Methods for soft landing of macromolecules

Abstract

The present disclosure, inter alia, includes methods for performing soft landing under cryogenic conditions with macromolecules such as proteins and protein complexes. In some embodiments, the methods herein may be useful for preparing samples for cryoelectron microscopy (cryo EM). In some embodiments, the methods may be performed in a cryogenic-compatible soft landing system as described herein. In other embodiments, a different cryogenic-compatible soft landing system may be employed for soft landing of the macromolecules.

Description

[0001] METHODS FOR SOFT LANDING OF MACROMOLECULES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to US Provisional Patent Application No. 63 / 519,550, filed August 14, 2023, which is incorporated by reference herein in its entirety.

[0004] FIELD

[0005] This disclosure relates to novel methods for soft landing of proteins, protein complexes, and other macromolecules under cryogenic conditions. Methods herein may be useful in preparing samples for cryogenic electron microscopy and other imaging techniques.

[0006] BACKGROUND

[0007] Soft landing (SL) is a technique for deposition of a gas phase ion onto a surface, where the ion adheres without the formation of a covalent bond and without significant modification to its structure (14). Most commonly, a charged molecular ion is generated from a solid or solution material by an electrospray source, or alternatively by an ionization method that preserves noncovalent interactions. Electric and magnetic fields and gas flows may then be used to manipulate the trajectory of gas phase ions of an analyte of interest (15, 16). For example, ions may be filtered for those that fall within a particular m / z value, or mobility instruments may be used to select ions based on their collisional cross sectional (CCS) area. A landing surface is then used to collect the desired ions containing the analyte. In many cases, this process takes place under vacuum. Soft landing has been successful with small molecules, for instance, to prepare surface-modified chips and microarrays and the like. It has also been successful with certain polymers such that rationally designed monomers can be built onto chips to provide various coatings with applications in the optical coating and electronics fields (14, 17-24). It would be useful to employ soft landing techniques also with more complex molecules, such as proteins, polynucleotides, protein complexes, viruses, and the like, for example, for a variety of biological assays and imaging, such as cryo EM imaging. However, the delicate folding and structure of such molecules makes soft landing particularly challenging. (See, e.g., refs. 19, 22, 23, 25, 26, 27, 28.) For example, many efforts in protein soft landing imaging have stalled because of denaturation of proteins as a function of charge and landing time (18, 26, 28). Hence, there is a need in the field for improved systems for soft landing of proteins and other macromolecules and macromolecular complexes. SUMMARY

[0008] The present disclosure, inter alia, includes methods for performing soft landing under cryogenic conditions with macromolecules such as proteins and protein complexes. In some embodiments, the methods herein may be useful for preparing samples for cryoelectron microscopy (cryo EM). In some embodiments, the methods may be performed in a cryogenic- compatible soft landing system as described herein. In other embodiments, a different cryogenic-compatible soft landing system may be employed for soft landing of the macromolecules.

[0009] Exemplary embodiments herein include the following, as well as others described elsewhere below in the sections that follow this Summary:

[0010] 1. A method of preparing and isolating positively charged macromolecular ions comprising an analyte, the method comprising: a. ionizing a sample comprising a macromolecular analyte with an electrospray source to form positively charged macromolecular ions; b. separating the positively charged macromolecular ions on the basis of their collisional cross sectional area, for example, in a DMA or nDMA; and c. directing positively charged macromolecular ions comprising the analyte to a landing pedestal under cryogenic conditions, and depositing the positively charged macromolecular ions comprising the analyte onto a grid located on the landing pedestal, wherein the grid has been pre-treated with plasma, or by deposition of hydrophilic molecules, or wherein the landing pedestal is potentiated so as to form a negatively charged grid.

[0011] 2. The method of embodiment 1, wherein the macromolecular analyte is a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide.

[0012] 3. The method of embodiment 1, wherein the macromolecular analyte is a polypeptide or polypeptide complex.

[0013] 4. The method of any one of embodiments 1-3, wherein the positively charged macromolecular ions have a +1 to +5 charge.

[0014] 5. The method of embodiment 4, wherein the positively charged macromolecular ions have a +1 charge.

[0015] 6. The method of any one of embodiments 1-5, wherein the positively charged macromolecular ions are deposited onto the grid in an aerosol stream of from 1 L / minute to 10 L / minute, or from 1 L / minute to 5 L / minute, or from 1 L / minute to 4 L / minute, or from 1 L / minute to 2 L / minute, or at 1 L / minute, 2 L / minute, 3 L / minute, or 4 L / minute. 7. The method of any one of embodiments 1-6, wherein cryogenic conditions comprise a temperature of -60 °C or lower, such as from -60 °C to -170 °C, -80 °C to -170 °C, -60 °C to -160 °C, -60 °C to -140 °C, -80 °C to -140 °C, or -120 °C to -140 °C.

[0016] 8. The method of any one of embodiments 1-7, wherein the grid has been pre-treated with plasma.

[0017] 9. The method of any one of embodiments 1-7, wherein the grid has been pre-treated by deposition of trehalose.

[0018] 10. The method of any one of embodiments 1-7, wherein the landing pedestal is held at a potential of -100V to -600V, or -200V to -600V, or -300V to -600V, or -400V to - 500V, or -450V to form a negatively charged grid.

[0019] 11. The method of any one of embodiments 1-10, wherein the positively charged macromolecular ions are deposited on the grid as a charged droplet or a charged macromolecule comprising a hydration shell.

[0020] 12. The method of embodiment 11, wherein the positively charged macromolecular ions are deposited in random orientations within the droplet or hydration shell.

[0021] 13. The method of any one of embodiments 1-12, wherein the grid is a removable grid.

[0022] 14. The method of any one of embodiments 1-13, wherein the grid comprises an amorphous carbon, graphene, or graphene oxide support film.

[0023] 15. The method of any one of embodiments 1-14, wherein the method further comprises removing the grid after deposition of the positively charged macromolecular ions on the grid, optionally wherein the grid is removed under a gas with a dew point less than the operating temperature of the landing chamber.

[0024] 16. The method of embodiment 15, further comprising submerging the removed grid with deposited ions in liquid nitrogen.

[0025] 17. The method of any one of embodiments 1-16, wherein the electrospray source is a charge reduced electrospray source.

[0026] 18. The method of any one of embodiments 1-17, wherein the separating of the positively charged macromolecular ions on the basis of their collisional cross sectional area is performed by an nDMA.

[0027] 19. The method of any one of embodiments 1-18, wherein the landing pedestal is enclosed within a landing chamber. 0. The method of embodiment 19, wherein the landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature. 21. The method of embodiment 19 or 20, wherein the electrospray source, an DMA or nDMA, and landing chamber are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing pedestal.

[0028] 22. The method of any one of embodiments 19-21, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the positively charged macromolecular ions from the DMA or nDMA to the landing pedestal.

[0029] 23. The method of any one of embodiments 19-22, wherein the landing chamber further comprises a focusing lens.

[0030] 24. The method of embodiment 23, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

[0031] 25. The method of any one of embodiments 1-24, wherein the landing pedestal is in contact with a cooling unit, wherein the cooling unit optionally comprises a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar.

[0032] 26. The method of embodiment 25, wherein the landing pedestal comprises a central tube allowing flow of gas cooled by the cooling unit.

[0033] 27. The method of embodiment 25 or 26, wherein the landing pedestal is in contact with the cooling unit indirectly through a landing chamber enclosing the landing pedestal being in direct contact with the cooling unit.

[0034] 28. The method of any one of embodiments 1-27, wherein the system further comprises an automatic temperature control element.

[0035] 29. The method of any one of embodiments 19-28, wherein the landing pedestal is fully enclosed within the landing chamber during landing.

[0036] 30. The method of any one of embodiments 19-28, wherein the bottom of the landing chamber is open to atmosphere during landing, and wherein the chamber is located above one or more cold traps, wherein the one or more cold traps are optionally made of metal, wherein the one or more cold traps are optionally in direct contact with liquid nitrogen, and wherein vaporized liquid nitrogen forms a nitrogen gas head space in the landing chamber during landing.

[0037] 31. The method of embodiment 30, wherein the landing chamber further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

[0038] 32. The method of any one of embodiments 1-18, wherein the landing pedestal is comprised within a landing apparatus, in which apparatus the landing pedestal is located above a first platform, the first platform being in contact with a liquid nitrogen source, and wherein the top of the landing pedestal is below or in line with a second platform, wherein the second platform and the first platform together are positioned so as to allow vaporized nitrogen gas from the liquid nitrogen source to displace atmospheric air at the top of the landing pedestal, wherein optionally the first and / or second platform is a cryo EM clipping station.

[0039] 33. The method of embodiment 32, wherein the landing apparatus further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

[0040] 34. The method of embodiment 32 or 33, wherein the electrospray source, an DMA or nDMA, and landing apparatus are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing apparatus.

[0041] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. All references cited herein are incorporated by reference into this disclosure in their entireties.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] This disclosure includes a number of figures, (Figs. 1-28), which help to illustrate particular embodiments or concepts described herein. These figures are summarized either in text provided with the figure or in the Examples section below. Abbreviations used within certain figures herein include the following: CAD = computer-aided design; SLD = soft landing device; nDMA = nano differential mobility analyzer; IM = ion mobility; EM = electron microscopy; MS = mass spectrometry; ESI = electrospray ionization; SEM = scanning electron microscopy. A color version of certain figures herein was submitted in priority application No. 63 / 519,550, which is incorporated by reference herein.

[0044] Fig. 1 provides an overview of certain major components of a soft landing device (SLD) described herein. In (1), a 210Po charge reduction ESI source (lonDX, Inc.) is coupled to (2) a nDMA (TSI, Inc.). Ions are directed into (3) a custom electrostatic focusing lens embedded within (4) a landing chamber. Different chambers were tested for atmospheric or atmospheric / cryogenic landing and various focusing lens were integrated during optimization of the SLD designs.

[0045] Figs. 2A-2B. The detection of (Fig. 2A) NSP4 or (Fig. 2B) ubiquitin proteins was achieved by coating the electrospray emitter with Sigmacote. Due to the small molecular weight of the proteins, a significant amount of electrospray oligomer artifact was additionally observed. The main fitted peaks of the monomer and dimers are shown in red, with the raw data shown in blue. Experiments of these and other small proteins infused with covalent coated capillary, bare fused silica, peek or buffer additives were generally not possible or extremely unreliable.

[0046] Fig 3 shows an exploded view of an atmospheric-only landing chamber.

[0047] Figs. 4A-4C show the pedestal top geometry for landing at atmospheric conditions. The top of the pedestals is made of brass, which is connected to an acetal plastic hollow ’A” tube. The total length of the hollow tube was adjusted for the atmospheric or the cryogenic landing chamber designs. Inside the tube is a wire to transmit current from the brass pedestal top, or from a grid seated on the brass pedestal top, to a detector. Fig. 4A shows the groove to fit autogrid style tweezers. Fig. 4B shows the groove to support tweezers for manipulating unclipped grids. Fig. 4C provides an illustration of a grid being inserted or removed from the pedestal top. The slot underneath the grid is to allow clearance for tweezers. The ridges ensure that the grid is securely seated and cannot fall off.

[0048] Figs. 5A-5C show three views of all components of the pedestal used for current detection at atmospheric conditions is shown. Figs. 5A and Fig. 5B show the same component in which the view is rotated, while Fig. 5C provides a sliced view. The acetal plastic hollow tube holds a wire connected to the brass top. A set-screw stopper can be adjusted to move the relative position of the pedestal to the focusing lens. The wire is connected to a BNC connector such that it may be subsequently connected to a detector.

[0049] Figs. 6A-6F. Fig. 6A shows a photo of the final ion focusing lens. Fig. 6B shows Simlon simulation of a protein transmitted through the lens from the chamber inlet to the pedestal at a DMA flow of 20 L / min and an aerosol in / out and ESI flow of 2 L / min. Fig. 6C shows a CAD drawing showing the funnel suspended in the cage in 3D. Fig. 6D shows a 2D view of the ion funnel seated in the cage. Fig. 6E shows a schematic of the atmospheric-only landing chamber, where the grey lines represent the ion funnel. Fig. 6F shows a CAD of the chamber inlet diffuser assembly, where a felt pad is inserted into a mesh backed ring.

[0050] Figs. 7A-7D. For the data shown in these figures, 200 nM aldolase was infused into the nDMA with a 50 pm ESI tip and analyzed at different nDMA settings through detection on the landing pedestal in the atmospheric system. The aldolase tetramer was detected at ~0.3 kV and the monomer at -0.17 kV. The ramp rate (RR) was calculated as the DMA voltage range divided by the ramp rate time (kV / s) and the flat time (FT) corresponded to the time the nDMA was held at baseline prior to initiating another scan. Fig. 7A shows comparisons of the impact of FT on peak shape and position at two different RR. Regardless of RR, increasing the FT resulted in a rightward shift to the peak position, indicating a minimum FT was required for the nDMA to discharge. Fig. 7B shows that signal intensity was maximized at slow RR, and eventually converged around a true peak intensity. Fig. 7C shows a comparison of the CV in the amplitude and area of the peaks at different RR and focusing voltages. At a slow enough ramp rate, the CVs converged to values < 10 % and became similar to quantitation performed with a normalized pedestal reading at a constant transmission voltage (on / off analysis). Fig. 7D is a plot of the raw data for the experiments performed in Fig. 7C. The maximal focusing voltage between the pedestal constant transmission mode, on / off experiments began to converge with the peak fitting analysis at slow ramp speeds. Likewise, the area and amplitude of the peak began to converge at slower speeds.

[0051] Fig. 8 shows signal for 200 nM thyroglobulin in constant nDMA transmission mode that was taken at focusing voltage zero pre and post a focusing lens voltage sweep experiment. Even after 10 min, the signal measured post sweep was significantly higher. This indicated that some buildup of charge remained on the pedestal without being fully discharged to ground.

[0052] Figs. 9A and 9B show photos of (Fig. 9A) the atmospheric-only and (Fig. 9B) the atmospheric and cryogenic system built for soft landing.

[0053] Fig. 10 provides an exploded view of the cryolanding chamber.

[0054] Figs. 11 A-l IB show a cryo-SLD cryolanding chamber and metal dish assembly, with landing chamber in (Fig. 11 A) the closed position for landing, and in (Fig. 1 IB) the open position for grid manipulation.

[0055] Fig. 12 is a detail of the cryolanding chamber exhaust assembly, where the reversible flow for pedestal sheath gas enters through the tee.

[0056] Fig. 13 is a cryogenic pedestal view detail. Cold gas flows up through the center tube to cool the pedestal.

[0057] Figs. 14A and 14B provide (in Fig. 14A) a schematic and (in Fig. 14B) detail cutaway of the cooling system that uses a resistive heating element submerged in liquid nitrogen. Note that the tapered cap also serves as pressure relief in case of excessive pressure build-up.

[0058] Fig. 15 shows a detail of a banana dewar designed to sit around the landing chamber in the metal dish. Filling it with liquid nitrogen enables transfer of the grid from the pedestal to a grid holder.

[0059] Figs. 16A-B show details of the grid box holder for grid transfer (Fig. 16A), and of the grid holder sitting in the banana dewar (Fig. 16B). Figs. 17A-17B show photos of the (Fig. 17A) hat to shield the cryogenic landing chamber during atmospheric mode for current measurement and (Fig. 17B) dome to trap liquid nitrogen headspace gas to provide a dry environment after lifting the landing chamber.

[0060] Fig. 18 shows a cryo-TEM image of lacey carbon grids landed for 30 min with GroEl at - 20 C. The large dark spots represent condensate from grid transfer into the TEM. The smaller spots are GroEl. The concentration of GroEl on the C film, rather than within the holes, is an indicator that the graphene film has been ripped.

[0061] Figs. 19A-19B show cryo-EM images of GroEl (Fig. 19A) on a 2000mesh Cu grid with 1 layer graphene prepared by cold-landing for 20 min at -20° C or (Fig. 19B) on a UltrAuFoil Rl.2 / 1,3 holey gold by traditional spotting and vitrification procedures. As shown in the insets in A, good contrast was observed, and the top-down ring / side barrel views were observed; also evident were locations where GroEl was landed on top of each other, which was not observed in Fig. 19B.

[0062] Fig. 20. Protein-less ice balls were analyzed to determine the structure of ice landed on- grid. The lack of diffraction spots in the averaged amplitude FFT spectrum suggested that a noncrystalline ice was formed.

[0063] Figs. 21A-C show photographs of a second cryogenic soft landing system (Figs. 21A-B showing a grid positioned in the system and the whole system, respectively), and an example of GroEL landed at liquid nitrogen temperature at 73,000 magnification (Fig. 36C). As shown in Fig. 36C, GroEL landed at liquid nitrogen showed distinct shapes from warmer temperatures at 73,000 magnification.

[0064] Figs. 22A-22C. Figs. 22A-22B show a third cryogenic instrument design in closed (Fig. 22A) and open (Fig. 22B) positions, in which a landing pedestal is not contained in a chamber, but that is instead positioned between two platforms, one platform located below the landing grid but above a liquid nitrogen cooling element such as a tank or dewar, and a second platform surrounding and in line with the landing grid, wherein liquid nitrogen gas forms a headspace between the two platforms sufficient to expel the surrounding atmospheric gas so that condensation on the pedestal and grid is reduced. Fig. 22C shows landing of GroEL particles in this system. Limited GroEl particles were observed due to low current, but importantly, the amount of condensate was minimal demonstrating the potential of this approach.

[0065] Figs. 23 A and 23B show data obtained from the second cryogenic landing system of Figs. 21 A-B. Fig. 23A shows condensate levels at lOL / minute while Fig. 38B shows levels at 20L / minute. Fig. 23 A compared to Fig. 23B shows that at 10 L / min flow, there are significantly larger ice crystals on the grid than at 20 L / min flow, when excluding the largest crystals which are a consequence of grid transfer steps.

[0066] Figs. 24A-24B show comparison of micrographs of AAV in 200 mM ammonium acetate landed on (Fig. 24A) C film or (Fig. 24B) plasma treated C film grids.

[0067] Figs. 25A-25C show comparison of class averages of 2D cryoEM images of AAV capsids in 200 mM ammonium acetate and 0.1% UDM landed on (Fig. 25 A) plasma treated (Fig. 25B) trehalose coated or (Fig. 25C) negatively charged carbon film grids. Some AAV capsids appear to feature dehydrated central cavities leading to lower pixel densities in the images, most evident in Fig. 25A and Fig. 25B. Most capsids landed in these experiments appear significantly deformed relative to the expected native shape of the capsid, though some subset of particles appear to have a grossly preserved capsid geometry (e.g. third image from left in Fig. 25A). Dark and bright annular rings around the edges of the capsids are due to imperfect correction for the electron microscope’s contrast transfer function (CTF) in this experiment.

[0068] Fig. 26 shows 2D class averages of images of GroEL particles soft-landed on a negatively charged grid demonstrate that GroEL assemblies land and freeze onto the grid surface in multiple orientations, including the stereotypical top view (heptameric, down the long 7-fold axis of the assembly) and side view (rectangular, down the shorter 2-fold axis). In the case of the top view (second and fourth panel), internal features of the native protein fold appear preserved, with seven-fold symmetric protein features and a hollow core similar to those seen in the equivalent views seen in conventional cryoEM. Averages of side views (first and third image) appear to partially resolve the two heptameric rings.

[0069] Fig. 27 shows 2D class averages of images of AAV capsids in ammonium acetate buffer soft-landed on a negatively charged grid. Both views feature a well-resolved ring of density corresponding to the protein capsid. The outer dark annulus is an artifact of CTF correction, and does not relate to the protein or droplet structure.

[0070] Fig. 28 shows a section through a 3D tomographic reconstruction of an AAV particle in 200 mM ammonium acetate and 0.1% UDM soft-landed onto a grid blotted with trehalose. A transparent blue sphere is overlay ed, which has the diameter of an intact AAV capsid. The grid’s carbon surface is approximated with the yellow line.

[0071] DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0072] Definitions

[0073] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0074] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only.

[0075] Terms such as “element” or “component” or “unit” may be used interchangeably to refer to parts of an apparatus or system.

[0076] As used herein, the transition term “consisting essentially of,” when referring to steps of a claimed process or method signifies that the process or method comprises no additional steps beyond those specified that would materially affect the basic and novel characteristics of the process or method. As used herein, the transition term “consisting essentially of,” when referring to a composition or product signifies that it comprises no additional components beyond those specified that would materially affect its basic and novel characteristics.

[0077] As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise.

[0078] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0079] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0080] The term “soft landing” herein refers to the deposition of a gas phase ion onto a surface, where the ion adheres to the surface noncovalently and without significant structural modification, such as significant protein unfolding or mis-folding in the case of a protein ion.

[0081] An “analyte” herein refers to a molecule or substance to be analyzed, such as a small molecule, peptide, polypeptide, macromolecule, or a complex of any of the above.

[0082] The term “small molecule” as used herein refers to an organic molecule having a molecular weight of 50 Daltons to 2500 Daltons.

[0083] The term “peptide” as used herein refers to a chain of fifty amino acids or less linked by peptide bonds, including amino acid chains of 2 to 50, 2 to 15, 2 to 10, 2 to 8, or 6 to 14 amino acids. A “polypeptide” or “protein” interchangeably herein typically refers to a chain of amino acids longer than that of a peptide.

[0084] A “macromolecule” as used herein refers to a molecule that is larger than a “small molecule” and includes, for example, biological polymers such as proteins, nucleic acids, and virus particles, and complexes comprising proteins and nucleic acids, as well as non-biological, synthetic polymeric molecules.

[0085] A “charged ion” herein, such as a “charged protein ion” or a “charged macromolecular ion” refers to a molecular ion that carries a charge, wherein the charge may be located on the molecule itself, or in or on the surface of a surrounding hydration shell. The charged ion may be in the form of a charged droplet, a charged molecule comprising a hydration shell (i.e., a hydrated ion), or a dry (i.e., anhydrous) ion. In a “charged droplet,” for example, the molecule may be surrounded by a aqueous shell, wherein either or both of the molecule and the surrounding aqueous shell may comprise the charge. Similarly, in the case of a “hydrated ion,” where the charged ion comprises a hydration shell, and thus is not anhydrous, either the molecule and / or the molecules of the aqueous shell may comprise the charge. Where the charged ion is in a “dry state” or “anhydrous state,” it does not contain a significant amount of surrounding water molecules.

[0086] “Cryogenic” herein is an adjective referring to the use of cold temperatures, such as due to cooling of equipment or samples with liquid nitrogen or in an appropriate freezing device that produces temperatures below 0 °C.

[0087] “Atmospheric” herein is an adjective referring to ambient air, i.e., the normal surrounding atmosphere, or its use. “Atmospheric conditions” herein refer to conditions under ambient pressure, or in which there is no device applied to deliberately change the pressure. “Non- atmospheric” conditions herein means conditions other than those at ambient pressure.

[0088] An “electrospray source” or “electrospray ionization source” or “ESI source” refers to an apparatus that applies electricity to a solution for the purposes of producing an aerosol comprising of charged droplets, usually containing an analyte, that may subsequently yield charged ions.

[0089] The term “charge reduction” as used herein refers to a device or method that can reduce the overall charge of a charged ion from an electrospray source.

[0090] A “charge reduced electrospray source” herein refers to an electrospray source that incorporates a charge reduction method or device.

[0091] A “differential mobility analyzer” or “DMA” refers to a device that separates charged ions on the basis of their collisional cross sectional area. In a DMA, ions travel within a sheath gas and take a trajectory directing them to an outlet when attracted to a center rod of opposite polarity at a given voltage. A “nano differential mobility analyzer” or “nDMA” is a type of DMA that uses a shortened center rod, and may be used, for example, to separate analytes with improved resolution.

[0092] A “chamber” in some embodiments herein refers to a space that is “enclosed.” A “chamber” herein can have any appropriate shape. As used herein, the term “enclosed,” such as referring to a landing pedestal in a chamber, means that the landing pedestal is configured to be inside the chamber space. A “fully enclosed” chamber herein refers to a chamber that is enclosed on all sides. In some other cases, the chamber may be enclosed on all but one side or face, or allowing for openings on one side or face, such as at the bottom face. In some cases, a chamber may be enclosed on either all but one side or on all sides, except for appropriate ports for tubing or connections that allow gas or charged ions to flow into and / or out of the chamber. Certain chambers herein are capable of operation under non-atmospheric conditions by being protected from entry of ambient air, either by being fully enclosed as noted above or by other means. In some embodiments, fully enclosing a chamber may help in keeping out ambient air so that the system is closed to atmosphere. In some embodiments, for example, a fully enclosed chamber may in some embodiments have an air-tight seal, such as via an O-ring. In other embodiments, a fully enclosed chamber does not have an airtight seal. For example, in certain cases an airtight seal might not be necessary to sufficiently shield a landing pedestal from ambient air. In other systems herein, a chamber may not be fully enclosed. For instance, it may be be enclosed, for example, on three sides and thus have one open side or face or an open portion on one side or face, such as at the bottom, or may otherwise not be fully enclosed, but, for example, nonetheless be capable of maintaining appropriate conditions on the landing pedestal and grid during operation, such as avoiding or reducing entry of ambient air.

[0093] A “pedestal” refers to a platform, such as a flat-shaped or grooved platform, which is placed on a rod or other support to hold it in place. A “landing pedestal” is a pedestal that can be used for deposition of charged ions in a system herein.

[0094] A “focusing lens” herein refers to a component of a system herein that may be useful in directing a particular charged ion toward a landing pedestal, and therefore increasing the ion flux in a certain area.

[0095] As used herein, the terms “deposition” or “depositing” or “landing” are used interchangeably when referring to placement of a charged ion on a landing pedestal.

[0096] A “grid” herein is a removable disk on the landing pedestal that is used for deposition of charged ions. The grid may be removed, for example, to be placed in a microscope, such as an electron microscope, for imaging of the analytes. Types of “grid” include an “Autogrid®”, which is also known as a “clipped grid”, and grids made of any type of materials and with any type of film. An “Autogrid®” or “clipped grid” refers to a type of grid placed on the landing pedestal that has already been secured in place into a metal ring with a clip, such as a C-clip to enable automated handling by downstream instruments, such as a TEM. Other grids that do not have such features are “unclipped” grids.

[0097] A “cold trap” component of a system herein refers to a component chilled relative to the temperature of the landing chamber gas temperature, such as a component placed into contact with a cooling mechanism, such as a tank or dewar containing liquid nitrogen or liquid ethane or similar substance, and that is capable of collecting condensate from the surrounding air or gas. In some cases, a cold trap is included to help reduce condensation on the landing pedestal or grid during landing under cryogenic conditions when a landing chamber is used that is not fully enclosed during landing. In some cases, a cold trap comprises a metal component such as a metal grid holder.

[0098] A “non-crystalline solid” as used herein refers to a solid form in which molecules are at least partially randomly oriented, and thus, at least partially amorphous in structure. In some embodiments, cryogenic electron microscopy requires a sample to be at least partially amorphous to avoid scattering of the electron microscopy beam, loss of signal, and / or damage of the macromolecules in the sample.

[0099] Other terms may be defined in the sections that follow.

[0100] Exemplary Systems and Components for Performing Soft Landing

[0101] Methods herein may be performed using a variety of soft landing systems. Such systems have several components, which may include those described in this section.

[0102] Electrospray and Charge Reduction

[0103] In some embodiments, a soft landing system utilizes an electrospray (or electrospray ionization, ESI) source to provide gas phase ions, such as charged macromolecular ions, such as charged protein ions. Examples of electrospray sources include a variety of commercially available electrospray devices, such as those adapted from mass spectrometry uses. In some cases, the electrospray source is a charge reduced electrospray source, while in other cases the overall system and methods are run so as to reduce the charge of the macromolecular ions generated in the electrospray ionization.

[0104] An exemplary charge reduced electrospray source, for example, is described in US Patent No. 6,649,907 Bl. In some embodiments, a sealed 210Po source, such as a 210Po static eliminator (e.g., from NRD, Long Island, NY or Amstat Industries, Inc., Lake Zurich, IL), can be used for charge reduction. 210Po decays into alpha particles, which yield a variety of charge reducing species through interactions with air and carbon dioxide. In some charge reduction systems, positively and negatively charged ions containing analyte enter a charge reduction chamber comprising 210Po decay particles, and with a sufficient electric field strength, may be reduced to as low as +1 and -1 charge and neutral charge species. (See also M. Scalf et al., Anal. Chem. 72(1): 52-60 (2000).) In other embodiments, a non-radioactive charge reducing device or an ESI source using an alternative alpha-particle emitting radioactive material may also be employed to reduce the charge of the charged ions.

[0105] In other embodiments, in lieu of a charge reduction electrospray source, one or more solvents or chemical additives known to reduce the positive or negative charge of ions generated from the electrospray source may be added. Examples include water, methanol, acetonitrile, and triethylamine.

[0106] In some embodiments, the charged ions to be landed in the landing chamber are positively charged. In some embodiments, the charged ions to be deposited in the landing chamber have a charge of +1 to +10, such as +1 to +5. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +3. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +2. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1. In other cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -10, such as -1 to -5. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -3. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -2. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1.

[0107] A low charge may be beneficial, for example, in ensuring that the charged ion in droplet form may be subjected to evaporation without causing a charged protein to be ejected and denatured at the air-water interface, for example. In addition, charge reduction occurs within microseconds, thereby minimizing the coulombic repulsion between droplets, resulting in a fewer ion losses. Likewise, the low charge ensures the droplet may proceed through evaporation processes over tens to hundreds of milliseconds without causing a charged macromolecular ion to be ejected, which could result in denaturation at the air-water interface (71). Finally, given a homogenous charge state population, the collisional cross section of a droplet containing a protein is directly proportional to the analyte size (Stoke’s law), rather than the ratio of that size versus total charge (72).

[0108] Differential Mobility Analysis Electrostatic fields may be applied to filter macromolecular charged ions generated by an electrospray source for ions that fall within a particular m / z value. In some embodiments, a differential mobility analyzer (DMA) may be applied to select ions based on their collisional cross sectional (CCS) area (64-66, 74). In a DMA, ions travel through a sheath gas towards a central rod of opposite polarity, resulting in specific trajectories related to an ion’s collisional cross sectional area. At a uniform charge, the CCS area is directly proportional to the size of the macromolecule to be analyzed. Furthermore, a particle diameter may be directly calculated from an nDMA voltage, where protein calibrants are not required due to the known first-order relationship (75). The extension of the DMA to separate very large particles (AAVs, types of aerosols) in the hundreds of nanometers of range has been previously demonstrated (76). The term “particles” herein, in the context of a charged macromolecular ion particle, means droplets or hydrated macromolecular ions or other discreet ion particles found in a vapor stream from electrospray, for example, and separated in a DMA.

[0109] In some cases, a DMA may be a commercially available DMA (e.g., TSI, Inc., Minnesota, USA). In some cases, a DMA may be a nano DMA (nDMA), which includes a shorter rod than a traditional DMA, and thus, may be useful for separating smaller macromolecular ions. In some embodiments, the DMA or nDMA is capable of separating particles of from 10 nm to 1000 nm in diameter, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm in diameter. Thus, in some embodiments, the system herein is capable of depositing particles of from 10 nm to 1000 nm in diameter, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm in diameter on the landing pedestal of a landing chamber, such as a cryogenic-compatible landing chamber.

[0110] A DMA or nDMA may be connected to the electrospray source using conductive tubing. In general, the DMA or nDMA and the electrospray source should be in close proximity to each other, as longer tubing length could result in a longer delay time between ion deflection and current measurement. In general, the apparatus for performing methods herein may comprise a means for connecting the electrospray source, a DMA or nDMA, and a landing pedestal and / or landing chamber to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing pedestal.

[0111] Landing Pedestal and Optional Landing Chamber In some embodiments, macromolecular ions, such as protein ions flow from the electrospray source through a DMA or nDMA or other device to filter the ions and isolate the ion containing the desired analyte, and to a landing pedestal on which the desired ions are landed. In some embodiments, the landing pedestal comprises a grid on which the charged macromolecular ions are deposited during landing.

[0112] In some embodiments, the landing pedestal is enclosed within a landing chamber, such as a cryogenic-compatible landing chamber, or the landing pedestal is contained by a landing apparatus such that atmospheric conditions surrounding the landing pedestal can be controlled. An atmospheric landing chamber, for example, allows the landing process to be run under atmospheric conditions, i.e., under atmospheric pressure conditions. In some cases, the system may be run under ambient air, while in other cases it may be run under a gas such as nitrogen, carbon dioxide, or a noble gas. In some cases, the relative humidity of the air or gas is less than 80%, for example, to avoid moisture retention by the charged ions. In some cases, the relative humidity is less than 75%, less than 70%, or less than 60%, or less than 50%, or less than 40% or less than 20%. Thus, in some embodiments, landing is performed under atmosphere rather than under a vacuum. Thus, in such embodiments, the system does not operate under vacuum and / or the landing chamber does not operate under vacuum, and thus, does not comprise a vacuum chamber. In other cases, however, the landing chamber is under vacuum, and thus is not run under atmospheric conditions.

[0113] In some cases, the system is configured to land ions in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal. In some cases, the gas in the landing chamber is sufficiently dry as to remove water molecules from the charged macromolecular ions as they travel to the landing pedestal or once deposited on the landing pedestal.

[0114] Furthermore, in some embodiments, systems used for methods herein are placed in a room with controlled atmospheric conditions, such as controlled temperature and relative humidity so that the relative humidity of the ambient air that may be used in the system retains the appropriate relative humidity level for optimal operation of the system.

[0115] In some embodiments, a landing chamber or landing apparatus also comprises a focusing lens to further direct the charged ions to the landing pedestal. In some cases, the focusing lens comprises an ion funnel with a preceding gas diffuser. An example of such a focusing lens is provided in Fig. 6A-6F. In some cases, the landing chamber or landing apparatus also includes a temperature control device (e.g., from Omega Engineering, Norwalk, CT). Exemplary Cryogenic-Compatible Landing Chambers and Apparatuses

[0116] In some embodiments, the landing chamber or landing apparatus comprising the landing pedestal and grid onto which the charged macromolecular ions are to be deposited is cryogenic- compatible, thus allowing the methods to be run under cryogenic conditions. In some embodiments, the landing pedestal is in contact with a cooling unit (e.g. through direct contact between a landing chamber or apparatus with the cooling unit) that is capable of cooling the landing pedestal to temperatures below zero degrees Celsius, such as, in some cases, -5 °C or lower, -10 °C or lower, -15 °C or lower, -20 °C or lower, -25 °C or lower, or -30 °C or lower. In some cases, the cooling unit comprises a heat resistant element submerged in a chilled fluid such as liquid nitrogen or liquid ethane or the like. In some cases, the chilled fluid is stored in a dewar. In some cases, the chilled fluid is liquid nitrogen, such as, to allow for nitrogen gas comprising headspace to develop around the landing pedestal and grid from evaporation of the liquid nitrogen. In some cases, the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit. Other means of cooling the chamber or apparatus may also be used in other embodiments. Examples include a Peltier stage cooling device and a cold finger condenser. For example, a Peltier device could be employed to direct cold gas into or around the pedestal. In some cases, the landing pedestal may be mounted on a Peltier stage.

[0117] In some embodiments a cryogenic-compatible landing chamber or apparatus allows the landing process to be run under atmospheric pressure conditions. In some cases, the system may be run under ambient air, while in other cases it may be run under a gas such as nitrogen, carbon dioxide, or a noble gas. In some cases, a cryogenic landing chamber or apparatus may be operated in cryogenic conditions under air or under a gas whose dew point is less than the desired chilling temperature. In some cases, the system is configured to land ions in in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal. In some cases, the gas in the landing chamber is sufficiently dry as to remove water molecules from the charged macromolecular ions as they travel to the landing pedestal or once deposited on the landing pedestal.

[0118] In some embodiments, the landing chamber or apparatus is operated in cryogenic conditions under air or under a gas whose dew point is less than a desired chilling temperature. Furthermore, in some embodiments, systems herein are placed in a room with controlled atmospheric conditions, such as controlled temperature and relative humidity so that the relative humidity of the ambient air that may be used in the system retains the appropriate relative humidity level for optimal operation of the system. In some cases, the landing chamber or apparatus also includes a temperature control device (e.g., from Omega Engineering, Norwalk, CT).

[0119] In some embodiments, the cryogenic-compatible landing chamber or apparatus also comprises a focusing lens to further direct the charged ions to the landing pedestal. In some cases, the focusing lens comprises an ion funnel with a preceding gas diffuser.

[0120] International Application No. PCT / US2023 / 029891, filed August 9, 2023, and incorporated by reference herein, for instance, describes three different landing chamber or apparatus designs that are compatible with methods herein. These three systems are also shown in figures herein. In one system, described for example in Figs. 10-13 herein, a cryogenic- compatible landing chamber is fully enclosed during landing in order to reduce entry of surrounding atmosphere, which could cause condensation inside the chamber and onto the grid, for example, if it were allowed to enter to a significant degree. In this embodiment, the chamber is fully closed during the landing and attaches to the rest of the system via a seal that can be released after landing in order to manipulate the landed molecule on the grid. For example, in this embodiment, the seal is an O-ring seal that can be used to avoid entry of outside air into the chamber so that the gas conditions inside the chamber can be controlled to avoid condensation on the grid. For instance, in this example, the chamber is sealed shut during landing aside from the ports for entry of ions and sheath gas and the like. As shown in that application disclosure, the bottom of the chamber is in connection with a cooling unit that may comprise a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar. After landing, the landing chamber can be opened or removed, so that one can manipulate the grid before or after landing. This system can be operated at temperatures down to -60 °C, or, in some cases, at a temperature of -5 °C or lower, -10 °C or lower, -15 °C or lower, - 20 °C or lower, -25 °C or lower, or -30 °C or lower, or -40 °C or lower, or at temperature ranges between -60 °C and, for example, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, or -40 °C, or at temperature ranges between -40 °C and, for example, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, or - 30 °C.

[0121] In another design described in International Application No. PCT / US2023 / 029891, filed August 9, 2023, a cryogenic-compatible chamber is partially open to the outside atmosphere during the landing process, such as at the bottom of the chamber, so long as condensation on or near the grid of the landing pedestal is sufficiently reduced. See, e.g., Figs. 21 A-B herein. For example, the chamber may enclose the landing pedestal on all sides but the bottom. For example, in this design, in contrast to that of Figs. 10-13, the bottom of the chamber is not sealed, and therefore is open to atmosphere during landing, and the bottom of the chamber rests above at least one cold trap. In some cases, one or more further cold traps are added below the bottom of the chamber. The cold trap(s) may be made of metal, which preferentially attracts condensate from the surrounding air onto the cold trap(s). Below the cold trap(s) rests a tank or dewar containing liquid nitrogen. In some cases, the liquid nitrogen may be filled part-way up the cold traps so that the cold trap(s) are in contact with the liquid nitrogen but so that the cold trap(s) are only partly submerged in the liquid nitrogen and thus are also exposed to the air. In other cases, the cold trap(s) are not in direct contact with the liquid nitrogen.

[0122] This system unsealed at the bottom still was nonetheless sufficient to control condensation on the landing pedestal and grid. This may be due in part to the movement of nitrogen gas from the liquid nitrogen located below the chamber up into the chamber itself due to its open bottom, sufficiently to retain a nitrogen headspace in the chamber, in part to condensate preferentially forming on the exposed surface of the cold trap(s), and in part due to the relatively dry gas flowing through the chamber, with a dew point much lower than the ambient air. For example, the nitrogen headspace may have lower humidity than the surrounding atmosphere, and may effectively displace the atmosphere from the chamber and also prevent outside air from entering the chamber to a significant degree during landing. For instance, depending on the gas chosen for the sheath gas, the dew point of the gas in the head space may be between -52 °C and -62 °C, and, while the dew point of the ambient air is expected to vary from day to day, it is often about 2 °C, which is considerably higher than that of the gas in the chamber head space. This system, compared with the fully enclosed chamber system described above, also can be operated at temperatures at or below -60 °C and in some cases down to liquid nitrogen temperature. As liquid nitrogen temperature is -196 °C, and as the system sits above the liquid nitrogen source, allowing for some heat to be lost, it is estimated that operating at liquid nitrogen temperature indicates an operational temperature of the landing pedestal would be higher than - 196 °C, such as from -130 °C to -170 °C, or -140 °C to -160 °C. Thus, this system may be more compatible with lower temperatures than the fully closed chamber system above. In some cases, this system can be operated at a temperature of -5 °C or lower, -10 °C or lower, -15 °C or lower, -20 °C or lower, -25 °C or lower, or -30 °C or lower, or -40 °C or lower, or at a temperature of - 60 °C or lower, such as -60 °C down to an operational liquid nitrogen temperature, such as -60 °C to -170 °C, -60 °C to -160 °C, -60 °C to -140 °C, -80 °C to -140 °C, -60 °C to -120 °C, or - 120 °C to -140 °C.

[0123] A third design also described in International Application No. PCT / US2023 / 029891 (Fig. 22A-B herein) involves a landing apparatus that positions a landing pedestal comprising a grid in between two platforms, one platform located below the landing grid but above a liquid nitrogen cooling element such as a tank or dewar, and a second platform surrounding and in line with the landing grid, wherein liquid nitrogen gas forms a headspace between the two platforms sufficient to expel the surrounding atmospheric gas so that condensation on the pedestal and grid is reduced. To manipulate the grid after landing, the top platform can be removed. In one example of such a design, the two platforms are cryo-EM clipping stations that are designed to fit around the landing platform. Using clipping stations in this way may allow for simpler manipulation of molecules landed on the grid, as they can be moved to a location on the bottom clipping station platform for storage or analysis. An example of clipping stations that may be configured as platforms in this design are MiTeGen® clipping stations (Ithaca, NY, USA). A photograph of such a device in closed and open configuration is shown in Figs. 22A-B.

[0124] Grids

[0125] In the above systems, the landing pedestal holds a removable grid onto which the charged macromolecular ions are deposited. The grid may be a clipped grid, e.g. clipped in between an Autogrid® ring and C-clip, while in other cases it may be left unclipped (an unclipped grid). The grid may be made from any of a variety of materials suitable for sample support in electron microscopy, such as copper, gold, molybdenum, or nickel. The grid supports a thin layer of electron-transparent material onto which macromolecular ions are landed. This thin foil (commonly referred to as “support film”) may be made from any of a variety of materials, including many that can be used for conventional cryogenic electron microscopy (cryoEM), such as amorphous carbon, graphene, graphene oxide, or silicon monoxide. Alternatively, non- metallic grids may be used, for example silicon grids with silicon nitride support films. In some cases, the grid may be pre-treated prior to soft landing or rendered negatively charged according to methods herein, discussed in detail below.

[0126] Additional Components of Exemplary Soft Landing Systems

[0127] In some embodiments, the systems herein also include a computer processor, which may be used to store software for control of the system. In some embodiments, the processor is connected to the internet to allow monitoring online. The system may also comprise appropriate software for controlling one or more of the electrospray source, DMA, and / or landing pedestal or surrounding chamber or apparatus. Thus, for example, in some embodiments, a computer processor and appropriate software may be used to direct the electrospray of a starting sample, to adjust the electrospray voltage, to control gas flow and / or voltage in a DMA, and to control the temperature and / or atmospheric conditions for landing, and removal or placement of grids before and after a sample is landed, or any combination of these features.

[0128] Methods of Performing Soft Landing The present disclosure encompasses methods of performing soft landing of macromolecules comprising, in some embodiments, (a) ionizing a sample comprising a macromolecular analyte with an electrospray source to form positively charged macromolecular ions; (b) separating the positively charged macromolecular ions on the basis of their collisional cross sectional area, for example, in a DMA or nDMA; and (c) directing positively charged macromolecular ions comprising the analyte to a landing pedestal under cryogenic conditions, and depositing the positively charged macromolecular ions comprising the analyte onto a grid located on the landing pedestal, wherein the grid has been pre-treated with plasma, or by deposition of hydrophilic molecules, or wherein the landing pedestal is potentiated so as to form a negatively charged grid.

[0129] The macromolecular analyte may be any of a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide, such as a protein-protein or protein-nucleic acid complex. In some cases, the macromolecular analyte is a polypeptide or polypeptide complex.

[0130] The electrospray source for performing the methods may be as described above, such as a charge reduced electrospray source. Alternatively charge reduction may be performed by appropriate solvent conditions.

[0131] For soft landing, it may be desirable for the ion containing the analyte to have as low a charge as possible. In some embodiments herein, the ion to be landed is positively charged, and the grid is polar or negatively charged. In some cases, the charged ions to be deposited on the grid have a charge of +1 to +10 or of +1 to +5. In some cases, the charged ions have a charge of +1 to +4 or +1 to +3. In some cases, the charged ions have a charge of +1 to +2. In some cases, the charged ions have a charge of +1.

[0132] The velocity of nanodroplets is a function of tubing diameter and gas flow, as below: 4Q v=^r

[0133] Where v is the nanodroplet velocity, Q is the flow rate, and d is the tubing diameter.

[0134] In some embodiments, the positively charged macromolecular ions are deposited onto the grid in an aerosol stream of from 1 L / minute to 10 L / minute, or from 1 L / minute to 5 L / minute, or from 1 L / minute to 4 L / minute, or from 1 L / minute to 3 L / minute, or from 1 L / minute to 2 L / minute, or at 1 L / minute, 2 L / minute, 3 L / minute, or 4 L / minute, calculated based on a 14 inch tubing diameter, or an equivalent gas flow rate to those above if the tubing diameter is smaller or larger. If the tubing diameter is reduced from ’A” to ’ / s”, for example, the gas flow rate would be reduced by a factor of four to reproduce the nanodroplet velocity. Thus, if a narrower tubing diameter were used, gas flow rates would be reduced by a corresponding factor squared to achieve similar nanodroplet velocity. Similarly, if the tubing diameter is larger than 14 inch, the gas flow rate would be increased according to the equation above. In some such cases, the charged ions to be deposited on the grid also have a charge of +1 to +10 or of +1 to +5. In some cases, the charged ions have a charge of +1 to +4 or +1 to +3. In some cases, the charged ions have a charge of +1 to +2. In some cases, the charged ions have a charge of +1.

[0135] In some embodiments, the method is run under cryogenic conditions comprising a temperature of -60 °C or lower, such as from -60 °C to -170 °C, -80 °C to -170 °C, -60 °C to - 160 °C, -60 °C to -140 °C, -80 °C to -140 °C, or -120 °C to -140 °C. In some cases, the method is performed with an ion having a +1 or +2 charge, with the macromolecular ions being deposited on the grid in an aerosol stream of 1-2 L / minute, and at a temperature of -60 °C or lower, such as from -60 °C to -170 °C, -80 °C to -170 °C, -60 °C to -160 °C, -60 °C to -140 °C, - 80 °C to -140 °C, or -120 °C to -140 °C. In some cases, the method is performed with an ion having a +1 or +2 charge, with the macromolecular ions being deposited on the grid in an aerosol stream of 1-2 L / minute, and at a temperature of -120 °C to -140 °C. In some embodiments, the thermocouple for measuring such temperature is located near the top of the landing pedestal, near the grid surface.

[0136] In some cases, the grid is a removable grid, such as a clipped or unclipped grid. The grid may be made from any of a variety of materials suitable for sample support in electron microscopy, such as copper, gold, molybdenum, or nickel. The grid supports a thin layer of electron-transparent material onto which macromolecular ions are landed. This thin foil (commonly referred to as “support film”) may be made from any of a variety of materials, including many that can be used for conventional cryogenic electron microscopy (cryoEM), such as amorphous carbon, graphene, graphene oxide, or silicon monoxide. Alternatively, non- metallic grids may be used, for example silicon grids with silicon nitride support films. In some embodiments, the method may further comprise removing the grid. In some embodiments, the grid is pre-treated such that it comprises a polar or negatively charged surface for landing of positively charged macromolecular ions. For example, in some cases, the grid is pre-treated with plasma. In some cases, the grid is pre-treated by deposition with hydrophilic molecules, such as saccharides, such as trehalose or sucrose, or other polar molecules such as glycerol or polypropylene glycol (PPG). In some embodiments, other molecules with cryoprotective or fixative properties or other protein-stabilizing solutes may also be used. In some cases, the grid is pre-treated with a saccharide, such as trehalose. In some cases, the charge of the grid surface is induced by applying an electrical potential to the pedestal, such as a DC bias of -IV to - 600V, or -100V to -600V, or -300V to -600V, or -400V to -500V, such as -450V, in order to form a negatively charged grid. In some cases, the positively charged macromolecular ions are deposited on the grid as a charged droplet or a charged macromolecule comprising a hydration shell. In some cases, the droplet or hydration shell is deposited as a non-crystalline solid on the grid upon landing and cooling. In some embodiments, particular solution components are added to the sample in order to encourage the deposited droplet or hydration shell surrounding the macromolecular ions to form a non-crystalline solid on the landing pedestal or grid, or that otherwise are known to help prevent formation of crystalline water. Examples include solvents such as methanol or ethanol or certain compounds such as silicon or silicon oxide. In addition, in some cases, the macromolecular ions may not have a preferred orientation, and may be arranged in random orientations within the droplet.

[0137] In some cases, the method further comprises removing the grid after deposition of the positively charged macromolecular ions on the grid. In some such cases, the grid is removed under a gas with a dew point less than the operating temperature of the landing pedestal (i.e., less than the operating temperature of the landing chamber or apparatus). This may avoid condensation on the deposited macromolecular ion sample on the grid to the extent possible. The method, in some embodiments, further comprises submerging the removed deposited grid in liquid nitrogen, for example, in preparation for analysis. In some embodiments, the charged macromolecular ion adheres to the landing pedestal or grid without formation of a covalent bond. In some embodiments, it adheres to the landing pedestal or grid without significant loss of structure, complexation, or folding modifications compared to its native state in solution.

[0138] In some embodiments, when preparing cryogenic samples, such as for cryoEM analysis, it may be necessary to reduce the amount of condensate in or around the landed ions on the grid as much as possible in the system and / or when transferring the grids to a cryoEM device for analysis. In some embodiments, one may first dry the landing pedestal with dried air, such as from a hair dryer or similar device, in order to reduce the relative humidity, prior to chilling the landing chamber and conducting a landing experiment in the system. After landing, in some embodiments, a grid containing the landed ions may be transferred from the landing pedestal to a chilled holder, which may be of any appropriate size or shape to hold the grid, under a gas with a dew point less than the chilled temperature, such as nitrogen gas, carbon dioxide, or a noble gas. See, e.g., Figs. 14-16B for an example of one means of transferring a chilled landing grid. In some cases, the grid with the landed ions may be stored in chilled conditions for later manipulation, such as under liquid nitrogen.

[0139] In some methods herein, the method is performed with a soft landing device comprising a landing chamber or apparatus such as that described above and elsewhere in the Examples and figures herein. In some cases, the landing chamber is operated under ambient air under atmospheric conditions. In other cases, it is operated under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%, less than 60%, less than 40%, or less than 20%. In some cases, the method is performed in a controlled temperature and humidity room in order to control the ambient air temperature and humidity.

[0140] For example, in some cases the electrospray source is a charge reduced electrospray source. In some cases, the step of separating the positively charged macromolecular ions on the basis of their collisional cross sectional area is performed by a DMA, such as an nDMA. In some cases, when performing methods herein, the DMA or nDMA transmission voltage needed for a particular analyte is known or can be accurately estimated prior to performing the method, for example, based on the calculated particle diameter for the analyte. In other cases, prior to performing the deposition experiments, it may be necessary to perform a scan over the expected transmission voltage range and to collect a spectrum of sample ions over the range of voltages in order to select the optimal voltage for use in the DMA or nDMA during the method.

[0141] In some systems used for performing these methods, the landing pedestal comprising the grid is enclosed within a landing chamber. In some cases, the landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature. In some cases, the system comprises an electrospray source, a DMA or nDMA, and a landing chamber. In some such cases, the source, DMA or nDMA, and the landing chamber are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing pedestal. In some cases, the system further includes a means of connecting the landing chamber to a DMA or nDMA to allow flow of the positively charged macromolecular ions from the DMA or nDMA to the landing pedestal. In some cases, the landing chamber further comprises a focusing lens, which may, in some embodiments, include an ion funnel with a preceding gas diffuser. In some cases, the landing pedestal is in contact with a cooling unit, wherein the cooling unit optionally comprises a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar. For instance, the cooling unit may be in direct contact with a landing chamber enclosing the landing pedestal, or it may be in direct contact with the bottom of the pedestal. In some cases, the landing pedestal comprises a central tube allowing flow of gas cooled by the cooling unit. In some cases, the system further comprises an automatic temperature control element, for example, to ensure that the temperature is below a particular level or within a particular, preferred range. The landing pedestal may be enclosed within a landing chamber or landing apparatus in some cases, such as to help control the atmosphere surrounding the landing pedestal and grid. In some cases, the landing pedestal is fully enclosed within the landing chamber during landing. In other cases, the landing pedestal is enclosed within a landing chamber, but the bottom of the landing chamber is open to atmosphere during landing. In such a case the chamber may be located above one or more cold traps, wherein the one or more cold traps are optionally made of metal, wherein the one or more cold traps are optionally in direct contact with liquid nitrogen. For example, this design allows vaporized liquid nitrogen to form a nitrogen gas head space in the landing chamber during landing, thus keeping ambient air away from the grid. In some embodiments, a landing chamber further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. Methods herein may also be performed in a system in which the landing pedestal is comprised within a landing apparatus, in which apparatus the landing pedestal is located above a first platform, the first platform being in contact with a liquid nitrogen source, and wherein the top of the landing pedestal is below or in line with a second platform, wherein the second platform and the first platform together are positioned so as to allow vaporized nitrogen gas from the liquid nitrogen source to displace atmospheric air at the top of the landing pedestal, wherein optionally the first and / or second platform is a cryo EM clipping station. In such a design, the landing apparatus might not form a chamber, yet is designed so as to maintain a controlled cryogenic atmosphere around the landing pedestal and grid. In some such cases, the landing apparatus further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. And in some embodiments of this system, the electrospray source, a DMA or nDMA, and landing apparatus are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing pedestal.

[0142] The Examples below provide further nonlimiting details of methods disclosed herein and soft landing devices on which they may be performed. The Examples below are intended to be exemplary only and are not intended to limit the scope of the disclosure.

[0143] EXAMPLES

[0144] Examples 1-5 describe exemplary soft landing device (SLD) systems on which the methods herein may be run and exemplary system components. The present disclosure is not intended to be limited to such SLD systems, and methods herein, accordingly, may also be run on other systems not described below.

[0145] Example 1: Electrospray source, nDMA, and focusing lens

[0146] A soft landing device (SLD) (Fig 1) comprised a charge reducing electrospray source, a nano differential mobility analyzer (nDMA) (TSI Inc., Shoreview, MN), and a landing chamber, and optionally a focusing lens. Several different landing chamber designs are described in subsequent examples.

[0147] Techniques for electrospray are extensively described in the literature (16). A charge reduced electrospray source included an inlet for a capillary with a pulled or ground electrospray emitter stabilized by differentially controlled air and carbon dioxide. The ESI tips sit just outside a charge reduction chamber, where a 210Po static eliminator (NRD, LLC, Long Island, NY) decays into alpha particles, which yield a variety of charge reducing species through interactions with the air and CO2. With the application of voltage at a liquid junction, positive and negatively charged liquid droplets enter this field, and, given sufficient field strength, are reduced to -1, +1, and neutral charged species.

[0148] Charge reduction theoretically offers a number of benefits to soft landing. The charge reduction occurs within microseconds, thereby minimizing the coulombic repulsion between droplets, resulting in a fewer ion losses. Likewise, the low charge ensures the droplet may proceed through evaporation processes over tens to hundreds of milliseconds without causing a charged protein to be ejected, which could result in denaturation at the air- water interface (70). Finally, given a homogenous charge state population, the collisional cross section of a droplet containing a protein is directly proportional to the protein size (Stoke’s law), rather than the ratio of that size versus total charge (71).

[0149] A comparison was made for the theoretical deposition energy of a +1 protein at atmosphere versus a +20 protein under vacuum in a Q Exactive™ UHMR (Thermo Fisher Scientific, Bremen, DE). At atmosphere, the +1 ion would land with 8 x 10'6eV, versus 63 eV for a +20 ion under vacuum moving at 226 m / s, such as in the recent example of Westphall et al. 2022 (27). Energy would have to be reduced under vacuum to a velocity of 100 m / s to fall under the classical “soft” definition of < 35 eV; without moving to lower charge, there is a limitation to how far velocity can be reduced due to the optics and gas expansion acceleration that occurs in a vacuum based system (72). Thus, the current design is nearly 6-orders of magnitude softer than those at the state-of-the-art, and inversely lower to the charge state of ions deposited by the time- resolved velocity spray atmospheric system (0-50-fold reduced). Differential mobility analyzers offer a low-resolution technique for separating charged droplets / analytes on the basis of collisional cross sectional area (63-65, 73). Ions travel through a sheath gas towards a central rod of opposite polarity, resulting in specific trajectories related to an ion’s collisional cross sectional area. All experiments described here are performed to detect positive-mode ions. The particle diameter may be directly calculated from the nDMA voltage, where protein calibrants are not required due to the known first-order relationship (74). While large proteins or complexes such as AAV capsids could be observed with standard fused silica capillary, proteins in the hundreds of kilodalton range required either polyacrylamide or methylated coated capillary to prevent nonspecific interactions. For proteins less than 45 kDa, Sigmacote (Sigma Aldrich, St. Louis, MO) was added to prevent binding to the capillary (Fig 2). No changes to the mean mobility or the hydrodynamic distribution were observed.

[0150] Example 2: Atmospheric landing chamber and focusing lens

[0151] A landing chamber designed for room temperature atmospheric landing was first constructed (Fig 3). The landing chamber was connected to the DMA outlet by rubber tubing, where ions were propelled through the line by a gas flow. The sheath gas exhaust from the DMA was plumbed into the landing chamber, such that it provided a secondary flow of inert gas to fill the chamber. Ions were initially directed through the landing chamber to a glass rod filled with steel wool soldered to a wire that transmitted signal via connection to a detector. The glass rod / pedestal was eventually replaced with a brass rod designed with a special seat to hold unclipped grids or clipped Autogrids®, with space to insert a tweezer to move grids (Fig 4-5). In a different format, brass rods with a smooth top were used, and copper or other types of conductive tape were used to secure grids to the pedestal. Ions were tested for landing directly onto the pedestal or a grid-on-pedestal setup by measuring the transmitted current.

[0152] A focusing lens was included in the atmospheric SLD that integrated an ion funnel with a preceding gas diffuser (Fig 6). Optimized focusing voltage was a function of the protein / particle mass / mobility, where higher voltages were required to transmit larger species. Different current measurement approaches were investigated to enable quantitation, where the pedestal current differential, normalized at each voltage, was considered the most reliable approach, followed by a comparison of the absolute amplitude of a protein across a voltage sweep scan (Fig 7). It was observed that for each protein, to obtain reliable current measurements, the nDMA voltage ramp rate and the nDMA voltage flat time (time to return to ground) must both be above certain thresholds, or else the peak shape and signal became unreliable (Fig 7). For measurements made at a constant nDMA voltage and across variable focusing lens voltages, normalization was required at each new focusing lens setting, demonstrating that the pedestal was not held at a true ground. The pedestal’s ability to build a charge throughout the course of experiments was further demonstrated by measuring the transmitted current pre and post a focusing voltage sweep. There was a significant difference in the two voltages detected pre / post experiment despite a grounding wire on the pedestal, suggesting that charge may be building within the chamber and unable to dissipate efficiently (Fig 8). Thus, when pedestals were later constructed of all metal for cryogenic landing, rather than for current measurement and atmospheric landing, the pedestals were additionally grounded.

[0153] Example 3: First cryogenic landing chamber

[0154] Many of the challenges of Cryo-EM would be solved by soft landing, including preferential orientation, heterogeneity, and contrast challenges. Given that a protein was sufficiently hydrated, rather than nearly dry as under vacuum systems, issues associated with gas-phase denaturation, where a native protein begins to turn “inside-out” in the mass spectrometer (87), could also be avoided. While a hydrated protein would still have air-water interfaces, soft landing is a fast technique. A traditional mass spectrometer’s cycle time is in the tens of microseconds for a time-of-flight instrument (88) and in the tens of milliseconds in an Orbitrap mass spectrometer (where cycle time excludes measurement of the image current in the Orbitrap) (89). For a differential mobility instrument, transient times are dependent on sheath, aerosol flows, and an ion’s collisional cross sectional area, and would therefore range from the high hundreds of microseconds to single millisecond range (90). In each of these cases, these transient times are below the expected time of migration for a protein in a droplet, which is largely thought to be 100-500 ms, depending on the protein, in traditional EM (91). Prior work related to “voltage assisted spray” has demonstrated that nearly no denaturation of apoferritin occurred when sprayed for 36 ms prior to freezing (55). A first cyrogenic-compatible landing chamber was centered around two concepts. The first was a hypothesis that proteins with nanometer sized water shells would form amorphous ice under relatively warm conditions (0 to - 60 C), although it was not clear at which density level. Nanolayer ice formation has been studied in a limited manner in nanolayer bulk solutions (92-98), and never in the case of droplets moving through a chilled field. Currently, Cryo-EM’ s standard vitrification must use liquid ethane preferably (5), or at least liquid nitrogen (99), temperatures to cool protein liquid films fast enough to form low density amorphous ice, so this would represent a major advancement. The second hypotheses was that vacuum systems were not required to land proteins on a dry chilled surface, rather, that an inert atmosphere within a landing chamber would suffice, and that grid extraction could be achieved by external atmosphere controls. Pictured in Fig 9 are photos of a device configured for either atmospheric use (Fig 9A) or cryogenic use (Fig 9B). The device remained in the same configuration through the outlet of the nDMA between the two systems. The bottom of the chamber was open and grooved for an Ciring seal (Fig 10). The cryo-landing chamber was seated on a sliding and locking rod (Fig 11). The inlet was connected to the nDMA “aerosol out” with rubber tubing, and the sheath gas was connected to the Swagelok union port at the top of the chamber. The dew point of the “aerosol out” gas from the nDMA, with an ESI spray flowing or off, was measured at -62 C or -50 C, respectively. Given a need to further dry the gas and reduce the dew point, a separate desiccant system was built and installed, though is currently not in use. The open-ended landing chamber slides down into place on an O-ring in a seated metal dish. In the center of the O-ring, a gas line assembly was made to allow gas to enter or exist the system (Fig 12). The gas line assembly was covered in three layers of foam insulation to minimize heat transfer to the environment. The gas assembly was made of metal from the liquid nitrogen rubber dewar cap up to the point where a right angle turn to reach the landing pedestal was required, at which point it was replaced with doubly insulated rubber tubing. Multiple types of insulation were tested to achieve the best insulation possible. Special cryogenic pedestals were designed (Fig 13) which allowed for a chilled gas to circulate and cool the landing surface. A cooling system was developed by submerging a resistant heating element in liquid nitrogen stored in a dewar (Figure 14). Real time correction of the temperature was made through a powered temperature control unit that supplied voltage to the element and monitored the temperature.

[0155] Operation of the cryogenic unit was critical to preventing buildup of condensate on the chilled grid / pedestal. As such, the critical points of the user operation include those described here. The nDMA gas was kept on but the nDMA voltage was turned off to prevent protein transmission. Prior to turning on the chilling gas, the cryo-landing chamber was slid up, and the rubber tubing between the metal gas line assembly and the pedestal was disconnected. A hair dryer was used to warm the metal dish / pedestal / assembly to room temperature and to ensure all parts were fully dry. A clipped or unclipped grid was loaded onto the pedestal using EM tweezers. The cryo-landing chamber was slid down, pressed into the O-ring, and locked in place. After a minute of allowing the nDMA exhaust gas to purge any atmosphere from the cryo- landing chamber, the rubber tubing was connected to the metal cooling gas assembly and the heating control was set to the desired temperature (for the following experiments -20 C). When temperature was reached (~30 sec), the nDMA transmission voltage was turned on, and landing was allowed to proceed for the desired time length (for the following experiments, generally 20 min). After landing was finished, the nDMA voltage was turned off. A “banana” dewar custom designed to fit the cryogenic metal dish was filled with liquid nitrogen (fig 15). A grid / AutoGrid box was seated into a custom box holder (fig 16), that was then placed in the dewar. The dewar was transferred with tongs or a special tool into the metal dish around the cryo-landing chamber (Fig 11). It was given ~l-2 min to fill the air with N2 headspace. The cryo-landing chamber was then unsealed from the O-ring, but not lifted. An independent N2 gas supply was connected via rubber tubing to the tee previously acting as the exhaust, and turned on at a low level (<4 L / min) as a reverse sheath gas. The cryo-landing chamber was then lifted, and a cap was slid in place to trap the liquid nitrogen headspace from evaporating (Fig 17B). Tweezers were used to pickup the grid and then rotate it such that the surface of the grid was parallel to the direction of the reverse sheath gas. The grid was quickly transferred into the grid box holder under liquid nitrogen, and subsequently stored. The timing of these steps as well as the orientation of the various caps used were critical to preventing condensate from occurring on the surface of the grid / pedestal. Given a desire to perform a system check prior to cryogenic landing, a set of cryogenic metal dish compatible pedestals for use at atmosphere that connected to a detector were also constructed. A custom hat was made for shielding to replace the door that was previously used in the atmospheric setup (Fig 17 A). The design has the same and enhanced functionality of the atmospheric device previously described. The device voltages and control were set either manually through power controllers, or through a computer interfaced to a national instrument control box.

[0156] Initially, Cryo-EM results with landing under cryogenic conditions proved challenging. It was observed that nearly all of the graphene or graphene oxide films, regardless of mesh size, ripped during landing when tested with lacy carbon or Quantifoil® AutoGrids (data partially shown, Fig 18). Interestingly, these grids did not rip in the atmospheric only system. Any application of a plasma treatment to the grid surface significantly exacerbated this problem. On these grids, we were able to observe that GroEl was landing with minimal condensate developing (any condensate shown is at levels expected with the standard grid transfer steps into the Glacios™ EM). 1 layer graphene over 2000 mesh Cu grids showed the most robust behavior with only about 50% (versus near 100%) of the films ripped. On these grids, GroEl was deposited over the graphene film with multiple different orientations, and the barrel versus top- down views could be distinguished. Interestingly, GroEl was also observed as multimers within a single frozen droplet, but not in the form of a complex. These orientations are highlighted in the inlay in Figure 19A. In addition to the GroEl -like particles and multimer particles, smaller GroEl species, potentially representing GroEl subunits were observed. These may be a contaminant, a grid feature, or a result of landing. Consequently, 2D averages were not obtained due to a lack of a single homogeneous sub population. The contrast observed in the cryo-EM images was qualitatively at least as good, if not above, that observed in control experiments (Figure 19B). Importantly, class averages were next performed on the protein-less ice balls that were not condensate (Figure 20). Without an x-ray experiment, it is impossible to determine the exact ice structure. But the lack of a high-intensity ring at the 1 / 3.7 A spatial frequency corresponding to the (002) diffraction spots of cubical and hexagonal ice in the averaged 2D amplitude spectrum of all ice balls suggests that some form of amorphous (unknown if low or high density) ice was achieved, and thus landing at -20 C, rather than under much colder conditions, is a suitable approach for soft landing cryo-EM.

[0157] Example 4: Second cryogenic-compatible landing chamber system

[0158] New hardware was developed for landing at liquid nitrogen temperatures. In comparison to the landing chamber of Example 3, the metal landing chamber dish with a Styrofoam dewar was removed. A hole of about 2 / 16 inches was then drilled in the center of the dewar, which contains liquid nitrogen during operation, in order to accommodate the landing pedestal. A tight seal around the pedestal prevented the liquid nitrogen from leaking, and allowed the pedestal to be moved up and down in height without the liquid nitrogen leaking. The tee valve exhaust holding the pedestal was shortened and the exhaust was capped with a nut. A photograph of this system is shown in Figs. 21 A-B, and an example of GroEL landed at liquid nitrogen temperature at 73,000 magnification is shown in Fig. 21C.

[0159] Before operation, the system was brought to room temperature and dried. The grid was placed on the pedestal and raised into place. At least two cold traps were added to the system. Cold traps consisted of large pieces of metal that were raised higher than the liquid nitrogen fill line such that any condensate would preferentially condense onto them. The landing chamber was placed above the metal cold traps. The landing chamber was operated in an open-to- atmosphere position, such that the bottom of the chamber, which is open to atmosphere, sat right above the cold traps. The liquid nitrogen was manually filled during landing, which ranged from 5-20 min in time. At the start of the landing time, the focusing lens was turned on and the DMA was set to transmit at the desired protein transmission voltage. After landing, the DMA voltage was turned off and the focusing lens was set to off. The pedestal was pulled down until it hovered above the liquid nitrogen fill line. The tweezers were prechilled, and used to grab the grid and dunk it in liquid nitrogen as the chamber was simultaneously lifted. The grid was then stored as previously described.

[0160] The key features that differentiate this second landing system from the cryogenic- compatible system described in Example 3 are that the landing chamber is open to atmosphere at the bottom instead of being closed to atmosphere, and the temperature of freezing that could be achieved. Remarkably, no increase in condensate on the grid was observed despite the chamber being open to atmosphere at the bottom, which is attributed to atmosphere being pushed away by the liquid nitrogen head space & the sheath gas. For example, because liquid nitrogen in the dewar below the chamber vaporizes, it forms a nitrogen gas head space inside the chamber above. Furthermore, the “open-to-air” landing approach should have an approximate 2-fold decrease in the landing kinetic energy. As the gas is no longer directed into a constricted exhaust port, it is no longer accelerated to the same degree, and thus the particles move slower prior to landing.

[0161] Further data, shown in Figs. 23 A-B, indicate that a combination of the sheath flow and the liquid nitrogen headspace in the chamber are important in controlling condensate in the openbottom, unsealed chamber system. For example, Fig. 23 A compared to Fig. 23B shows that at 10 L / min flow, there are significantly larger ice crystals on the grid than at 20 L / min flow, when excluding the largest crystals which are a consequence of grid transfer steps.

[0162] Example 5: Third cryogenic-compatible landing system

[0163] A further alternative system for landing was also designed that was relatively easy to operate, and that does not include a focusing lens component. It consisted of using two MiteGen® (Ithaca, NY, USA) clipping stations stacked on top of each other, and a 6 mm grid pedestal positioned halfway up the hole of the second station. A photograph of the system in closed and open configurations is shown in Figs. 22A-B. The landing pedestal consisted of a 6 mm threaded rod held in place with a thread adapter on a PTFE plate, and pushed through a foam dewar. By positioning the rod halfway up the second (top) station, the walls of the holder acted as boundaries to keep the grid secure during landing, to ensure that liquid nitrogen did not fill the void, and to limit condensate. Into the top of the hole of the second (top) clipping station, tubing from the nDMA was inserted, such that there was about 2 mm between the ion exit and the grid. The holes of each clipping station were therefore directly on top of each other, so the grid was located in line with the entry of the aerosol out gas flow. The top clipping station conveniently acted as a cold trap for the system, and liquid nitrogen was filled to a level a few millimeters below the height of the pedestal. During grid removal, the top station was lifted, and the landing grid was simultaneously pushed onto the bottom station before it was picked up with tweezers and stored. Because, in this system, clipping stations were used as platforms surrounding the landing pedestal and grid, this system has the possibility of being more easily automated such that, after landing, the grid may be moved to a location on the bottom station, for example, for storage. Fig. 22C shows GroEL particles from landing in this system. While only few particles were observed, the amount of condensate was minimal, demonstrating the potential of this system design.

[0164] Example 6: Impact of Grid Surface Properties on Soft-Landed Protein Complexes

[0165] Experiments were performed in the SLD of Example 4 on soft landing of GroEL particles or AAV capsids with different alterations of grid surface properties. Protein droplets were deposited onto grids in that SLD system in an aerosol stream of 2.5 L / minute (tubing radius 14 inch) and with a grid temperature of about -130 °C. When imaged by cryoEM, the proteins have hydration shells with radii of up to about 2 nm. The impact of modifying the hydrophobic surface of EM grids by treatment with plasma or by deposition of the polar molecule trehalose, or the impact of rendering the grid negatively charged by applying potentiation (e.g., with a DC bias) to the landing pedestal, on the landed droplets was assessed. Pelco easiGlow™ was used for plasma treatment in air with programmable features: negative polarity, 2 minute program time, 0.39 mbar pressure, and 15mA set current. Grids were blotted by placing the grids onto 3% trehalose (w / w, EMD Millipore Billerica MA) for one minute, then excess liquid was blotted away with filter paper. Grids were potentiated from a high voltage power supply (Bertran Associates Inc Series 225) by attaching a lead to the brass pedestal on which the grid sits.

[0166] The impact of grid surface properties on the shape of frozen droplets is demonstrated in Fig. 24, contrasting soft-landed AAV capsids in 200 mM ammonium acetate on unmodified amorphous carbon support film (nominally 3-4 nm -thick continuous amorphous carbon film on copper grid, model CF150-CU-UL, Electron Microscopy Sciences), which is expected to present a hydrophobic surface, with the same sample landed on grids with plasma-treated C foil before the landing experiment. Deposition onto plasma-treated foils led to higher density of frozen nanodroplets seen by cryoEM compared to landing onto untreated foils. In addition, the proteincontaining nanodroplets were significantly smaller and more spherical with the plasma treatment. We believe that the transition to a hydrophilic or charged surface increased the wettability of the droplet on the surface, decreased splattering on the surface and likely increased the freezing rate, resulting in more spherical and clearly identifiable protein-containing droplets. The contrast between the frozen protein-containing nanodroplets (Fig. 24B) and the background (thin support foil) was also significantly greater than that seen in conventional cryoEM experiments between biological macromolecules and the surrounding vitreous ice layer in which they are embedded.

[0167] Grids (CF150-CU-UL, Electron Microscopy Sciences) were next modified by plasma treatment, coating with trehalose, or potentiation with a DC bias (-450V), and the effects of these modifications were compared, as shown in Figs. 25A-C. Variations were observed in the average cryoEM image intensity in the core of AAV capsids, which we interpret as variations in the degree of filling of capsids with buffer solvent. In these experiments, capsids soft-landed onto grids when a DC bias was applied (referred hereafter as “negatively charged grid”) appeared more hydrated than those landed on a trehalose-coated surface, which in turn appeared more hydrated than those landed on a plasma-treated surface. These effects suggest that the interplay between wettability, freezing rate, and evaporative cooling is an important consideration in tuning the system to produce ideal particles for cryoEM, in cases where preserved internal hydration is important to preserve the protein native state.

[0168] The contrast of the landed proteins in Fig. 25 was significantly increased compared to traditional cryoEM preparation of proteins in bulk solution. From 2D cryoEM images, however, significant deformations of AAV capsids had occurred in many of the droplets , particularly when landed onto plasma-treated or trehalose-coated surfaces. (Figs. 25A-B.) To investigate this further, we collected a series of tilted cryoEM images of AAV capsids soft-landed onto trehalose-coated carbon foil grids. This allowed us to obtain 3D tomographic reconstructions of the capsid nanodroplets (e.g., Figure 28), which show that the radius of curvature of the top of the droplet is consistent with an intact AAV capsid surrounded by a very thin hydration layer, on the order of a nanometer. The bottom of the AAV appears partially flattened onto the carbon surface. This is consistent with a process (we presume on the millisecond timescale) whereby the nanodroplet wets the surface, the capsid partially collapses, and the nanodroplet then freezes without experiencing significant rebound.

[0169] To test whether a sufficient number of apparently intact soft-landed AAV capsids soft- landed onto surfaces held at a negative DC potential could be averaged to yield high-resolution average structure, we collected cryoEM images of a “negatively-charged grid” under imaging conditions suitable for high-resolution structure determination from conventional cryoEM grids. A subset (11,551 of 72,637) of capsid images appeared intact in 2D and yielded class averages featuring a well-resolved symmetric ring of density corresponding to the protein capsid (Figure 27). But we were unable to derive high-resolution reconstructions, suggesting that the high- resolution features of the native capsid structures were not preserved in this experiment.

[0170] Nanodroplets containing GroEL were also soft-landed onto a negatively-charged grid, leading to cryoEM images in which the characteristic overall shape of GroEL particles was recognizable (Figure 26). In averages of top-view images (Figure 26, first panel), internal heptameric features and an open core were observed, and in averages of side-view images a low- density (dark) equatorial line separating the two heptamers of GroEL were visible. Together, these observations suggest that key features of the native protein fold were preserved in the core of GroEL.

[0171] In addition, we did not find evidence for a preferred orientation of soft-landed GroEL particles (either top or side views). This is in contrast with the well-known preferred orientation of GroEL particles when observed by conventional cryoEM and may indicate a major advantage of soft landing over conventional cryoEM in that the landed particles may have a random orientation. On the other hand, average images of GroEL did not contain any high-resolution features outside of its core, suggesting the fine native structure had not been preserved in these experiments, perhaps due to dehydration.

[0172] In the absence of protein dehydration, protein structural damage in the soft landing system of Example 4 could be a result of an inelastic collision between the protein and the grid surface. The velocity of the nanodroplets can be modulated by the gas flow in which the ion is carried into and through the landing chamber. To test whether modulating nanodroplet velocity would affect the structural integrity of AAV capsids, we compared cryoEM images of “negatively-charged” grids onto which capsid nanodroplets had been landed with gas flows of 1, 2.5, or 3.5 L / min. Following 2D classification of -8-15,000 images of capsid-containing nanodroplets from each grid, we counted the number of images contributing to classes featuring approximately symmetric AAV capsids. By that measure, approximately 33%, 10% and 7% of capsids were approximately symmetrical when landed with gas flows of 1, 2.5, and 3.5 L / min respectively, suggesting that reduced ion velocity can contribute significantly to preserving protein structure during soft landing.

[0173] Additional changes that may reduce protein damage on landing include, for example, shortening the landing chamber to reduce the distance of the grid pedestal from the liquid nitrogen; using a liquid nitrogen-filled pedestal; and chilling the sheath gas in the landing chamber to cool nanodroplets in flight, before landing. Other potential modifications to the soft landing system include decreasing the angle of droplet landing, determining optimal degree of pedestal charge, tuning the protein’s water shell thickness, and modifying buffer components.

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Claims

What is claimed is:

1. A method of preparing and isolating positively charged macromolecular ions comprising an analyte, the method comprising: a. ionizing a sample comprising a macromolecular analyte with an electrospray source to form positively charged macromolecular ions; b. separating the positively charged macromolecular ions on the basis of their collisional cross sectional area, for example, in a DMA or nDMA; and c. directing positively charged macromolecular ions comprising the analyte to a landing pedestal under cryogenic conditions, and depositing the positively charged macromolecular ions comprising the analyte onto a grid located on the landing pedestal, wherein the grid has been pre-treated with plasma, or by deposition of hydrophilic molecules, or wherein the landing pedestal is potentiated so as to form a negatively charged grid.

2. The method of claim 1, wherein the macromolecular analyte is a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide.

3. The method of claim 1, wherein the macromolecular analyte is a polypeptide or polypeptide complex.

4. The method of any one of claims 1-3, wherein the positively charged macromolecular ions have a +1 to +5 charge.

5. The method of claim 4, wherein the positively charged macromolecular ions have a +1 charge.

6. The method of any one of claims 1-5, wherein the positively charged macromolecular ions are deposited onto the grid in an aerosol stream of from 1 L / minute to 10 L / minute, or from 1 L / minute to 5 L / minute, or from 1 L / minute to 4 L / minute, or from 1 L / minute to 2 L / minute, or at 1 L / minute, 2 L / minute, 3 L / minute, or 4 L / minute.

7. The method of any one of claims 1-6, wherein cryogenic conditions comprise a temperature of -60 °C or lower, such as from -60 °C to -170 °C, -80 °C to -170 °C, - 60 °C to -160 °C, -60 °C to -140 °C, -80 °C to -140 °C, or -120 °C to -140 °C.

8. The method of any one of claims 1-7, wherein the grid has been pre-treated with plasma.

9. The method of any one of claims 1-7, wherein the grid has been pre-treated by deposition of trehalose.

10. The method of any one of claims 1-7, wherein the landing pedestal is held at a potential of -100V to -600V, or -200V to -600V, or -300V to -600V, or -400V to - 500V, or -450V to form a negatively charged grid.

11. The method of any one of claims 1-10, wherein the positively charged macromolecular ions are deposited on the grid as a charged droplet or a charged macromolecule comprising a hydration shell.

12. The method of claim 11, wherein the positively charged macromolecular ions are deposited in random orientations within the droplet or hydration shell.

13. The method of any one of claims 1-12, wherein the grid is a removable grid.

14. The method of any one of claims 1-13, wherein the grid comprises an amorphous carbon, graphene, or graphene oxide support film.

15. The method of any one of claims 1-14, wherein the method further comprises removing the grid after deposition of the positively charged macromolecular ions on the grid, optionally wherein the grid is removed under a gas with a dew point less than the operating temperature of the landing chamber.

16. The method of claim 15, further comprising submerging the removed grid with deposited ions in liquid nitrogen.

17. The method of any one of claims 1-16, wherein the electrospray source is a charge reduced electrospray source.

18. The method of any one of claims 1-17, wherein the separating of the positively charged macromolecular ions on the basis of their collisional cross sectional area is performed by an nDMA.

19. The method of any one of claims 1-18, wherein the landing pedestal is enclosed within a landing chamber.

20. The method of claim 19, wherein the landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature.

21. The method of claim 19 or 20, wherein the electrospray source, an DMA or nDMA, and landing chamber are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing pedestal.

22. The method of any one of claims 19-21, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the positively charged macromolecular ions from the DMA or nDMA to the landing pedestal.

23. The method of any one of claims 19-22, wherein the landing chamber further comprises a focusing lens.

24. The method of claim 23, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

25. The method of any one of claims 1-24, wherein the landing pedestal is in contact with a cooling unit, wherein the cooling unit optionally comprises a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar.

26. The method of claim 25, wherein the landing pedestal comprises a central tube allowing flow of gas cooled by the cooling unit.

27. The method of claim 25 or 26, wherein the landing pedestal is in contact with the cooling unit indirectly through a landing chamber enclosing the landing pedestal being in direct contact with the cooling unit.

28. The method of any one of claims 1-27, wherein the system further comprises an automatic temperature control element.

29. The method of any one of claims 19-28, wherein the landing pedestal is fully enclosed within the landing chamber during landing.

30. The method of any one of claims 19-28, wherein the bottom of the landing chamber is open to atmosphere during landing, and wherein the chamber is located above one or more cold traps, wherein the one or more cold traps are optionally made of metal, wherein the one or more cold traps are optionally in direct contact with liquid nitrogen, and wherein vaporized liquid nitrogen forms a nitrogen gas head space in the landing chamber during landing.

31. The method of claim 30, wherein the landing chamber further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

32. The method of any one of claims 1-18, wherein the landing pedestal is comprised within a landing apparatus, in which apparatus the landing pedestal is located above a first platform, the first platform being in contact with a liquid nitrogen source, and wherein the top of the landing pedestal is below or in line with a second platform, wherein the second platform and the first platform together are positioned so as to allow vaporized nitrogen gas from the liquid nitrogen source to displace atmosphericair at the top of the landing pedestal, wherein optionally the first and / or second platform is a cryo EM clipping station.

33. The method of claim 32, wherein the landing apparatus further comprises a focusing lens, optionally wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

34. The method of claim 32 or 33, wherein the electrospray source, an DMA or nDMA, and landing apparatus are connected to allow electrospray gas flow from the source, through the DMA or nDMA, and to the landing apparatus.

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