the medium 2. Alternatively, the fluorescent probes 4 can already be embedded in the medium 2 when the macromolecule is embedded in the medium 2. When determining 24 the spatial positions 24 of the fluorescent probes 4 relative to the macromolecule 1, it is possible to distinguish between different fluorescent probes 4 with different chemical and physical properties and, consequently, different affinities to different regions 6 and 8 of the surface 3 of the macromolecule 1.The determination of an interface 25, which delineates the determined spatial positions relative to the macromolecule, follows. This interface essentially corresponds to the surface of interest 3 of the macromolecule 1. However, when creating 26 a three-dimensional map of the spatial distribution of the surface 3, properties of the fluorescent probes 4 are taken into account. This concerns the dimensions of the fluorescent probes 4, which lead to an offset between their determined spatial positions and the surface of interest 3, even when the fluorescent probes 4 bind directly to the surface 3. Furthermore, different affinities of the fluorescent probes 4 are considered in order to include different regions 6 and 8 of the surface 3 of the macromolecule 1 with different physical and / or chemical properties in the map.When creating the surface map 26, structural information available from other sources for macromolecule 1 can also be taken into account, for example, information obtained from a structural formula or primary structure using X-ray crystallography, NMR, cryo-EM, or artificial intelligence. Macromolecule 1 is, in particular, a protein or a protein complex. The resulting surface map 3 therefore includes essential information on the tertiary structure of the respective protein or the quaternary structure of the respective protein complex.

[0054] Fig. 11 shows a simplified example of map 30 of macromolecule 1 according to Fig. 1 , which is obtained as a result of the inventive method. Despite the again only two-dimensional representation in Fig. 11 Is the received map actually three-dimensional? It contains various symbols, numbered 31 to 33. Fig. 11Various regions of a plotted profile 34 on the surface 3 are marked, in which the macromolecule exhibits different physical and / or chemical properties at its surface. A weaker expression of these properties is indicated by unfilled symbols 31 to 33, and a stronger expression of these properties is indicated by filled symbols 31 to 33. The expression of the physical and / or chemical properties can also be represented in a more differentiated manner in the map 30, for example, by specifying absolute or relative binding constants, which can be determined by tracking fluorescent probes 4 moving in the medium 2 over time, each with its own defined physical and / or chemical properties, within the framework of the method according to the invention.Together with the spatial arrangement 34, the physical and / or chemical properties, which include in particular polarity, hydrophilicity and lipophilicity, but also bond strengths for bonds by van der Waals forces, hydrogen bonds, electrostatic attraction or the like, determine the ability or tendency of the macromolecule 1 to form bonds with other molecules or macromolecules in individual areas of its surface 3. REFERENCE MARK LIST

[0055] 1 Macromolecule 2 Medium 3 Surface 4 Fluorescent probe 5 Solid line 6 Region 7 Dashed line 8 Region 9 Fluorescent state 10 Fluorophore 11 Activity or affinity group 12 Coupling site 13 Base 14 Diffusion path 15 Double arrow 16 Light intensity distribution 17 Zero 18 Anode 19 Cathode 20 Position marker 21 Flow 22 Embedding 23 Introducing 24 Determining spatial positions 25 Determining an interface 26 Creating 27 Determined spatial position 28 Negative image 29 Space 30 Map 31-33 Symbol 34 Course

Claims

1. Method for mapping the surface (3) of a macromolecule (1) comprising the steps of: - introducing (23) at least one fluorescent probe (4) into a medium (2) in which the macromolecule (2) is or will be embedded, - determining a plurality of spatial positions of the at least one fluorescent probe (4) with regard to the macromolecule (1) by localization of the at least one singularized fluorescent probe (4) at a single standard deviation of not more than 2 nm, wherein fluorescence light photons emitted by the singularized fluorescent probe (4) are registered, - determining a bounding surface bounding the determined spatial positions (27) with regard to the macromolecule (1), and - generating a three-dimensional map (30) of at least a part of the surface (3) of the macromolecule (1) from the bounding surface.

2. Method of claim 1, characterized in that so many spatial positions of the at least one fluorescent probe (4) are determined that an average surface density of the spatial positions (27) determined at the surface (3) of the macromolecule (1) is at least 25 or at least 50 or at least 100 or at least 200 positions per 100 square nanometers.

3. Method of claim 1 or 2, characterized in that a local surface density of the determined spatial positions (17) is registered along the bounding surface and entered into the three-dimensional map (30).

4. Method of any of the preceding claims, characterized in that, in generating the three-dimensional map (30), properties of the at least one fluorescent probe (4) are considered, which are selected from a group including dimensions of the at least one fluorescent probe (4) and physical or chemical affinities of the at least of one fluorescent probe (4) to special areas of the surface (3).

5. Method of claim 4, characterized in that binding affinities and / or binding constants for molecules with predetermined physical and / or chemical affinities are derived from the determined spatial positions (27) and the properties of the at least one fluorescent probe (4), and entered into the three-dimensional map (30).

6. Method of any of the preceding claims, characterized in that the at least one fluorescent probe (4) is provided with an affinity group (11) which is selected such that it has an increased or inclusive affinity for special areas (6, 8) of the surface (3) of the macromolecule (1) with special chemical and / or physical properties.

7. Method of any of the preceding claims, characterized in that a spatial arrangement of the macromolecule (1) - is predetermined by coupling the macromolecule (1) in a fixed orientation to a fixed coupling site (12) in the medium (2), and / or - is fixed by means of the medium (2) in which it is embedded, and / or - is determined by determining at least one spatial marker position of at least one position marker (20) attached to the macromolecule (1).

8. Method of any of the preceding claims, characterized in that the step of determining the plurality of the spatial positions of the at least one singularized fluorescent probe (4) with regard to the macromolecule (1) is implemented by localization by means of MinFlux or MinSTED.

9. Method of any of the preceding claims, characterized in that the at least one fluorescent probe (4) is singularized by adjusting a low spatial concentration of the at least one fluorescent probe (4) in the medium (2).

10. Method of any of the preceding claims, characterized in - that the spatial positions of the at least one fluorescent probe (4) are reached within the medium (2) by relative movements with regard to the macromolecule (1), - wherein, optionally, - a diffusion velocity of the at least one fluorescent probe (4) within the medium (2) is adjusted by a composition and / or a temperature of the medium (2) and / or dimensions and / or chemical and / or physical properties of the fluorescent probe (4), and / or - the relative movements are elicited by: - an external excitation of the medium (2) for vibrations and / or - a flow of the medium (2) and / or - movements of the macromolecule (1) with regard to the medium (2) and / or - an electric or magnetic field.

11. Method of any of the preceding claims, characterized in that the spatial positions of the at least one fluorescent probe (4) are achieved by means of singularizing alternating subsets of a total number of equal fluorescent probes (4) included in the medium (2), wherein, optionally, the step of singularizing is implemented - with switching on and / or off switchable fluorescent probes (4), and / or - with activating photoactivatable fluorescent probes (4), and / or - with using fluorogenic fluorescent probes (4"').

12. Method of any of the preceding claims, characterized in that different fluorescent probes (4', 4") with different affinities to different areas of the surface (3) of the macromolecule (1) are introduced in the medium (2), wherein the step of determining the plurality of the spatial positions and, optionally, the step of determining the bounding surface bounding the spatial positions is separately implemented for the different fluorescent probes (4', 4").

13. Method of any of the preceding claims, characterized in - that several copies of the macromolecule (1), each together with at least one fluorescent probe (4), are embedded in the medium (2), - that a plurality of spatial positions of the respective at least one singularized fluorescent probe (4) are determined with regard to each of the several copies of the macromolecule (1), and - that the bounding surface is determined such that it bounds the determined spatial positions (27) of all singularized fluorescent probes (4) with regard to all copies of the macromolecule (1).

14. Method of any of the preceding claims, characterized in that, in the step of generating the three-dimensional map (30) of at least a part of the surface (3) of the macromolecule (1) from the bounding surface, already present structural information on the macromolecule (1) is considered.

15. Method of any of the preceding claims, characterized in that the step of determining the spatial positions of the at least one fluorescent probe (4) with regard to the macromolecule (1) and the step of determining the bounding surface bounding the spatial positions with regard to the macromolecule (1) are repeated after - a structural change in the macromolecule (1) has been caused by a change of physical or chemical surrounding conditions, and / or - a further macromolecule (1) has been attached to the macromolecule (1), and / or - a molecule complex including the macromolecule (1) has at least partially been dissolved.